Escherichia coli

Eur J Biochem, 1989 Sep 15, 184(2), 345 - 52
Interaction of elongation factor Tu from Escherichia coli with aminoacyl-tRNA carrying a fluorescent reporter group on the 3' terminus; Ott G et al.; Transfer ribonucleic acids containing 2-thiocytidine in position 75 ({s2C}tRNAs) were prepared by incorporation of the corresponding cytidine analogue into 3'-shortened tRNA using ATP(CTP):tRNA nucleotidyltransferase . {s2C}tRNA was selectively alkylated with fluorescent N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine (1,5-I-AEDANS) on the 2-thiocytidine residue . The product {AEDANS-s2C}aminoacyl-tRNA, forms a ternary complex with Escherichia coli elongation factor Tu and GTP, leading to up to 130% fluorescence enhancement of the AEDANS chromophore . From fluorescence titration experiments, equilibrium dissociation constants of 0.24 nM, 0.22 nM and 0.60 nM were determined for yeast {AEDANS-s2C}Tyr-tRNATyr, yeast Tyr-tRNATyr, and the homologous E . coli Phe-tRNAPhe, respectively, interacting with E . coli elongation factor Tu.GTP . The measurement of the association and dissociation rates of the interaction of {AEDANS-s2C}Tyr-tRNATyr with EF-Tu.GTP and the temperature dependence of the resulting dissociation constants gave values of 55 J mol-1 K-1 for delta S degrees' and -34.7 kJ mol-1 for delta H degrees' of this reaction.

Biochem Biophys Res Commun, 1989 Sep 15, 163(2), 851 - 9
Tetramer formation of a variant type human transthyretin (prealbumin) produced by Escherichia coli expression system; Furuya H et al.; A variant of human transthyretin(TTR, prealbumin) with methionine for valine substitution at position 30 is a major component of amyloid fibrils found in patients of familial amyloidotic polyneuropathy(FAP) type I, an autosomal dominant genetic disease . But the molecular nature of the variant TTR has been obscure, because most of plasma TTR from FAP patients is a mixture of variant and wild type TTR and no pure preparation of the variant has been available . For this reason, we constructed a system in which the variant type TTR was efficiently synthesized . In this system, the recombinant variant TTR was first synthesized as a fusion protein with E . coli outer membrane protein A (ompA) signal peptide, processed to eliminate the signal peptide and finally secreted to the culture medium . The final concentration of the recombinant variant TTR in the medium was about 5 mg/l . SDS polyacrylamide gel electrophoresis and gel filtration analysis suggested that the recombinant variant TTR can form tetramer as seen for native one . Purification of the protein was accomplished by only two steps of chromatography.

Biochim Biophys Acta, 1989 Sep 15, 992(3), 397 - 9
Branched-chain-amino-acid aminotransferase assay using radioisotopes; Airas RK; A method for the assay of the activity of branched-chain-amino-acid aminotransferase from Escherichia coli has been developed . Radioactive isoleucine is used, the radioactive oxoacid formed in the enzymic reaction is converted to its p-nitrophenylhydrazone, and the hydrazone is extracted into toluene based scintillation liquid . The small reaction tubes containing the toluene layer and the reaction mixture as a water layer are placed into liquid scintillation vials and counted for radioactivity . The radioactive amino acid remaining in the water layer causes only a rather low background.

J Biol Chem, 1989 Sep 15, 264(26), 15674 - 80
Crystallographic quaternary structural analysis of AMP nucleosidases from Escherichia coli and Azotobacter vinelandii; Giranda VL et al.; Adenosine monophosphate nucleosidases from Azotobacter vinelandii and Escherichia coli have been studied crystallographically to determine their quarternary structures . Preliminary characterization of the A . vinelandii enzyme shows that the crystals are monoclinic, C2 with a = 347 A, b = 204 A, c = 114 A, and beta = 91.7 degrees . The asymmetric unit contains 12 or 9 subunits of Mr 54,000 . Self-rotation functions with data from the AMP nucleosidases from A . vinelandii and from E . coli (Giranda, V . L., Berman, H . M., and Schramm, V . L . (1986) J . Biol . Chem . 261, 15307-15309) are consistent with the monomers arranged as hexamers with point symmetry 32 . The hexamers are arranged in the unit cells so that crystallographic 2-fold axes are coincident with the local 2-folds of the point group 32.

J Biol Chem, 1989 Sep 15, 264(26), 15535 - 41
Purification and characterization of SopA and SopB proteins essential for F plasmid partitioning; Mori H et al.; Mini-F plasmid has the trans-acting genes sopA and sopB and the cis-acting site sopC which are essential for accurate partitioning of plasmid DNA molecules into both daughter cells . In this study, we purified independently SopA and SopB proteins, analyzed the in vitro DNA-binding activity of these proteins by the gel retardation assay, and determined the precise binding sites of DNA by the footprinting method . SopA binds to four repeated sequences (CTTTGC) located in the promoter-operator region of the sopAB operon . The SopA binding activity is enhanced by the addition of SopB protein . SopB protein itself does not bind to this DNA region . These results suggest that the complex of SopA and SopB proteins autoregulate the expression of the sopA-sopB operon . On the other hand, SopB protein binds to the sopC region, in which 12 direct repeats of 43-base pairs nucleotides exist . SopB protein recognizes the inverted repeats of 7 base pairs in each direct repeats . SopA protein does not affect the SopB binding activity to the sopC DNA segment.

J Biol Chem, 1989 Sep 15, 264(26), 15169 - 72
Essential role of T4 phage deoxycytidylate hydroxymethylase in a multienzyme complex for deoxyribonucleotide synthesis; Thylen C et al.; Several enzymes of deoxyribonucleoside triphosphate (dNTP) biosynthesis interact in T4 phage-infected Escherichia coli to form a multienzyme aggregate, the T4 dNTP synthetase complex . To test the specificity of enzyme interactions seen in vitro with this complex, we analyzed bacteria infected with four T4 gene 42 amber mutants, which specify truncated forms of dCMP hydroxymethylase, one of the constituent enzymes in the complex . Mutants that specify a nearly full length gene 42 product can form an intact complex, as revealed by two criteria: kinetic coupling among constituent enzymes in crude extracts of infected bacteria, and co-elution of enzyme activities from a gel filtration column . By these criteria, mutations that specify truncated proteins less than half the size of the full length protein cause disruption of the complex . These findings suggest that an enzymatically inactive form of dCMP hydroxymethylase can contribute toward assembly of an intact complex, so long as the incomplete protein is of sufficient size to fold normally, allowing interaction with other proteins in the complex.

Biochem Biophys Res Commun, 1989 Sep 15, 163(2), 780 - 7
A novel ubiquitous protein 'chaperonin' supports the endosymbiotic origin of mitochondrion and plant chloroplast; Gupta RS et al.; The deduced amino acid sequences for a major mitochondrial protein (designated P1, related to the 'chaperonin' family of proteins) from human and Chinese hamster cells show extensive similarity (greater than 60% identity observed over the entire length) with a related protein present in evolutionarily as divergent organisms as Escherichia coli, Coxiella burnetii, Mycobacterium species, cyanobacteria as well as in yeast mitochondria and higher plant chloroplasts . Of the different groups of bacteria for which sequence data is available, maximum similarity of the mammalian/yeast P1 protein is observed with the corresponding protein from purple bacteria (especially C . burnetii) while the protein from plant chloroplasts exhibited highest similarity with the corresponding protein from cyanobacteria . The sequence data for this protein thus support the contention that the endosymbiont that gave rise to mitochondrion was a member of purple bacteria, while plant chloroplast originated from a member of the cyanobacterial lineage.

Eur J Biochem, 1989 Sep 15, 184(2), 361 - 5
Synthesis of a gene for the protein kinase domain of the epidermal growth factor receptor and its expression in Escherichia coli; Farrow SN et al.; A gene encoding the protein kinase domain of the epidermal growth factor receptor has been chemically synthesised, cloned and expressed in Escherichia coli . The 942-base-pair gene was constructed by enzymatic ligation of 56 oligonucleotides and cloned into an expression vector downstream of the E . coli trp promoter . Production of active gene product was confirmed by means of a protein kinase assay, demonstrating that the enzymatic activity of the protein kinase domain of the epidermal growth factor receptor is retained after expression in E . coli.

J Biol Chem, 1989 Sep 15, 264(26), 15475 - 82
Synthesis in Escherichia coli of GTPase-deficient mutants of Gs alpha; Graziano MP et al.; We have reduced the GTPase activity of the alpha subunit of Gs, the guanine nucleotide-binding regulatory protein that stimulates adenylyl cyclase, by introduction of point mutations analogous to those described in p21ras . Mutants G49V and Q227L differ from the wild type protein in the substitution of Val for Gly49 and Leu for Gln227, respectively (analogous to positions 12 and 61 in p21ras) . Wild type and mutant proteins were synthesized in Escherichia coli, purified, and characterized . The rate constants for dissociation of GDP from G49V recombinant Gs alpha (rGs alpha) (0.47/min) and Q227L rGs alpha (0.23/min) differ by no more than 2-fold from that observed for the wild type protein (0.5/min) . In marked contrast, the rate constants for hydrolysis of GTP by G49V rGs alpha (0.78/min) and Q227L rGs alpha (0.03-0.06/min) are 4-fold and roughly 100-fold slower than that for wild type rGs alpha (3.5/min) . These reductions in the rate of hydrolysis of GTP result in significant fractional occupancy of these proteins by GTP in the presence of the nucleotide, 0.37 for G49V rGs alpha and 0.78 for Q227L rGs alpha, compared to 0.05 for wild type rGs alpha . When reconstituted with cyc- (Gs alpha-deficient) S49 cell membranes or purified adenylyl cyclase, both mutant proteins stimulate adenylyl cyclase activity in the presence of GTP to a much greater extent than does wild type Gs alpha; their maximal ability to activate the enzyme is largely unaltered . The fractional ability of a given Gs alpha polypeptide to active adenylyl cyclase in the presence of GTP correlates well with the fractinal occupancy of the protein by the nucleotide . The mutant subunits appear to interact normally with G protein beta gamma subunits, and their ability to activate adenylyl cyclase is enhanced by interaction with beta-adrenergic receptors . These results indicate that the structural analogy that has been inferred between the guanine nucleotide-binding domains of G proteins and the p21ras family is at least generally correct . They also provide confirmation of the kinetic model of G protein function and document mutations that permit the expression in vivo of constitutively activated G protein alpha subunits.

J Biol Chem, 1989 Sep 15, 264(26), 15398 - 403
Thermodynamic and spectroscopic characteristics of the cytochrome bc1 complex . Role of quinone in the behavior of cytochrome b562; Salerno JC et al.; Cytochrome b562 does not behave as a single independent thermodynamic component in preparations of purified quinol cytochrome c reductase . This effect is much more pronounced in quinone sufficient preparations; in such preparations, the epr spectrum of the cytochrome is Eh sensitive, with a peak shift from g = 3.42 to 3.48 occurring as the potential is lowered from 100 mV to 0 mV . The peak shift is dependent on the presence of quinone and can be restored to quinone-depleted preparations by supplementation with ubiquinol 2 if phospholipid depletion is not too severe . The results suggest that cytochrome b562 is strongly interacting with the Qc quinone binding site.

J Biol Chem, 1989 Sep 15, 264(26), 15376 - 83
Kinetic characterization of the unisite catalytic pathway of seven beta-subunit mutant F1-ATPases from Escherichia coli; al-Shawi MK et al.; We have studied the kinetics of "unisite" ATP hydrolysis and synthesis in seven mutant Escherichia coli F1-ATPase enzymes . The seven mutations are distributed over a 105-residue segment of the catalytic nucleotide-binding domain in beta-subunit and are: G142S, K155Q, K155E, E181Q, E192Q, M209I, and R246C . We report forward and reverse rate constants and equilibrium constants in all seven mutant enzymes for the four steps of unisite kinetics, namely (i) ATP binding/release, (ii) ATP hydrolysis/synthesis, (iii) Pi release/binding, and (iv) ADP release/binding . The seven mutant enzymes displayed a wide range of deviations from normal in both rate and equilibrium constants, with no discernible common pattern . Notably, steep reductions in Kd ATP were seen in some cases, the value of Kd Pi was high, and K2 (ATP hydrolysis/synthesis) was relatively unaffected . Significantly, when the data from the seven mutations were combined with previous data from two other E . coli F1-beta-subunit mutations (D242N, D242V), normal E . coli F1, soluble and membranous mitochondrial F1, it was found that linear free energy relationships obtained for both ATP binding/release (log k+1 versus log K1) and ADP binding/release (log k-4 versus log K-4) . Two conclusions follow . 1) The seven mutations studied here cause subtle changes in interactions between the catalytic nucleotide-binding domain and substrate ATP or product ADP . 2) The mitochondrial, normal E . coli, and nine total beta-subunit mutant enzymes represent a continuum in which subtle structural differences in the catalytic site resulted in changes in binding energy; therefore insights into the nature of energy coupling during ATP hydrolysis and synthesis by F1-ATPase may be ascertained by detailed studies of this group of enzymes.

Biochem Biophys Res Commun, 1989 Sep 15, 163(2), 739 - 45
Expression of human poly(ADP-ribose) polymerase with DNA-dependent enzymatic activity in Escherichia coli; Ikejima M et al.; A cDNA for human poly(ADP-ribose) polymerase was inserted into a plasmid, transfected and expressed in E . coli . A lysate of the E . coli cells containing the expression plasmid reacted with antibody against human poly(ADP-ribose) polymerase and synthesized poly(ADP-ribose) . The partially purified poly(ADP-ribose) polymerase expressed in E . coli had the same molecular weight and enzymological properties as human placental poly(ADP-ribose) polymerase, including affinity for NAD, turnover number and DNA-dependency for activity . This expression system should be useful for structure-function analysis of poly(ADP-ribose) polymerase.

Eur J Biochem, 1989 Sep 15, 184(2), 367 - 74
Human immunodeficiency virus reverse transcriptase expressed in transformed yeast cells . Biochemical properties and interactions with bovine tRNALys; Sallafranque-Andreola ML et al.; Human immunodeficiency virus (HIV) reverse transcriptase has been purified from yeast transformed by an autoreplicating plasmid containing the retroviral DNA polymerase gene . The previously described purification procedure for the yeast-expressed reverse transcriptase {Barr, P.J., Power, M.D., Chun Ting Lee-Ng, Gibson, H . & Luciw, P . (1987) Bio/Technology 5, 486-489} has been substantially modified, leading to an increased yield and a higher degree of purity . Several biochemical properties of the enzyme are described (template specificity, effect of DNA synthesis inhibitors); interestingly, HIV reverse transcriptase is highly resistant to N-ethylmaleimide . A complex between the human retroviral enzyme and the bovine tRNALys was shown, using a direct approach, by glycerol gradient centrifugation, as well as by the protective and specific effect of the tRNALys against enzyme inactivation by thermal denaturation and trypsin digestion . A competitive type of inhibition of HIV reverse transcriptase by tRNALys, but not by tRNAVal, is observed when viral RNA or activated DNA are used as templates.

Eur J Biochem, 1989 Sep 15, 184(2), 353 - 9
Isolation and characterisation of a cDNA clone for a chlorophyll synthesis enzyme from Euglena gracilis . The chloroplast enzyme hydroxymethylbilane synthase (porphobilinogen deaminase) is synthesised with a very long transit peptide in Euglena; Sharif AL et al.; A cDNA expression library was constructed from light-grown Euglena gracilis poly(A)-rich RNA in lambda gt11 . Antibodies to Euglena hydroxymethylbilane synthase, the third enzyme in the porphyrin biosynthetic pathway, were used to screen the library and a clone encoding part of the sequence of hydroxymethylbilane synthase was identified . This was used to rescreen the library and a full-length clone was isolated, which encoded not only the entire mature protein (Mr 36,927), but also an N-terminal extension of 139 amino acids . The deduced Mr of the whole polypeptide is 51,744, which corresponds to the size of the protein immunoprecipitated from the translation products of Euglena poly(A)-rich RNA . The mature protein is 60-70% similar to hydroxymethylbilane synthase from human erythrocytes and Escherichia coli . The sequence of the N-terminal extension has similarities to both the transit peptides of chloroplast proteins and those for the endoplasmic reticulum . This is the first report both of a cDNA clone for an enzyme of the chlorophyll biosynthetic pathway and of a putative transit peptide for a nuclear-encoded Euglena protein.

J Immunol, 1989 Sep 15, 143(6), 2006 - 12
The mapping of epitopes of the 18-kDa protein of Mycobacterium leprae recognized by murine T cells in a proliferation assay; Harris DP et al.; The 18-kDa protein of Mycobacterium leprae was purified from recombinant plasmids pUL108 and pML-3 grown in Saccharomyces cerevisiae and Escherichia coli, respectively . Significant lymphoproliferative responses were observed when T cells from immunized mice were challenged in culture with purified 18-kDa protein . Synthetic peptides have been prepared that span most of the 148 amino acid residues that constitute the sequence of the 18-kDa protein and used to map epitopes recognized by T cells . When mice were immunized with 18-kDa protein and lymph node cells subsequently prepared and challenged in microculture proliferative assays by using synthetic peptides, only one region of the intact protein appeared stimulatory . This T cell epitope was located between residues 116 and 121, adjacent to an epitope between residues 110 and 115 which we have previously shown to bind the L5 mAb . Immunization of mice with peptides, and subsequent challenge of lymph node cells in assays by using the 18-kDa protein as Ag revealed that residues 111-125 were the most effective in priming responses . Furthermore, the ability of 18-kDa primed lymph node cells to recognize determinants on both M . leprae and Mycobacterium tuberculosis indicates that in addition to possessing an M . leprae-specific B cell determinant, the 18-kDa protein contains a cross-reactive T cell epitope(s).

Biochim Biophys Acta, 1989 Sep 14, 998(1), 32 - 42
Isolation and characterization of biologically active murine interleukin-1 alpha derived from expression of a synthetic gene in Escherichia coli; Daumy GO et al.; A murine interleukin-1 alpha (mIL-1 alpha) gene coding for amino acids 115 to 270 of the precursor protein (Lomedico, P.T., Gubler, U., Hellmann, C.P., Dukovich, M., Giri, J.G., Pan, Y.E., Collier, K., Semionow, R., Chua, A.O . and Mizel, S.B . (1984) Nature 312, 458-462) was chemically synthesized and expressed in Escherichia coli . mIL-1 alpha, in the form of insoluble inclusion bodies, accounted for approx . 30% of total cellular protein produced by the recombinant strain . A simple isolation protocol was developed in which inclusion body material was first solubilized in 3 M guanidine hydrochloride, and the mIL-1 alpha was then simultaneously purified and allowed to fold to its active conformation by dialysis against distilled water . This procedure yielded pure, biologically active mIL-1 alpha with 41% recovery of the mIL-1 alpha present in the guanidine hydrochloride extract . The purified preparation had the expected amino acid composition, a molar absorptivity of 28,200 M-1.cm-1 and a pI of 5.2 . No methionyl-mIL-1 alpha was detected by N-terminal sequence analysis, and the endotoxin level was less than 10 pg per micrograms of mIL-1 alpha . The specific biological activity was 3.10(7) units/mg in a co-mitogenic thymocyte proliferation assay . In addition to full-length mIL-1 alpha, the preparation contained N-terminally truncated mIL-1 alpha species (mainly des-4 and des-6 amino acid forms) . The truncated species were isolated and found to have the same biological activity as the complete polypeptide . Thus, the active fragment of mIL-1 alpha appears to consist of a proteinase-sensitive N-terminal region which is not essential for activity, and a proteinase-resistant core which harbors the essential determinants of its cytokine function.

Nucleic Acids Res, 1989 Sep 12, 17(17), 6781 - 94
The recR locus of Escherichia coli K-12: molecular cloning, DNA sequencing and identification of the gene product; Mahdi AA et al.; The recR gene of Escherichia coli, which is associated with recBC-independent mechanisms of recombination and DNA repair, has been located between dnaZX and htpG on a 6.4 kb EcoRI fragment of DNA that has been cloned and analysed in lambda and plasmid vectors . Nucleotide sequencing of this interval revealed two open reading frames that constitute an operon lying immediately downstream of dnaZX . The second of these two reading frames was identified as recR . It encodes a polypeptide with a predicted molecular weight of 21,965 Daltons that migrates on SDS gels as a 26 kDa protein . The first gene of the operon encodes a polypeptide of 12,015 daltons . Its function is not known.

Nucleic Acids Res, 1989 Sep 12, 17(17), 6865 - 81
Characterization of yeast mitochondrial RNase P: an intact RNA subunit is not essential for activity in vitro; Morales MJ et al.; We have previously described a mitochondrial activity that removes 5' leaders from yeast mitochondrial precursor tRNAs and suggested that it is a mitochondrial RNase P . Here we demonstrate that the cleavage reaction results in a 5' phosphate on the tRNA product and thus the activity is analogous to that of other RNase Ps . A mitochondrial gene called the tRNA synthesis locus encodes an A + U-rich RNA required for this activity in vivo . Two regions of this RNA display sequence similarity to conserved sequences in bacterial RNase P RNAs . This sequence similarity coupled with the analogous activities of the enzymes has led us to conclude that the RNAs are homologous and that the tRNA synthesis locus does code for the mitochondrial RNase P RNA subunit . The smallest and most abundant transcript of the tRNA synthesis locus is 490 nucleotides long . However, during purification of the holoenzyme, RNA is degraded and pieces of the original RNA are sufficient to support RNase P activity in vitro.

FEBS Lett, 1989 Sep 11, 255(1), 15 - 20
Recombinant proricin binds galactose but does not depurinate 28 S ribosomal RNA; Richardson PT et al.; Preproricin transcripts microinjected into Xenopus oocytes were expressed and the product was segregated by the oocyte endoplasmic reticulum and core glycosylated . Recombinant proricin was soluble, stabilised by intramolecular disulfide bonds and biologically active in that it could bind to immobilized lactose (selectin 2) or immobilized asialofetuin . Affinity-purified proricin did not catalyse the depurination of 28 S ribosomal RNA unless it was reduced, when slight but significant activity was observed . Gel filtration of the reduced proricin fraction showed that this depurination activity was not associated with proricin . The activity was apparently due to ricin A chain released by reduction from mature ricin which was, in turn, generated from proricin, presumably via endogenous oocyte endoprotease activity.

Science, 1989 Sep 8, 245(4922), 1104 - 7
Generation of a catalytic antibody by site-directed mutagenesis; Baldwin E et al.; A hybrid Fv fragment of the dinitrophenyl-binding immunoglobulin A (IgA), MPOC315, has been generated by reconstituting a recombinant variable light chain (VL) produced in Escherichia coli with a variable heavy chain (VH) derived from the antibody . The Tyr34 residue of VL was substituted by His in order to introduce a catalytic imidazole into the combining site for the ester hydrolysis . The His mutant Fv accelerated the hydrolysis of the 7-hydroxycoumarin ester of 5-(2,4-dinitrophenyl)-aminopentanoic acid 90,000-fold compared to the reaction with 4-methyl imidazole at pH 6.8 and had an initial rate that was 45 times as great as that for the wild-type Fv . The hydrolyses of aminopropanoic and aminohexanoic homologs were not significantly accelerated . Thus a single deliberate amino acid change can introduce significant catalytic activity into an antibody-combining site, and chemical modification data can be used to locate potential sites for the introduction of catalytic residues.

J Biol Chem, 1989 Sep 5, 264(25), 14741 - 7
Purification and characterization of pituitary bovine somatotropin; Wood DC et al.; Bovine somatotropin (bST) has been isolated from pituitary glands and compared in a variety of chemical analyses and bioassays with somatotropin derived from recombinant Escherichia coli . Comparison of pituitary extracts and purified bST by Western blot analysis of two-dimensional gels suggested that the immunoreactive somatotropin species present in the extract were also present in the purified material, with no significant losses or degradation as a result of the purification method . NH2-terminal sequence analysis indicated the presence of equal quantities of Ala-Phe-Pro-Ala-Met-Ser-Leu-Ser- and Phe-Pro-Ala-Met-Ser-Leu-Ser- sequences . The Met-Ser-Leu-Ser-NH2-terminal sequence, a degradation product observed in NIH standard lots, was not detected . Assay of bioactivity in a bovine liver receptor-binding assay and in a female rat growth assay showed pituitary bST and recombinant methionyl-bovine somatotropin to be equipotent . Tryptic maps and sequence analysis of pituitary-derived somatotropin suggest the presence of isoaspartate derivatization at Asp128.

J Biol Chem, 1989 Sep 5, 264(25), 15130 - 5
Conversion of monofunctional DNA adducts of cis-diamminedichloroplatinum (II) to bifunctional lesions . Effect on the in vitro replication of single-stranded DNA by Escherichia coli DNA polymerase I and eukaryotic DNA polymerases alpha; Hoffmann JS et al.; Reaction of cis-diamminedichloroplatinum (II) with single-stranded M13 phage DNA in vitro produced monofunctional platinum-DNA adducts on guanine and bifunctional lesions with either two guanine bases (GG) or one adenine and one guanine (AG) . When DNA containing a majority of monofunctional platinum-DNA lesions was dialyzed against 10 mM NaCIO4 at 37 degrees C, conversion of monoadducts to bifunctional lesions was observed . We examined the effect of post-treatment formation of bifunctional lesions on DNA synthesis by Escherichia coli DNA polymerase I and highly purified eukaryotic DNA polymerase alpha from Drosophila melanogaster and calf thymus . Arrest sites on the platinated template were determined by polyacrylamide gel electrophoresis . Monofunctional lesions did not appear to block DNA synthesis . Inhibition of replication increased as bifunctional platinum-DNA lesions formed during post-treatment incubation; GG adducts inhibited replication more than AG . These results suggest that bifunctional GG platinum-DNA adducts may be the major toxic damage of cisplatin.

J Biol Chem, 1989 Sep 5, 264(25), 14902 - 8
Comparison of the human immunodeficiency virus type 1 and 2 proteases by hybrid gene construction and trans-complementation; Le Grice SF et al.; To determine the cleavage specificity of the proteases of the type 1 and 2 human immunodeficiency viruses (HIV-1, HIV-2), we interchanged this domain of the polymerase (pol) genes and analyzed the maturation programs of the chimeric polyproteins in an Escherichia coli expression system . In both cases, release of reverse transcriptase and integrase was observed, together with the respective 10-kDa protease form resulting from autocatalysis, although the maturation proceeded less efficiently compared to the homologous system . In further experiments, the ability of both HIV-1 and HIV-2 proteases to release in vivo gag p24 from an in-frame fusion of the full length gag and protease precursors was analyzed . In either case, p24 was released, albeit with greater efficiency in the heterologous gene construction . In vitro mixed lysate experiments with the HIV-1 gag precursor furthermore demonstrate that both enzymes respond to the aspartyl proteinase inhibitor pepstatin A . Taken together, these results illustrate that although different cleavage recognition sequences exist for HIV-1 and -2, they are amenable to the proteases of both viruses, but additionally that subtle differences in the mode of action of both enzymes are observable.

J Biol Chem, 1989 Sep 5, 264(25), 14698 - 703
Isolation and characterization of lactose permease mutants with an enhanced recognition of maltose and diminished recognition of cellobiose; Collins JC et al.; In the present study, lactose permease mutants were isolated which have an enhanced recognition toward maltose (an alpha-glucoside) and diminished recognition for cellobiose (a beta-glucoside) . Nine mutants were isolated from a strain encoding a wild-type permease (pTE18) and nine from a strain encoding a mutant permease which recognizes maltose (pB15) . All 18 mutants were subjected to DNA sequencing, and it was found that all mutations are single base substitutions within the lac Y gene effecting single amino acid substitutions within the protein . From the pTE18 parent, substitutions involved Tyr-236 to Phe or His; Ser-306 to Thr; and six independent mutants in which Ala-389 was changed to Pro . From pB15, Tyr-236 was changed to Phe or Asn, Ser-306 to Thr or Leu, Lys-319 to Asn, and His-322 to Tyr, Asn, or Gln . All 18 mutants exhibited enhanced recognition for maltose (compared with the pTE18 strain) and a diminished recognition for cellobiose . In addition, all mutants showed a diminished recognition toward beta-galactosides as well . The Phe-236, His-236, Leu-306, Asn-319, Tyr-322, Asn-322, and Gln-322 mutants were completely defective in the uphill accumulation of methyl-beta-D-thiogalactopyranoside whereas the Asn-236, Thr-306, and Pro-389 mutants could effectively accumulate methyl-beta-D-thiogalactopyranoside against a concentration gradient . The mutants obtained in this study, together with previous lactose permease mutants, tend to be found on transmembrane segments, and those which are on the same transmembrane segment are often found three or four amino acids away from each other . This pattern is consistent with a protein structure in which important amino acid side chains project from several transmembrane segments in such a way as to form a hydrophilic channel for the recognition and transport of H+ and galactosides . It is proposed that the mechanism for H+/lactose cotransport is consistent with a "flanking gate" model in which the protein contains a single recognition site for galactosides within the channel which is flanked on either side by gates.

J Biol Chem, 1989 Sep 5, 264(25), 14638 - 45
Attenuation in the regulation of the pyrBI operon in Escherichia coli . In vivo studies of transcriptional termination; Levin HL et al.; The attenuation model for transcriptional regulation of the Escherichia coli pyrBI operon is based on the assumption that transcription terminates upstream of the structural genes at a rho-independent terminator when cells contain high levels of UTP . When, however, the cells are limited for pyrimidines, the presence of ribosomes translating the short leader peptide is presumed to cause an alteration in the secondary structure of the terminator in a way that allows RNA polymerase to transcribe the entire operon . These two premises of transcriptional regulation were tested by using exonuclease protection assays to map the 3' ends of transcripts extracted from cells containing either ample or depleted concentrations of pyrimidines . The results support the model since 99% of the pyrBI transcripts terminated at the (G + C)-rich region of dyad symmetry upstream of the structural genes when cells were grown in excess uracil . In addition, a significant portion (36%) of the pyrBI transcripts extracted from cells containing reduced pyrimidine concentrations extended past the dyad into the structural genes . This observation correlated with the amounts of aspartate transcarbamoylase synthesized in cells under the various conditions . The mapping technique was also used to determine the position of the 5' ends of the transcripts to measure contributions of two potential start sites (P1 and P2) to the pool of pyrBI transcripts . The results show that under all conditions no more than 3% of the total transcripts had 5' ends corresponding to the upstream promoter, P1 . In cells lacking P1 virtually all transcripts from P2 terminated at the (G + C)-rich hairpin when the cellular level of pyrimidines was high . Conversely 57% of the transcripts extended past the terminator when cells were grown in UMP . The S1 nuclease technique also provided a measure of the steady state level of transcripts originating at P2 . In cells depleted of pyrimidines there was a 5-10-fold increase in these transcripts depending on the number of copies of pyrBI . This increase, which is independent of attenuation, is caused by a different regulatory mechanism which as yet has not been identified.

J Biol Chem, 1989 Sep 5, 264(25), 14624 - 6
NMR study of human mutant hemoglobins synthesized in Escherichia coli . Consequences of tyrosine alpha 42 substitutions; Ishimori K et al.; The hydroxyl group of Tyr alpha 42 in human hemoglobin forms a hydrogen bond with the carboxylate of Asp beta 99 which is considered to be one of the most important hydrogen bonds for stabilizing the "T-state." However, no spontaneous mutation at position 42 of the alpha subunit has been reported, and the role of the tyrosine has not been tested experimentally . Two artificial human mutant hemoglobins in which Tyr alpha 42 was replaced by phenylalanine or histidine were synthesized in Escherichia coli, and their proton NMR spectra were studied with particular attention to the hyperfine-shifted and hydrogen-bonded proton resonances . The site-directed mutagenesis of the Tyr alpha 42----Phe removes the hydrogen bond described above and prevents transition to the T-state so that the mutant Hb is rather similar to the "R-state" even when deoxygenated . On the other hand, the mutation from tyrosine to histidine causes less drastic structural changes, and its quaternary and tertiary structures are almost the same as native deoxy-Hb A . This may be attributed to the formation of a new hydrogen bond between His alpha 1(42) and Asp beta 2(99) . These observations indicate that the hydrogen bond formed between Tyr alpha 42 and Asp beta 99 is required to convert unliganded Hb to the T-state.

J Biol Chem, 1989 Sep 5, 264(25), 14860 - 4
The importance of the link between Glu204 of the catalytic chain and Arg130 of the regulatory chain for the homotropic and heterotropic properties of Escherichia coli aspartate transcarbamoylase; Stebbins JW et al.; Recent x-ray crystallographic studies of aspartate transcarbamoylase bound with CTP have detected molecular asymmetry in the interface between the catalytic and regulatory subunits (Kim, K . H., Pan, Z., Honzatko, R . B., Ke, H.-M., and Lipscomb, W . N . (1987) J . Mol . Biol . 196, 863-875) . In three of the six interfaces, a salt link occurs between Arg130 of the regulatory chain and Glu204 of the catalytic chain; however, these same residues are 15 A apart in the other three interfaces . In order to determine if this is important for the function of the enzyme, two mutant versions of aspartate transcarbamoylase were created by site-specific mutagenesis . Glu204 of the catalytic chain was converted to a glutamine (Glu204c----Gln) and Arg130 of the regulatory chain was converted to a glycine (Arg130r----Gly) . The thermal stability of the Arg130r----Gly enzyme is dramatically reduced, whereas the thermal stability of the Glu204c----Gln enzyme is unaltered compared to the wild-type enzyme . The maximal velocity of both mutant enzymes is identical with that of the wild-type enzyme, however both mutant enzymes have altered substrate affinity and regulatory properties . Based on these studies, the link between Glu204 of the catalytic chain and Arg130 of the regulatory chain is important for the heterotropic properties of the enzyme . Furthermore, the interface between the domain of the regulatory chain which binds zinc and the domain of the catalytic chain which binds aspartate may be more important for CTP inhibition than ATP activation . These data also suggest that heterotropic cooperativity is very sensitive to alterations in the catalytic-regulatory interface . However, no clear relationship has been observed between the structural asymmetry and the function of the enzyme.

J Mol Biol, 1989 Sep 5, 209(1), 171 - 5
Densely packed beta-structure at the protein-lipid interface of porin is revealed by high-resolution cryo-electron microscopy; Sass HJ et al.; Porin is an integral membrane protein that forms channels across the outer membrane of Escherichia coli . Electron microscopic studies of negatively stained two-dimensional porin crystals have shown three stain accumulations per porin trimer, revealing the locations of pores spanning the membrane . In this study, reconstituted porin lattices embedded in glucose were investigated using the low-dose technique on a cryo-electron microscope equipped with a helium-cooled superconducting objective lens . The specimen temperature was maintained at 5 K to yield an improved microscopic and specimen stability . Under these conditions, we obtained for the first time electron diffraction patterns from porin lattices to a resolution of 3.2 A and images showing optical diffraction up to a resolution of 4.9 A . Applying correlation averaging techniques to the digitized micrographs, we were able to reconstruct projected images of the porin trimer to a resolution of up to 3.5 A . In the final projection maps, amplitudes from electron diffraction and phases from these images were combined . The predominant feature is a high-density narrow band (about 6 A in thickness) that delineates the outer perimeter of the trimer . Since the molecule consists of almost exclusively beta-sheet structure, as revealed by spectroscopic data, we conclude that this band is a cylindrical beta-pleated sheet crossing the membrane nearly perpendicularly to its plane . Another intriguing finding is a low-density area (about 70 A2) situated in the centre of the trimer.

J Biol Chem, 1989 Sep 5, 264(25), 15144 - 50
Myosin heavy chain kinase from developed Dictyostelium cells . Purification and characterization; Ravid S et al.; We purified to homogeneity the Dictyostelium discoideum myosin heavy chain kinase that is implicated in the heavy chain phosphorylation increases that occur during chemotaxis . The kinase is initially found in the insoluble fraction of developed cells . The major purification step was achieved by affinity chromatography using a tail fragment of Dictyostelium myosin (LMM58) expressed in Escherichia coli (De Lozanne, A., Berlot, C . H., Leinwand, L . A., and Spudich, J . A . (1988) J . Cell Biol . 105, 2990-3005) . The kinase has an apparent molecular weight of 84,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis . The apparent native molecular weight by gel filtration is 240,000 . The kinase catalyzes phosphorylation of myosin heavy chain or LMM58 with similar kinetics, and the extent of phosphorylation for both is 4 mol of phosphate/mol . With both substrates the Vmax is about 18 mumol/min/mg and the Km is 15 microM . The myosin heavy chain kinase is specific to Dictyostelium myosin heavy chain, and the phosphorylated amino acid is threonine . The kinase undergoes autophosphorylation . Each mole of kinase subunit incorporates about 20 mol of phosphates . Phosphorylation of myosin by this kinase inhibits myosin thick filament formation, suggesting that the kinase plays a role in the regulation of myosin assembly.

J Biol Chem, 1989 Sep 5, 264(25), 15074 - 82
Characterization of the spoT gene of Escherichia coli; Sarubbi E et al.; The Escherichia coli spoT gene encodes a guanosine-3',5'-bispyrophosphate (ppGpp) 3'-pyrophosphohydrolase known to be responsible for cellular (ppGpp) degradation . The DNA sequence of the spoT region is presented . The spoT gene is deduced to be 702 codons long, with a probable UUG initiation codon, and a deduced mass of 79,342 daltons . Two spoT mutations (spoT202 and spoT203) have been localized to an open reading frame by complementation of function as well as by genetic marker rescue . The ability to overexpress the spoT gene is limited, but enough ppGppase activity can be made to reverse ppGpp accumulation during the stringent response to amino acid starvation . The spoT gene is located within a larger spo operon and is flanked by two smaller genes . The first gene in the operon encodes omega, a protein that copurifies with RNA polymerase (Gentry, D . R., and Burgess, R . R . (1986) Gene (Amst.) 48, 33-40) . The spoT gene is the second gene in the operon; it is followed by a third open reading frame deduced to encode a protein with a mass of 25,343 daltons . Insertion of a kanamycin resistance gene in the omega gene reduces spoT gene expression as judged by lowered ppGppase activity, relA-dependent reduction of growth rate, and abolition of spoT mutant complementation activity . These effects are reversed by expression of the spoT gene, but not the omega gene, in trans . Transcription of the spo operon occurs in a clockwise direction on the E . coli chromosome and is probably directed by at least two promoters.

J Biol Chem, 1989 Sep 5, 264(25), 15012 - 21
Adenosine-5'-phosphosulfate kinase from Escherichia coli K12 . Purification, characterization, and identification of a phosphorylated enzyme intermediate; Satishchandran C et al.; Adenosine-5'-phosphosulfate kinase (ATP:adenylylsulfate 3'-phosphotransferase), the second enzyme in the pathway of sulfate activation, has been purified (approximately 300-fold) to homogeneity from an Escherichia coli K12 strain, which overproduces the enzyme activity (approximately 100-fold) . The purified enzyme has a specific activity of 153 mumol of 3'-phosphoadenosine 5'-phosphosulfate (PAPS) formed/min/mg of protein at 25 degrees C . The enzyme is remarkably efficient with a Vmax/Km(APS) of greater than 10(8) M-1 s-1, indicating that at physiologically low substrate concentrations the reaction is essentially diffusion limited . Upon incubation with MgATP a phosphorylated enzyme is formed; the isolated phosphorylated enzyme can transfer its phosphoryl group to adenosine 5'-phosphosulfate (APS) to form PAPS or to ADP to form ATP . The phosphorylated enzyme exists as a dimer of identical 21-kilodalton subunits, while the dephosphorylated form primarily exists as a tetramer . Divalent cations are required for activity with Mg(II), Mn(II), Co(II), and Cd(II) activating . Studies of the divalent metal-dependent stereoselectivity for the alpha- and beta-phosphorothioate derivatives of ATP indicate metal coordination to at least the alpha-phosphoryl group of the nucleotide . Steady state kinetic studies of the reverse reaction indicate a sequential mechanism, with a rapid equilibrium ordered binding of MgADP before PAPS . In the forward direction APS is a potent substrate inhibitor, competitive with ATP, complicating kinetic studies . The primary kinetic mechanism in the forward direction is sequential . Product inhibition studies at high concentrations of APS suggest an ordered kinetic mechanism with MgATP binding before APS . At submicromolar concentrations of APS, product inhibition by both MgADP and PAPS is more complex and is not consistent with a solely ordered sequential mechanism . The formation of a phosphorylated enzyme capable of transferring its phosphoryl group to APS or to MgADP suggests that a ping-pong pathway in which the rate of MgADP dissociation is comparable to the rate of APS binding might contribute at very low concentrations of APS . The substrate inhibition by APS is consistent with APS binding to the enzyme, to form a dead-end E.APS complex.

J Biol Chem, 1989 Sep 5, 264(25), 14806 - 11
Purification and characterization of recombinant human parathyroid hormone-related protein; Hammonds RG Jr et al.; Full-length human parathyroid hormone-related protein (PTHrP-(1-141} as well as a carboxyl-terminal shortened form (PTHrP-(1-108} have been expressed from recombinant DNA-derived clones . These proteins were expressed in Escherichia coli as fusion proteins so that cyanogen bromide cleavage yields the desired product . Both proteins were purified and then characterized by sodium dodecyl sulfate gel electrophoresis, amino-terminal amino acid sequencing, peptide mapping, and mass spectral analysis . Recombinant PTHrP-(1-141), PTHrP-(1-108), synthetic PTHrP-(1-34), and naturally derived PTHrP are all equipotent in the stimulation of cyclic AMP levels in the osteoblast-like cell line UMR 106-01 . However, PTHrP-(1-141) and -(1-108) are two to four times more active than PTHrP-(1-34) in the stimulation of plasminogen activator activity from this cell line . PTHrP-(1-141) reacts equipotently with PTHrP-(1-34) in a radioimmunoassay using an antiserum prepared against PTHrP-(1-34) . PTHrP-(1-141), -(1-108), and -(1-84) were used as PTHrP-specific mobility standards on sodium dodecyl sulfate gel electrophoresis to determine the approximate length of two forms of naturally derived PTHrP . The data show that PTHrP purified from the lung tumor cell line BEN contains a major form of about 108 amino acids and another form of about 141 amino acids.

J Biol Chem, 1989 Sep 5, 264(25), 14769 - 74
Sequence of the cloned Escherichia coli K1 CMP-N-acetylneuraminic acid synthetase gene; Zapata G et al.; The Escherichia coli CMP-N-acetylneuraminic acid (CMP-NeuAc) synthetase gene is located on a 3.3-kilobase (kb) HindIII fragment of the plasmid pSR23 which contains the genes for K1 capsule production (Vann, W . F., Silver, R . P., Abeijon, C., Chang, K., Aaronson, W., Sutton, A., Finn, C . W., Lindner, W., and Kotsatos, M . (1987) J . Biol . Chem . 262, 17556-17562) . The CMP-NeuAc synthetase gene expression was increased 10-30-fold by cloning of a 2.7-kb EcoRI-HindIII fragment onto the vector pKK223-3 containing the tac promoter . The complete nucleotide sequence of the gene encoding CMP-NeuAc synthetase was determined from progressive deletions generated by selective digestion of M13 clones containing the 2.7-kb fragment . CMP-NeuAc synthetase is located near the EcoRI site on this fragment as indicated by the detection of an open reading frame encoding a 49,000-dalton polypeptide . The amino- and carboxyl-terminal sequences of the encoded protein were confirmed by sequencing of peptides cleaved from both ends of the purified enzyme . The nucleotide deduced amino acid sequence was confirmed by sequencing several tryptic peptides of purified enzyme . The molecular weight is consistent with that determined from sodium dodecyl sulfate-gel electrophoresis . Gel filtration and ultracentrifugation experiments under nondenaturing conditions suggest that the enzyme is active as a 49,000-dalton monomer but may form aggregates.

Biochemistry, 1989 Sep 5, 28(18), 7313 - 8
Lysine-60 in the regulatory chain of Escherichia coli aspartate transcarbamoylase is important for the discrimination between CTP and ATP; Zhang Y et al.; Lysine-60 in the regulatory chain of aspartate transcarbamoylase has been changed to an alanine by site-specific mutagenesis . The resulting enzyme exhibits activity and homotropic cooperativity identical with those of the wild-type enzyme . The substrate concentration at half the maximal observed specific activity decreases from 13.3 mM for the wild-type enzyme to 9.6 mM for the mutant enzyme . ATP activates the mutant enzyme to the same extent that it does the wild-type enzyme, but the concentration of ATP required to reach half of the maximal activation is reduced approximately 5-fold for the mutant enzyme . CTP at a concentration of 10 mM does not inhibit the mutant enzyme, while under the same conditions CTP at concentrations less than 1 mM will inhibit the wild-type enzyme to the maximal extent . Higher concentrations of CTP result in some inhibition of the mutant enzyme that may be due either to hetertropic effects at the regulatory site or to competitive binding at the active site . UTP alone or in the presence of CTP has no effect on the mutant enzyme . Kinetic competition experiments indicate that CTP is still able to displace ATP from the regulatory sites of the mutant enzyme . Binding measurements by equilibrium dialysis were used to estimate a lower limit on the dissociation constant for CTP binding to the mutant enzyme (greater than 1 x 10(-3) M) . Equilibrium competition binding experiments between ATP and CTP verified that CTP still can bind to the regulatory site of the enzyme . For the mutant enzyme, CTP affinity is reduced approximately 100-fold, while ATP affinity is increased by 5-fold.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1989 Sep 5, 28(18), 7289 - 97
Crystallographic studies of the mechanism of xylose isomerase; Farber GK et al.; The mechanism of xylose isomerase (EC 5.3.1.5) has been studied with X-ray crystallography . Four refined crystal structures are reported at 3-A resolution: native enzyme, enzyme + glucose, enzyme + glucose + Mg2+, and enzyme + glucose + Mn2+ . One of these structures (E.G.Mg) was determined in a crystal mounted in a flow cell . The other structures were equilibrium experiments carried out by soaking crystals in substrate containing solution . These structures and other studies suggest that, contrary to expectation, xylose isomerase may not use the generally expected base-catalyzed enolization mechanism . A mechanism involving a hydride shift is consistent with the structures presented here and warrants further investigation . Additional evidence in support of a hydride shift comes from comparing xylose isomerase with triosephosphate isomerase which is known to catalyze an analogous reaction via an enediol intermediate . Evidence is presented that suggests that aldose-ketose isomerases can be divided into two groups . Phospho sugar isomerases generally do not require a metal ion for activity and show exchange of substrate protons with solvent . In contrast, simple sugar isomerases all require a metal ion and show very low solvent exchange . These observations are rationalized on the basis of the need for stereospecific sugar binding.

J Biol Chem, 1989 Sep 5, 264(25), 14591 - 3
Putative metal finger structure of the human immunodeficiency virus type 1 enhancer binding protein HIV-EP1; Maekawa T et al.; The region containing two copies of the sequence GGGACTTTCC in the human immunodeficiency virus type 1 (HIV-1) long terminal repeat, that is an NF-kappa B binding site, functions as an enhancer element for HIV transcriptional regulation . By a Southwestern method we have isolated a cDNA encoding the HIV-1 enhancer binding protein (HIV-EP1) from a human B-cell lambda gt11 library . DNase I footprinting analysis using the HIV-EP1 protein expressed in Escherichia coli showed that HIV-EP1 specifically bound to the HIV-1 enhancer . HIV-EP1 protein contains a domain with two tandem "zinc finger" sequences initially described in the Xenopus transcription factor IIIA . This represents the first demonstration of the structural feature of the protein that binds to the HIV-1 enhancer.

Biochemistry, 1989 Sep 5, 28(18), 7401 - 8
Site-directed mutagenesis of histidine-13 and histidine-114 of human angiogenin . Alanine derivatives inhibit angiogenin-induced angiogenesis; Shapiro R et al.; The roles of His-13 and His-114 in the ribonucleolytic and angiogenic activities of human angiogenin have been investigated by site-directed mutagenesis . Replacement of either residue by alanine (H13A and H114A) decreases enzymatic activity toward tRNA by at least 10,000-fold and virtually abolishes 10,000-fold and virtually abolishes angiogenic activity in the chick embryo chorioallantoic membrane assay . Both the H13A and H114A mutant proteins compete effectively with angiogenin in the latter assay; only a 5-fold molar excess of H13A over unmodified protein is required for complete inhibition . The His----Ala substitutions, however, do not have any significant effect on the interaction of angiogenin with human placental ribonuclease inhibitor, an extremely potent inhibitor of angiogenin (Ki approximately 7 x 10(-16 M) previously shown to interact with another active-site residue, Lys-40 . The effects of more conservative replacements-glutamine at position 13 and asparagine at position 114--were also examined . While the enzymatic activity of the H114N mutant was at least 3300-fold less than for the unmodified protein, the H13Q derivative had only 300-fold reduced activity toward tRNA and cytidylyl(3'----5') adenosine . Both substitutions substantially decreased angiogenic activity . The parallel effects on ribonucleolytic and biological activities observed with all four mutant proteins provide strong evidence that the latter activity of angiogenin is dependent on a functional enzymatic active site . The capacity of the H13A and H114A derivatives to compete with angiogenin in the chorioallantoic membrane assay suggests several additional features of the biological mode of action of this protein.

J Mol Biol, 1989 Sep 5, 209(1), 65 - 77
Rates of aminoacyl-tRNA selection at 29 sense codons in vivo; Curran JF et al.; We have placed aminoacyl-tRNA selection at individual codons in competition with a frameshift that is assumed to have a uniform rate . By assaying a reporter in the shifted frame, relative rates for association of the 29 YNN codons and their cognate aminoacyl-tRNAs were obtained during logarithmic growth in Escherichia coli . For five codons, three beginning with C and two with U, these relative rates agree with relative in vitro rates for elongation factor Tu-mediated aminoacyl-tRNA binding to ribosomes and subsequent GTP hydrolysis . Therefore, the frameshift assay probably measures this process in vivo . Observed rates for aminoacyl-tRNA selection span a 25-fold range . Therefore, the time required to transit different codons in vivo probably differs substantially . Codons very frequently used in highly expressed genes generally select aminoacyl-tRNAs more quickly than do rarely used codons . This suggests that speed of aminoacyl-tRNA selection is a significant factor determining biased use of synonymous codons . However, the preferential use of codons appears to be marked only for codons with the highest rates of aminoacyl-tRNA selection . Rapid selection in vivo is usually effected by elevation of the tRNA concentration for codons with moderate intrinsic speed (rate constant), not by choosing intrinsically fast codons . Despite a preference for high rate, there are quickly translated codons that are not commonly used, and common codons that are translated relatively slowly . Other factors are therefore more important than speed for some codons . Strong preference for rapid aminoacyl-tRNA selection is not observed in weakly expressed genes . Instead, there is a slight preference for slower aminoacyl-tRNA selection . The rate of aminoacyl-tRNA selection by a YNC codon is always greater than the rate of the corresponding YNU codon even though in many YNC/U pairs both codons react with the same elongation factor Tu/GTP/aminoacyl-tRNA complex . Thus, for these tRNAs, the differences between in vivo rate constants of tRNAs are dependent on the nature of anticodon base-pairing . However, no more general relationship is evident between codon/anticodon composition and rate of aminoacyl-tRNA selection . The frameshift method can be extended to all codons.

J Biol Chem, 1989 Sep 5, 264(25), 15120 - 9
Transcriptional activation of pBR322 DNA can lead to duplex DNA unwinding catalyzed by the Escherichia coli preprimosome; Parada CA et al.; Mechanisms that could operate to initiate pBR322 DNA replication in the absence of RNase H and DNA polymerase I are described . Two different pathways leading to extensive unwinding of pBR322 DNA have been observed under DNA replication reaction conditions in vitro . In the presence of RNA polymerase and DNA gyrase, specifically initiated RNA II (the leading-strand primer precursor) can form an RNA-DNA hybrid with the template that starts just upstream of the origin of DNA replication and continues for about 3 kilobases . Subsequent digestion of the RNA in this RNA-pBR322 DNA hybrid results in the formation of a highly unwound DNA termed form I . If DNA gyrase is absent during the RNA polymerase-catalyzed elongation of RNA II, a stable RNA-pBR322 DNA hybrid can still form that is localized to the origin region of the genome . Formation of this hybrid activates the primosome assembly site present on the lagging-strand DNA template, by displacing it to a single-stranded conformation, thereby allowing preprimosome assembly . Once assembled, the DNA helicase activity of the preprimosome, in the presence of the single-stranded DNA binding protein and DNA gyrase but in the absence of any further transcription, can also result in extensive unwinding of pBR322 DNA . The product of this reaction, form I DNA, is more unwound than form I DNA . The formation of both form I and form I DNA is inhibited by the presence of excess RNA I, as well as by RNase H at concentrations sufficient to catalyze the normal processing of RNA II required for initiation of leading-strand DNA synthesis . These results suggest that RNA II-pBR322 DNA hybrid formation is essential to permit preprimosome assembly during pBR322 DNA replication under conditions where both RNase H and DNA polymerase I are absent.

Biochim Biophys Acta, 1989 Sep 4, 984(2), 188 - 92
How many Na+-dependent carriers for L-alanine and L-proline in the eel intestine? Studies with brush-border membrane vesicles; Vilella S et al.; Using brush-border membrane (BBM) vesicles prepared from the intestine of the European eel, the specificity of L-alanine and L-proline Na+-dependent transport was investigated by measuring the uptake of isotopically labelled substrates . In the presence of Na+ ions, cross-inhibition between alanine and proline transports was observed; in addition alpha-(methylamino)isobutyric acid (MeAIB) inhibited proline but had no effect on alanine uptake . These results can be explained by the presence, in eel intestinal BBM vesicles, of at least two distinct agencies for Na+-dependent proline and alanine translocation . The first system is specific for alanine and short-chain neutral amino acids; the second system, specific for imino acids and the N-methylated analogues, is regulated by alanine concentration.

Biochim Biophys Acta, 1989 Sep 4, 1013(1), 21 - 7
The assessment of relative surface hydrophobicity as a factor involved in the activation of human polymorphonuclear leukocytes by uropathogenic strains of Escherichia coli; Steadman R et al.; The initiation of the respiratory burst and the degranulation of human polymorphonuclear leukocytes (PMN) in response to stimulation by uropathogenic strains of Escherichia coli is dependent on the expression of Type 1 fimbriae by those strains . These PMN responses correlate with an increasing tendency of the interacting E . coli strain to be retained on hydrophobic columns . The present work assessed the measurement of relative surface hydrophobicity in relation to PMN activation . Type 1 fimbriate organisms bound most readily to Octyl-Sepharose columns and were strongly agglutinated in the salt aggregation test . In contrast, the same organisms partitioned to the dextran-rich (hydrophilic) phase of aqueous two-polymer phase systems . Electron microscopic observation of the organisms eluted from the Octyl-Sepharose columns and of the organisms recovered from both phases of the aqueous two-phase systems demonstrated, however, that both Type 1 and P-fimbriate organisms were retained on the columns and partitioned into the dextran-rich phase as a consequence of their being fimbriate and failed to identify this as a major factor in the activation of PMN . In addition electron microscopy demonstrated that each P-fimbriate population had fewer organisms expressing fimbriae than did Type 1 fimbriate populations, confirming the importance of phase variation as a factor affecting the physicochemical characteristics of a bacterial population.

Rev Esp Enferm Apar Dig, 1989 Sep, 76(3), 203 - 9
{Immunosuppression with cyclosporin after orthoptic liver transplant in pigs}; Arias J et al.; The immunosuppressive effect of cyclosporine A (CsA) was studied in six pigs that underwent orthotopic liver transplant (OLT) . The drug was administered i.v . in low doses (1.5-4 mg/Kg-1/12 h-1) and associated with wide spectrum antibiotics . The mean survival of the animals was 23.4 +/- 11.2 days . All animals had fever, jaundice and hypertrichosis, and the preoperative corporal weight remained constant . Liver abscesses by E . coli were common autopsy findings . Livers also presented biliary regeneration and cholestasis with a slight lymphocytic infiltration . OLT in pigs without immunosuppressive treatment causes and elevation in serum gamma-GTP, GOT, LDH, alkaline phosphatase and bilirubin . In the post-mortem findings, the livers did not present abscesses but evidenced intense atrophy with hepatocellular necrosis and abundant lymphocytic infiltration.

Masui, 1989 Sep, 38(9), 1188 - 94
{The effects of ONO 3708, a new thromboxane receptor antagonist, on cardiovascular response during the early phases of endotoxin shock in anesthetized dogs}; Sasao J et al.; The effects of ONO 3708, a new thromboxane A2 receptor antagonist, on cardiovascular and airway responses, at an early phase during endotoxin shock were investigated in anesthetized dogs . The i.v . infusion (1mg.kg-1) of E.coli endotoxin caused an increase in mean pulmonary artery pressure (MPAP) from 9.9 +/- 1.0 to 19.1 +/- 2.3 mmHg at 5 min, and at 15 min after infusion, elevated MPAP returned toward the control level . Pretreatment with ONO 3708 abolished these effects of endotoxin on pulmonary artery pressure at an early phase . The change in airway pressure reached a maximum of 14.4 +/- 1.7 cmH2O from 10.0 +/- 1.9 at 5 min, followed by a gradual decline toward a baseline value at 30 min in the control group . ONO 3708 significantly attenuated increase in airway pressure induced by E . coli endotoxin . But pretreatment with ONO 3708 could not prevent decrease in systemic arterial pressure and cardiac output induced by endotoxin . These results suggest that role of thromboxane A2 on the cardiovascular response during endotoxin shock is played only on pulmonary vascular changes, and ONO 3708 has a beneficial effect at least during the early phase of endotoxin shock.

J Parenter Sci Technol, 1989 Sep-Oct, 43(5), 246 - 9
Pyrogens in small-volume parenterals prepared in hospital pharmacy; Lacasa C et al.; Three pyrogen assay methods {the rabbit method, Limulus Amebocyte Lysate (LAL) gelation and chromogenic substrate, and bioburden prior to sterilization} were evaluated in three standard batches of small-volume parenteral preparations; i.e., water for injection, sodium chloride 0.9% injection, and sodium bicarbonate 8.4% injection . The same preparations were also assayed after contamination with Escherichia coli followed by sterilization . All methods gave the same results with the water for injection and the sodium chloride 0.9% . With sodium bicarbonate 8.4%, only the rabbit method was valid . For sodium chloride 0.9%, the chromogenic substrate method was valid; the gelation method was not valid . Endotoxins from the manufacturing plant were less pyrogenic in rabbits than were standard endotoxins or those from hospital E . coli.

J Interferon Res, 1989 Sep, 9 Suppl 1, S61 - 5
High levels of circulating neutralizing antibody in normal animals to recombinant mouse interferon-beta produced in yeast; Sedmak JJ et al.; Plasma from normal outbred Swiss mice not previously given interferon (IFN) neutralized a glycosylated (high-mannose) recombinant murine IFN-beta made in yeast (rMuIFN-beta y) . The neutralization antibody titers were as high as 1:6,000 versus rMuIFN-beta y, whereas the titers obtained with native murine IFN-beta (MuIFN-beta) were 100-fold less; a recombinant murine IFN-beta make in Escherichia coli (rMuIFN-beta ec) was not neutralized at 1:10 dilution of plasma . An ELISA using rMuIFN-beta y demonstrated that it is gamma immunoglobulins in normal mouse plasma that bind rMuIFN-beta y . Normal rabbit serum also had very high endogenous neutralizing activity (1:5,300) against rMuIFN-beta y . Significant neutralizing activity also was detected in newborn bovine and normal human serum as well as pooled human immune serum globulin . Thus, endogenous neutralizing activity directed presumably at the carbohydrate residues of highly glycosylated recombinant proteins produced in yeast may limit their clinical utility.

J Appl Physiol, 1989 Sep, 67(3), 963 - 9
Blunted febrile response to intravenous endotoxin in starved rats; Shido O et al.; The effects of fasting on the febrile responses to intravenous injection of bacterial lipopolysaccharide (LPS; endotoxin) of Escherichia coli were investigated in rats . Ad libitum-fed rats (C) produced a biphasic fever with an increase in the temperature difference between brown adipose tissue and colon and shivering activity (SA) . Measurement by a direct calorimeter showed no particular changes in heat loss . Rats starved for 4 days (F4) responded to intravenous LPS with a monophasic fever accompanied by an increase in SA only . However the maximal rise in colonic temperature (Tco) did not differ from C rats . Subsequent 2-day fasting reduced SA and the maximal fever height . Endogenous pyrogen (EP) injected intravenously produced a prompt rise in Tco followed by prolonged hyperthermia in C rats . In the F4 rats, there was no such sustained rise in Tco as a result of intravenous EP . The response in Tco to intravenous prostaglandin E2 (PGE2) was the same in fed and starved rats . The administration of LPS, EP, and PGE2 into the lateral ventricle evoked a similar extent of hyperthermia in C and F4 rats . Because the second phase of fever has been shown to occur after pyrogens are translated into a febrile stimulus within the blood-brain barrier, it is assumed that the functional changes of the blood-brain barrier such as in the permeability of pyrogens or in the sensitivity of pyrogen receptors resulted in the absence of the second phase of fever in starved rats.

Eur J Immunol, 1989 Sep, 19(9), 1619 - 24
Visualizing interleukin 2 gene expression at the single cell level; Emilie D et al.; To analyze the expression of the interleukin (IL)2 gene at the single cell level, we have constructed a chimeric gene in which the regulatory sequences from the mouse IL2 gene were fused 5' proximal to the coding region of the Escherichia coli beta-galactosidase (lacZ) gene . Once stably introduced into a T cell hybridoma, the IL 2-lacZ reporter gene was shown to display a pattern of induction similar to the one observed for the resident IL 2 gene . Two points emerged from monitoring IL 2 expression for the resident IL2 gene . Two points emerged from monitoring IL 2 expression at the single cell level . First, upon activation the expression of the IL2-lacZ gene appears asynchronous . Second, at the peak of induction the percentage of beta-galactosidase+ cells never reached 100% and the level of beta-galactosidase reached among positive cells was highly heterogeneous within a cloned population of T cells . In combination with the possibility to sort viable lymphocytes according to lacZ expression, the IL2-lacZ reporter gene described herein should provide a way to isolate somatic cell mutants deficient in signal transduction by the T cell antigen receptor complex.

J Bacteriol, 1989 Sep, 171(9), 5148 - 54
Binding-protein-dependent alanine transport in Rhodobacter sphaeroides is regulated by the internal pH; Abee T et al.; The properties of an L-alanine uptake system in Rhodobacter sphaeroides were studied and compared with those of H+/lactose symport in R . sphaeroides 4P1, a strain in which the lactose carrier of Escherichia coli has been cloned and functionally expressed (F . E . Nano, Ph.D . thesis, University of Illinois, Urbana, 1984) . Previous studies indicated that both transport systems were active only when electron transfer took place in the respiratory or cyclic electron transfer chain, while uptake of L-alanine also required the presence of K+ (M . G . L . Elferink, Ph.D . thesis, University of Groningen, Groningen, The Netherlands, 1986) . The results presented in this paper offer an explanation for these findings . Transport of the nonmetabolizable L-alanine analog 2-alpha-aminoisobutyric acid (AIB) is mediated by a shock-sensitive transport system . The apparently unidirectional uptake of AIB results in accumulation levels which exceed 7 x 10(3) . The finding of L-alanine-binding activity in the concentrated crude shock fluid indicates that L-alanine is taken up by a binding-protein-dependent transport system . Transport of the nonmetabolizable lactose analog methyl-beta-D-thiogalactopyranoside (TMG) by the lactose carrier under anaerobic conditions in the dark was observed in cells and membrane vesicles . This indicates that the H+/lactose symport system is active without electron transfer . Uptake of AIB, but not that of TMG, is inhibited by vanadate with a 50% inhibitory concentration of 50 microM, which suggests a role of a phosphorylated intermediate in AIB transport . Uptake of TMG and AIB is regulated by the internal pH . The initial rates of uptake increased with the internal pH, and and pKa values of 7.2 for TMG and 7.8 for AIB . At an internal pH of 7, no AIB uptake occurred, and the rate of TMG uptake was only 30% of the rate at an internal pH of 8 . In a previous study, we found that K+ plays an essential role in regulating the internal pH (T . Abee, K . J . Hellingwerf, and W . N . Konings, J . Bacteriol . 170:5647-5653, 1988) . The dependence of solute transport in R . sphaeroides on both K+ and activity of an electron transfer chain can be explained by an effect of the internal pH, which subsequently influences the activities of the lactose-and binding-protein-dependent L-alanine transport system.

Virology, 1989 Sep, 172(1), 370 - 3
Evidence that nucleocapsid disassembly and a later step in virus replication are inhibited in transgenic tobacco protoplasts expressing TMV coat protein; Osbourn JK et al.; Tobacco mosaic virus (TMV)-like pseudovirus particles containing mRNA for Escherichia coli beta-glucuronidase (GUS) were electroporated into mesophyll protoplasts from control or TMV coat protein (CP)-transgenic tobacco (Nicotiana tabacum cv . Xanthi) . GUS-particles were expressed 100-fold less efficiently in CP-transformed than in control protoplasts whereas unencapsidated GUS mRNA was expressed only 2.8-fold less efficiently . Lower transient expression of packaged GUS mRNA is probably due to inhibited disassembly of nucleocapsids in CP-transgenic protoplasts . Control and U1 CP-transformed protoplasts are equally susceptible to infection by cowpea strain TMV (Cc), as well as unencapsidated Cc or U1 RNA . In contrast, native or in vitro reconstituted U1 TMV particles result in 5- to 6-fold fewer infected CP-transgenic than control protoplasts . When Cc RNA was transcapsidated in U1 CP in vitro, the hybrid virions were equally infectious in both classes of protoplasts . We conclude that although compatible U1 protein-protein interactions significantly inhibit (GUS) nucleocapsid disassembly in CP-transgenic protoplasts, the endogenous CP must also interfere with a later stage of infection involving the homologous viral RNA.

Mikrobiologiia, 1989 Sep-Oct, 58(5), 746 - 50
{The effect of substrates and irradiation with low intensity visible light on the rate of division of Escherichia coli}; Tiflova OA et al.; The effect of He--Ne laser irradiation (lambda = 632.8 nm, D = 4.10(3) J/m2) on the growth of Escherichia coli cultures was studied in a minimal medium containing glucose, glycerol of arabinose . The irradiated cultures immediately started to divide with a virtually identical specific growth rate (kappa = 0.78, 0.8 and 0.8 h-1) whatever the growth rate and the latent period of the parent cultures were . The ratio between cell numbers in the irradiated and non-irradiated cultures was highest 1 h after the irradiation: 1.25, 1.3 and 1.5 for the glucose, glycerol and arabinose cultures, respectively . Apparently, the irradiation with visible light of a low intensity creates an additional proton gradient and thus stimulates a new replication and division cycle in the population of cells whose membranes do not have delta pH necessary for the initiation of these processes . The system under study can serve as a model for studying the delta pH-dependent stages in the regulation of cell division.

Mikrobiologiia, 1989 Sep-Oct, 58(5), 709 - 15
{The role of intracellular pool of polyamines in the regulation of metabolism in Escherichia coli during aerobic-anaerobic transitions}; Tkachenko AG et al.; The transition from aerobic to anaerobic conditions of Escherichia coli cultivation is accompanied by a drop in the free pool of putrescine in the cell as well as in the medium and by a decrease in the intracellular concentration of spermidine . This process stems from at least two facts: (1) the activity of ornithine decarboxylase, a key enzyme in the synthesis of polyamines, falls down due to a decrease in the level of ATP in the cell; (2) the free pool of polyamines is bound to cell compartments, in particular, to nucleoid DNA . The results make it possible to consider the system of polyamine synthesis as a point in the regulatory interaction between the energetic and constructive types of E . coli metabolism.

Biotechniques, 1989 Sep, 7(8), 840 - 50
Automated methods for single-stranded DNA isolation and dideoxynucleotide DNA sequencing reactions on a robotic workstation; Mardis ER et al.; Automated procedures have been developed for both the simultaneous isolation of 96 single-stranded M13 chimeric template DNAs in less than two hours, and for simultaneously pipetting 24 dideoxynucleotide sequencing reactions on a commercially available laboratory workstation . The DNA sequencing results obtained by either radiolabeled or fluorescent methods are consistent with the premise that automation of these portions of DNA sequencing projects will improve the reproducibility of the DNA isolation and the procedures for these normally labor-intensive steps provides an approach for rapid acquisition of large amounts of high quality, reproducible DNA sequence data.

J Gen Microbiol, 1989 Sep, 135 ( Pt 9), 2365 - 78
Cloning and expression of Treponema pallidum antigens in Escherichia coli; Bailey MJ et al.; A library of Treponema pallidum genomic DNA fragments produced by partial Sau3A digestion was established in Escherichia coli K12 using the plasmid vector pAT153 . The library was screened using immune syphilitic rabbit serum and six recombinant phenotypes expressing eight treponemal polypeptides were detected . With two exceptions, all the recombinant gene products were the same size as polypeptides detected on Western immunoblots of T . pallidum . The genes encoding three novel gene products, with molecular masses in SDS-PAGE of 42, 17 and 15.5 kDa, which had not been cloned previously from T . pallidum were also identified . Monoclonal antibodies which reacted with four of the eight recombinant polypeptides were generated.

Plasmid, 1989 Sep, 22(2), 132 - 42
Shuttle plasmids constructed by the transformation of an Escherichia coli cloning vector into two Deinococcus radiodurans plasmids; Smith MD et al.; An Escherichia coli plasmid that confers kanamycin resistance (Kmr) was inserted into the large Deinococcus radiodurans cryptic plasmids pUE10 and pUE11, yielding pS28 and pS19 . The method of insertion involved both in vitro splicing and the natural transformation of D . radiodurans and yielded full-length clones in E . coli of pUE10 and pUE11 . Both pS28 and pS19 replicated and expressed Kmr in E . coli and D . radiodurans . In both pS28 and pS19, D . radiodurans plasmid sequences were immediately upstream from the Kmr determinant . Transformation experiments suggested that Kmr expression in D . radiodurans was initiated in upstream D . radiodurans sequences . Restriction maps of pS28 and pS19 showed that each plasmid contained three MraI sites . Both pS28 and pS19 transformed the MraI-producing D . radiodurans strain R1 at low frequencies . D . radiodurans strain Sark, which naturally contains pUE10 and pUE11, was transformed by pS28 and pS19 at much higher frequencies . A Sark derivative that was cured for pUE10 was isolated by screening Sark/pS28 subisolates for loss of kanamycin resistance.

Physiol Behav, 1989 Sep, 46(3), 535 - 9
Verapamil and indomethacin attenuate endotoxin-induced anorexia; Langhans W et al.; To characterize the mechanism of the anorexia during infection, we investigated the effect of E . coli lipopolysaccharide (LPS) on feeding in rats under various conditions: LPS (125, 100, 75, and 50 micrograms/kg body weight = b . wt.) injected intraperitoneally (IP) reduced food intake by decreasing meal frequency without affecting meal size . The Ca++-channel blocker verapamil (5 mg/kg b . wt., IP) or the antipyretic and antiphlogistic drug indomethacin (2.5 mg/kg b . wt., IP), but not combined alpha- and beta-adrenergic receptor blockade by IP phentolamine plus propranolol (500 micrograms/kg b . wt., each) attenuated the anorectic effect of LPS (125 or 100 micrograms/kg b . wt.) . The results suggest that a phospholipase A2-sensitive mechanism contributes to the anorexia during injection.

Biull Eksp Biol Med, 1989 Sep, 108(9), 369 - 72
{Morphofunctional characteristics of the duodenum in the early stages of experimental escherichiosis}; Parkhomenko IuG et al.; Using the model of experimental colibacillosis in mice morphological, immunomorphological and morphometrical studies of duodenum 1 to 24 hours after inoculation were performed . Typical dynamics of cellular composition in the intestinal wall, accompanied with increased lymphoplasmacellular infiltration, and dystrophic changes in cells of submucosal and intermuscular neuroplexuses were found . The data obtained testify, that the infectious process at the acute state of experimental colibacillosis has an immune character (immediate hypersensitivity of local type), characterised by increasing in immunoglobulin-containing cells and including in this process of mast and endocrine cells.

Mol Biol (Mosk), 1989 Sep-Oct, 23(5), 1340 - 9
{The effect of supercoiling of DNA from colicinogenic plasmids on the expression of col, imm and lys genes}; Panchenko IuA et al.; The expression of colicin genes is controlled by the SOS-system (Lex A repressor) and the adenylate-cyclase system (cAMP-CAP complex) . The effect of plasmid DNA supercoiling on the expression of the operons of colicins E1, E2, and E3 has been studied by using E . coli minicells . It has been shown for the colicin E1 operon that it is the promoter that is influenced by supercoiling: an increase in negative supercoiling elevates the expression and, vice versa, DNA relaxation reduces the expression . The effect of supercoiling on gene activity of the colicin E1 immunity protein has not been observed, which may be due to the specific orientation of this gene . With the two other colicins supercoiling affects the expression of all genes which constitute the operon . The regulation of the colicin operon expression has been confirmed to occur at three levels: by the LexA protein, by the cAMP-CAP complex, and by the plasmid DNA supercoiling.

Mol Biol (Mosk), 1989 Sep-Oct, 23(5), 1248 - 62
{Pattern recognition in the computer analysis of nucleotide sequences}; Aleksandrov NN et al.; We have used an algorithm from the pattern recognition theory "generalized portrait" to find a distinguishing vector for Escherichia coli promoters . We have made an attempt to solve closely linked problems for choosing significant signs of that signal, multiple alignment and for calculation of the recognition vector (matrix) . The promoters with known strength have been ranged with this vector . The analysis of the occurrence of predicted promoters has been carried out . The promoters search program for IBM-compatible computers is available from the authors.

J Biochem (Tokyo), 1989 Sep, 106(3), 460 - 7
Purification and characterization of the active serine: pyruvate aminotransferase of rat liver mitochondria expressed in Escherichia coli; Oda T et al.; In the previous study (Oda, T., et al . (1985) Eur . J . Biochem . 150, 415-421), we isolated a cDNA clone which expressed in Escherichia coli a specific size of product having the activity of rat liver serine:pyruvate aminotransferase (SPTm) . This specific product (SPT10) was purified to homogeneity through three different column chromatographies . The amino acid composition and N-terminal amino acid sequence of the purified enzyme agreed with those predicted from the nucleotide sequence of cDNA and showed that SPT10 consists of the whole amino acid sequence of mature SPTm and several extra amino acid residues at the N-terminus . The catalytic and physical properties of SPT10, such as substrate specificity, Km for alpha-keto acids, electric charge, and quaternary structure, were all very similar to those of SPTm . Using several cDNA clones which lack a 5'-terminal sequence corresponding to a portion of the N-terminal amino acid sequence of SPTm, we examined the expression profile of the specific product in bacteria transformed with each cDNA clone . The products encoded by these cDNAs were segregated into inclusion bodies and were neither catalytically active nor easily solubilized by sonication . In contrast, the inclusion bodies were not formed in the bacteria transformed with the cDNA clone for SPT10.

J Biochem (Tokyo), 1989 Sep, 106(3), 436 - 41
Production and characterization of recombinant human neutrophil chemotactic factor; Furuta R et al.; A putative mature human neutrophil chemotactic factor (NCF) corresponding to the C-terminal 72 amino acids of its precursor was directly produced in Escherichia coli by recombinant DNA technology . Human NCF was present in both the soluble and insoluble protein fractions of the homogenate of host cells, and it was partially purified as a water-soluble polypeptide from both fractions, separately . The partially purified NCF preparation was highly purified to an endotoxin-free homogeneous polypeptide by means of CM-Sepharose CL-6B column chromatography and gel filtration on Toyopearl HW-55 . No difference between the human NCF preparations purified from both starting materials could be found concerning purity, primary structure, solubility, molecular weight, and chemotactic activity for human neutrophils . The amino acid sequence of recombinant human NCF was identical to the sequence deduced from the cDNA sequence . A methionine residue due to the translation initiation codon was removed . Recombinant human NCF was found to be biologically active and to exhibit chemotactic activity for human neutrophils in vitro and cause a neutrophil infiltration in vivo in mice.

Genes Dev, 1989 Sep, 3(9), 1462 - 71
Identification of the sigma E subunit of Escherichia coli RNA polymerase: a second alternate sigma factor involved in high-temperature gene expression; Erickson JW et al.; The rpoH gene of Escherichia coli encodes sigma 32, the 32-kD sigma-factor responsible for the heat-inducible transcription of the heat shock genes . rpoH is transcribed from at least three promoters . Two of these promoters are recognized by RNA polymerase containing sigma 70, the predominant sigma-factor . We purified the factor responsible for recognizing the third rpoH promoter (rpoH P3) and identified it as RNA polymerase containing a novel sigma-factor with an apparent Mr of 24,000 . This new sigma, which we call sigma E, is distinct from the known sigma factors in molecular weight and promoter specificity . sigma E holoenzyme will not recognize the sigma 70- or sigma 32-controlled promoters we tested, but it does transcribe the htrA gene, which is required for viability at temperatures greater than 42 degrees C . The in vivo role of sigma E is not known . The transcripts from the sigma E-controlled rpoH P3 and htrA promoters are most abundant at very high temperature, suggesting the sigma E holoenzyme may transcribe a second set of heat-inducible genes that are involved in growth at high temperature or in thermotolerance.

Genetika, 1989 Sep, 25(9), 1541 - 50
{Analysis of expression of the gene from the BRa puff of Chironomus thummi encoding the low molecular weight secretory protein}; Bogachev SS et al.; The plasmid containing F6.2 gene from the BRa of Chironomus thummi within the pUR 292 vector was constructed . Chimeric protein containing beta-galactosidase-F6.2 polypeptide was produced in Escherichia coli BMH71-18 . The protein obtained has immunological similarity with secretion protein of 67,000 D from Ch . thummi salivary gland . The gene for sp67 is active in main and in special lobe of the gland . Based on the data obtained and on the previous results of the authors, conclusion is made that BRa is a complex locus containing several types of genes.

Jpn J Surg, 1989 Sep, 19(5), 556 - 62
Thromboxane as a possible hepatotoxic factor increased by endotoxemia in obstructive jaundice; Hanai T et al.; In a study using rats, we investigated whether liver damage induced by endotoxemia in obstructive jaundice is associated with thromboxane (TX) in order to acertain whether its vasoconstrictive and platelet aggregating properties play a role in reducing liver blood flow . The rats were divided into the following 5 groups; a control group, an endotoxin (Et) group, a bile duct ligation (BDL) group, a bile duct ligation and endotoxin (BDL + Et) group and an OKY046 (Thromboxane synthetase inhibitor) treated bile duct ligation + endotoxin (OKY-BDL + Et) group . The blood TXB2 levels in the Et, BDL and BDL + Et groups were higher than those in the control group . The liver TXB2 levels in the Et and BDL + Et groups were also higher than those in the control group . Liver phospholipids and liver blood flow decreased in the BDL + Et group, whereas in the OKY-BDL + Et group they returned close to the control group levels by decreasing the TXB2 levels in both the liver and blood to normal . These results suggest that the high level of TX in the blood and liver tissue may further aggrevate the liver during endotoxemia in obstructive jaundice by inhibiting liver blood flow.

Gig Sanit, 1989 Sep, (9), 26 - 9
{Colicin genotype characteristics of pathogenic Escherichia circulating in the environment}; Bei TV; Colicin identification of pathogenic Escherichia, isolated from the environmental objects, showed that determination of colicinogenicity signs and sensitivity to standard colicins in pathogenic E . to a more considerable degree depended on strain sulfur-group membership than on the amanating objects . The analysis of colicin genotype characteristics of pathogenic E., circulating in environment, provided much information and could be used in identifying a source of infection and ways of escherichiosis pathogen transmission.

Z Naturforsch {C}, 1989 Sep-Oct, 44(9-10), 838 - 44
{Physiologic significance of "stringent control" in Escherichia coli under extreme starvation}; Mach H et al.; The viability of three isogenic relA+/relA strain pairs of Escherichia coli (CP78/CP79; NF161/NF162; CP107/CP143) was studied during prolonged starvation for amino acids, glucose or phosphate . After amino acid limitation we found a prolonged viability of all relA+ strains which synthesized ppGpp . We suggest that some ppGpp-mediated pleiotropic effects of the stringent response (e.g . glykogen accumulation, enhanced protein turnover) might be involved in this prolongation of survival . After glucose or phosphate starvation there was no difference in the relA+/relA strains either in the ppGpp content or in the survival.

Arzneimittelforschung, 1989 Sep, 39(9), 1073 - 80
Preparation and biological activity of new substituted antimalarial diaminodiphenylsulfones; Pieper H et al.; Starting from 4,4'-diamino-diphenylsulfone (DDS) as a lead structure, new 2-substituted analogues as well as new 2-substituted 4-alkylamino-4'-amino diphenylsulfones have been designed and synthetized in different ways . This has led to compounds the inhibitory activity of which against 7,8-dihydropteroic acid synthase of plasmodia and mycobacteria is clearly superior to that of sulfadoxine and in most cases to that of DDS . Of special interest is 4'-amino-4-n-propylamino-2-methyl-diphenylsulfone . Together with inhibitors of 7,8-dihydrofolate reductase in vitro and in vivo it possesses a marked synergistic inhibitory activity against plasmodia . In contrast to DDS in doses up to 200 mg/kg p.o . (cat) no methemoglobin formation is observed . The compound has been selected for further studies.

Mol Gen Genet, 1989 Sep, 218(3), 431 - 6
Purification and characterization of the sopB gene product which is responsible for stable maintenance of mini-F plasmid; Watanabe E et al.; The mini-F plasmid has the trans-acting sopA, sopB genes and the cis-acting sopC DNA which are essential for plasmid partitioning . In this paper, we report the purification of the sopB gene product from extracts of cells harboring a pBR322 derivative carrying the sopB gene . The purity of the final preparation was more than 95%, as determined by densitometry . The amino acid sequence of the amino-terminal region of the protein for the 17 residues identified was identical to that predicted from the DNA sequence of the sopB gene . Therefore, it was concluded that the protein was the sopB gene product . Using anti-SopB serum, the SopB protein was detected in the cell lysates of F+, F', and Hfr strains . The SopB protein bound to the plasmid DNA of a pBR322 derivative carrying the sopC DNA segment, but not to the vector plasmid pBR322.

Mol Gen Genet, 1989 Sep, 218(3), 397 - 401
Context specific misreading of phenylalanine codons; Precup J et al.; It has previously been shown that the phenylalanine codon UUC encoding residue 8 of the Escherichia coli argI gene product, ornithine transcarbamylase, is misread as leucine at a high frequency during phenylalanine starvation . However, no misreading of the UUU encoding residue 3 was observed under these conditions . Using oligonucleotide-directed, site-specific mutagenesis, we have constructed mutants where these codons have been changed . Using these mutant argI genes we see a high level of mistranslation at position 8 during phenylalanine starvation whether the codon is UUU or UUC . With either codon at position 3 we see no leucine substitution . We also constructed a gene with a leucine codon at position 3 . The product of this latter mutated gene is stable and active, indicating that preferential turnover of mistranslated protein is not obscuring an otherwise high rate of misreading . This would seem to indicate that it is the context rather than the particular phenylalanine codon which is important in determining these misreading levels.

Biochem Int, 1989 Sep, 19(3), 593 - 601
Potentiation of cysteine proteinase-inhibitor activity of full-length Ha-ras oncogene products by denaturation-renaturation; Hiwasa T et al.; We have investigated protease-inhibitory activity against cathepsin L of human and murine Ha-ras oncogene products (p21s) produced by Escherichia coli . The inhibitory activity of full-length p21s was much weaker than that of truncated p21s, however, the inhibitory activity was potentiated after denaturation with 8 M urea and 2 M NaCl followed by renaturation . These suggest that p21 can be folded into multiple three-dimensional conformations which have different protease-inhibitory activities.

Anal Biochem, 1989 Sep, 181(2), 336 - 40
Purification of plasmid-expressed proteins which lack functional assay systems; Marvel CC et al.; A general method for the purification of proteins whose genes are cloned into plasmid vectors, but whose biochemical and functional characteristics are unknown, is described . A cell-free transcription-translation system from Escherichia coli K-12 is used to synthesize in vitro radiolabeled protein expressed from recombinant plasmid vectors . The radiolabeled proteins are then fractionated and used as markers for the purification of nonradiolabeled proteins without recourse to functional assays . Biochemical analysis of the purified proteins can reveal information about their cellular localization, binding parameters, and physical, enzymatic, or regulatory properties . This information complements in vivo genetic analysis with the goal of identifying the gene and the function of its protein product . An example using this technique in which the product of the usg gene in the hisT operon of E . coli has been purified and biochemically characterized is described.

Onderstepoort J Vet Res, 1989 Sep, 56(3), 207 - 9
Adverse effects of a proposed equine sublethal endotoxin model; Stadler P et al.; Commercially available Escherichia coli 055: B5 lipopolysaccharide was administered intravenously experimentally at a dosage of 10 micrograms/kg to 2 horses . Various clinical and clinico-pathological parameters were monitored before and after the endotoxin administration . Because of a hopeless prognosis, and for humane reasons, euthanasia was applied on both horses 6 h after administration . Values recorded for the different parameters, including the blood lactate level, were consistent with a lethal condition . It would appear that an intravenous dose of 10 micrograms/kg of endotoxin is potentially lethal to horses.

Klin Wochenschr, 1989 Sep 1, 67(17), 843 - 6
Molecular genetics of the human Na+/glucose cotransporter; Hediger MA et al.; Recent success in expression cloning has revealed the primary structure of the Na+/glucose cotransporter from rabbit small intestine, and this has subsequently led to the cloning of the Na+/glucose cotransporters from human small intestine and human kidney . Close homology is evident between the rabbit and human intestinal Na+/glucose cotransporters at the DNA level, and the predicted amino acid and secondary structure levels . The Na+/glucose cotransporter amino acid sequence from human kidney is 57% identical with that from human small intestine . Significant homology also exists between these Na+/glucose cotransporters and the E . coli Na+/proline cotransporter (putP) . The rabbit intestinal Na+/glucose cotransporter has 11 potential membrane spanning regions and 2 hydrophilic regions containing highly charged residues . The amino acid sequence shows two potential N-glycosylation sites (N-X-T/S) . Using an in vitro translation approach we were able to determine that only one of these (Asn 248) is glycosylated . Expression experiments with Xenopus oocytes using the N-glycosylation inhibitor tunicamycin indicate that glycosylation of Asn 248 is required for functional expression of the transporter . The N-X-T/S sequence at Asn 248 is conserved in the human intestinal and the human renal Na+/glucose cotransporter . Chromosomal localization studies map the human intestinal Na+/glucose cotransporter gene (SGLT1) to the q11.2----qter region of chromosome 22 and the human renal Na+/glucose cotransporter gene (SGLT2) to the q-arm of chromosome 16.(ABSTRACT TRUNCATED AT 250 WORDS)

J Membr Biol, 1989 Sep, 110(3), 227 - 33
Solubilization and reconstitution of the Rickettsia prowazekii ATP/ADP translocase; Plano GV et al.; The ATP/ADP translocase protein of Rickettsia prowazekii, an obligate intracellular parasite that had been grown in the chick yolk sac, was solubilized and reconstituted into liposomes composed of Escherichia coli phospholipid by an octylglucoside dilution procedure . Proteoliposomes prepared from membranes of Renografin-purified R . prowazekii translocated ATP by an obligate exchange mechanism . Influx of extravesicular ATP required intravesicular transportable nucleotide and efflux of intravesicular ATP required transportable extravesicular nucleotide in the medium . The transport activity was insensitive to carboxyatractyloside and bongkrekic acid, inhibitors of mitochondrial ADP/ATP translocation . Proteoliposomes prepared from membranes of standard (non-Renografin-purified) R . prowazekii exhibited both an inhibitor-sensitive mitochondrial translocase activity and an inhibitor-resistant rickettsial translocase activity . Proteoliposomes prepared from uninoculated yolk sac membranes exhibited only the inhibitor-sensitive mitochondrial translocase activity . The substrate specificity of each reconstituted translocase was determined and shown to correspond with that reported for intact mitochondria or rickettsiae . Following influx of ATP the steady-state value for intravesicular labeled ATP was dependent on the concentration of intravesicular nucleotide available for exchange.

Gene, 1989 Sep 1, 81(1), 193 - 4
Nucleotide sequence of the Escherichia coli tRNA(3Leu) gene; Wahab SZ et al.; A 300-nucleotide sequence was determined which includes the tRNA(3Leu) coding region and the flanking sequences.

Can J Microbiol, 1989 Sep, 35(9), 843 - 9
{Modification of the envelope and the protein content of Escherichia coli surviving in sea water}; Gauthier MJ et al.; A toxigenic strain of Escherichia coli displayed important structural modifications when placed in seawater which naturally lacked nutritive elements, as observed by electron microscopy . These include cell wall and cell body distortion, modification of the membranes, central segregation of the chromosome, and retraction of the cytoplasm . These modifications were accompanied by a decrease in cell protein content of approximately 40% . Certain cytoplasmic membrane proteins were lost, and new ones appeared . The development of these changes was considerably slower in cells that had previously been grown in a seawater medium . This suggests that osmotic regulation mechanisms, which enable E . coli to survive much longer in marine conditions, may have a protective influence.

Biochem J, 1989 Sep 1, 262(2), 581 - 9
Mechanism of DNA strand nicking at apurinic/apyrimidinic sites by Escherichia coli {formamidopyrimidine}DNA glycosylase; Bailly V et al.; Escherichia coli {formamidopyrimidine}DNA glycosylase catalyses the nicking of both the phosphodiester bonds 3' and 5' of apurinic or apyrimidinic sites in DNA so that the base-free deoxyribose is replaced by a gap limited by 3'-phosphate and 5'-phosphate ends . The two nickings are not the results of hydrolytic processes; the {formamidopyrimidine}DNA glycosylase rather catalyses a beta-elimination reaction that is immediately followed by a delta-elimination . The enzyme is without action on a 3'-terminal base-free deoxyribose or on a 3'-terminal base-free unsaturated sugar produced by a beta-elimination reaction nicking the DNA strand 3' to an apurinic or apyrimidinic site.

Appl Environ Microbiol, 1989 Sep, 55(9), 2424 - 7
Production of 4-methylumbelliferyl heptanoate hydrolase by Escherichia coli exposed to seawater; Fiksdal L et al.; The production of an enzyme, 4-methylumbelliferyl heptanoate hydrolase, in Escherichia coli exposed to enriched and nonenriched seawater was studied . In all media, except for seawater with no or very small amounts of organic material and seawater enriched with peptone, 4-methylumbelliferyl heptanoate hydrolase activity increased by 2 to 3 orders of magnitude within 2 days . Increased enzyme activity was assumed to be related to cells not undergoing lysis but adapting to conditions of nutrient limitation.

Appl Environ Microbiol, 1989 Sep, 55(9), 2414 - 5
Convenient preparative synthesis of {14C}trehalose from {14C}glucose by intact Escherichia coli cells; Brand B et al.; At high osmolarity, Escherichia coli synthesizes trehalose intracellularly, irrespective of the nature of the carbon source . Synthesis proceeds via the transfer of UDP-glucose to glucose 6-phosphate, yielding trehalose 6-phosphate, followed by its dephosphorylation to trehalose (H.M . Giaeyer, B.O . Styrvold, I . Kaasen, and A.R . Strom, J . Bacteriol . 170:2841-2849, 1988) . This reaction was exploited to preparatively synthesize {14C}trehalose from exogenous {14C}glucose by using intact bacteria of a mutant (DF214) that could not metabolize glucose . The total yield of radiochemically pure trehalose from glucose was routinely more than 50%.

Tijdschr Diergeneeskd, 1989 Sep 1, 114(17), 890 - 8
{Escherichia coli mastitis in cattle . II . Pathogenesis and symptomatic therapy}; Lohuis JA et al.; The pathogenesis, course run by the disease, and symptomatic treatment of mastitis due to Escherichia coli in dairy cattle are reviewed in relation to the functioning of cellular and humoral defence mechanisms . The systemic symptoms of disease during mastitis caused by E . coli are attributed to the release and subsequent absorption from the udder of endogenous inflammatory mediators, rather than the direct absorption of endotoxins into the circulation . The course run by the disease during mastitis due to E . coli varies considerably . Reasons for these variations include genetic variation, stage of lactation and the function of humoral and cellular defence mechanisms.

Res Vet Sci, 1989 Sep, 47(2), 178 - 84
Effects of allopurinol in experimental endotoxin shock in horses; Lochner F et al.; The effect of allopurinol pretreatment 12 hours before an intraperitoneal challenge with a sublethal dose of Escherichia coli endotoxin (50 micrograms kg-1) was evaluated in 18 horses . The horses were divided among three equal groups: 1-endotoxin alone; 2-5 mg allopurinol kg-1 bodyweight plus endotoxin; and 3-50 mg allopurinol kg-1 bodyweight plus endotoxin . A variety of evaluation parameters were used . No differences among the groups were noted in rectal temperature, heart rate, respiration rate, haematological values, blood PaO2, blood PaCO2, blood pH or blood bicarbonate . Significant (P less than 0.05) differences between the groups were noted as regards the changes in capillary refill time, base excess, blood glucose, blood lactate, blood beta-glucuronidase and recumbency time . The protection afforded by 5 mg allopurinol kg-1 appeared to be superior to that with 50 mg allopurinol kg-1.

Mol Biol Evol, 1989 Sep, 6(5), 526 - 38
Statistical tests for detecting gene conversion; Sawyer S; Statistical tests for detecting gene conversion are described for a sample of homologous DNA sequences . The tests are based on imbalances in the distribution of segments on which some pair of sequences agrees . The methods automatically control for variable mutation rates along the genome and do not depend on a priori choices of potentially monophyletic subsets of the sample . The tests show strong evidence for multiple intragenic conversion events at two loci in Escherichia coli . The gnd locus in E . coli shows a highly significant excess of maximal segments of length 70-200 bp, which suggests conversion events of that size . The data also indicate that the rate of these short conversion events might be of the order of neutral mutation rate . There is also evidence for correlated mutation in adjacent codon positions . The same tests applied to a locus in an RNA virus were negative.

Dtsch Tierarztl Wochenschr, 1989 Sep, 96(8), 419 - 21
{Verification of the protective effect of a toxoid vaccine against edema disease of weaned piglets in an infection model}; Awad-Masalmeh M et al.; 105 piglets (56 vaccinated and 49 control animals) were utilized in 6 consecutive experiments . Each used litter was divided randomly into vaccine and control animals . One week prior to weaning each of the 56 piglets of the vaccine groups received 5 mg of nonpurified toxin treated with glutaraldehyd subcutaneously whereas to the remaining 49 control animals an extract of apathogenic E . coli was administered . During the first 12 hours post weaning each of the 105 piglets was challenged perorally with 10(10) cfu of edema principle toxin producing germs of E . coli serogroup O 139 . 23 animals of the control groups (46.9%) and one animal of vaccine groups (1.8%) died due to the infection between days four and five post challenge . These control animals showed classical clinical symptoms as well as pathological findings typical for edema disease . In contrast, such findings as mentioned before could not be observed in the vaccinated piglets . The remaining part of the control animals and eight of those vaccinated ones exhibited edema disease symptoms . The vaccinated animals have shed the challenge strain one to three days, while the survivals of control groups shed those germs for two to six days . The vaccinated piglets showed a better growth rate than the remaining control animals . Presented data suggest that our toxoid immunizing procedure can be used successfully against edema disease of swine.

Circ Shock, 1989 Sep, 29(1), 1 - 12
Serum amino acids in experimentally induced endotoxic shock: the prognostic significance of hyperalaninemia; Metzler B et al.; Serum amino acid levels were determined in 40 male rabbits before and 2, 6, and 24 hr after shock induction with 50 micrograms kg-1 endotoxin from Escherichia coli 0 111 and compared with amino acid levels of 32 age-matched control animals . Whereas total amino acid concentration in the control group decreased continuously over the 24 hr period due to fasting of the animals, the corresponding concentration in the endotoxin-treated animals remained at the initial level for the first 6 hr after shock induction . During this period, the release of amino acids from peripheral tissues was apparently enhanced, as evidenced by elevated levels of serum phenylalanine and tyrosine . Endotoxin administration exerted a salient effect on serum alanine levels . In all endotoxin-treated rabbits, alanine concentration was increased significantly 2 hr (P less than 0.00001) and 6 hr (P less than 0.00001) after injection of the toxin, whereas alanine levels in the control group remained essentially unchanged . In most animals, peak alanine concentrations were observed at the 6 hr sampling time . Blood glucose measurements indicated that by this time the hyperglycemic period, which is typical of the initial phase of endotoxic shock, was already finished . A high level of alanine following shock induction alluded to the impending death of the animal . In all rabbits in which alanine concentration rose by more than 600 mumols l-1 during the first 2 hr after endotoxin administration, the increase correlated significantly with the rate of lethality (P less than 0.0001) . In contrast, alanine levels of surviving animals increased only moderately . It was concluded from these results that monitoring serum alanine levels might be a valuable and practical tool to assess the severity and outcome of endotoxic shock.

Am J Pathol, 1989 Sep, 135(3), 427 - 33
Interleukin-6 immunoreactivity in human tumors; Tabibzadeh SS et al.; The cytokine, interleukin-6 (IL-6), has emerged as a likely mediator of many of the systemic alterations observed in patients with cancer (fever, increased erythrocyte sedimentation rate, and alterations in plasma protein composition) and may also mediate local effects such as alteration in proliferation of tumor cells, increased tumor cell motility, and decreased intercellular adhesions between tumor cells . The distribution of IL-6 immunoreactivity in different human tumors was studied . IL-6 immunoreactivity was detected by the avidin-biotin-complex (ABC) procedure using a polyclonal rabbit antiserum raised against an E coli-derived human IL-6 (rIL-6) . Preimmune rabbit serum used as a control did not yield specific staining and preadsorption of the IL-6 antiserum with rIL-6 abolished specific staining . Strong-to-moderate IL-6 immunoreactivity was observed in the neoplastic elements present in primary squamous cell carcinomas, in adenocarcinomas of mammary, colonic, ovarian, and endometrial origin, in various adenocarcinomas metastatic to lymph nodes, and in soft tissue tumors including leiomyosarcoma and neurofibrosarcoma . Weak-to-moderate IL-6 immunostaining was observed in Hodgkin's and non-Hodgkin's lymphomas . This study demonstrates that most human tumors stain positively for IL-6, adding weight to the hypothesis that IL-6 is a key cytokine that participates in the host-tumor interaction.

Proc Natl Acad Sci U S A, 1989 Sep, 86(18), 6888 - 92
Codon choice and gene expression: synonymous codons differ in translational accuracy; Dix DB et al.; Ribosomes programmed by different synonymous codons also differ in discriminating among near-cognate aminoacylated tRNAs . In the initial step of the recognition reaction ribosomes programmed by UUC discriminate less well than ribosomes programmed by UUU against ternary complexes containing three types of Leu-tRNA, and ribosomes programmed by CUC discriminate less well than ribosomes programmed by CUU against ternary complexes containing Phe-tRNA . Furthermore, in the proofreading step ribosomes programmed by UUC discriminate less well than ribosomes programmed by UUU against two of three near-cognate Leu-tRNAs, and ribosomes programmed by CUC discriminate less well than ribosomes programmed by CUU against near-cognate Phe-tRNA . The codon-induced change in reaction rate with near-cognate ternary complexes is greater than that with cognate ternary complexes: the most efficient codon is, therefore, the least accurate . Because the efficient, but inaccurate, codon UUC is used preferentially in highly expressed mRNAs of Escherichia coli, maximization of translational accuracy apparently has not been significant in the evolution of this particular biased codon choice in E . coli.

Proc Natl Acad Sci U S A, 1989 Sep, 86(18), 6873 - 7
Codon discrimination and anticodon structural context; Lustig F et al.; Site-directed mutagenesis has been used to change the nucleotide C in the wobble position of tRNA(1Gly) (CCC) to U . The mutated tRNA was tested for its ability to read glycine codons in an in vitro protein-synthesizing system programmed with the phage message MS2-RNA that had been modified by site-directed mutagenesis so as to make it possible to monitor conveniently the reading of all four glycine codons . The results showed that while the efficiency of tRNA(1Gly) (UCC) was comparable to that of mycoplasma tRNA(Gly) (UCC) in the reading of the codon GGA, the mycoplasma tRNA(Gly) was far more efficient than the tRNA(1Gly) (UCC) in the reading of the codons GGU and GGC . Thus, the anticodon UCC, when present in the structural context of the tRNA(1Gly) molecule, behaved as predicted by the wobble rules while in the structural context of the mycoplasma tRNA(Gly) it read without discrimination between the nucleotides in the third codon position, in violation of the wobble restrictions . The result with the codon GGG showed that the anticodon UCC, when present in tRNA(1Gly), was considerably less efficient in reading this codon than it was in the structural context of the mycoplasma tRNA(Gly) . It would therefore seem that the anticodon UCC, when present in a certain tRNA, can be an efficient wobbler, while in the molecular environment of another tRNA it is markedly restricted in its ability to wobble.

Eur J Biochem, 1989 Sep 1, 184(1), 47 - 52
Reactivity of the P-site-bound donor in ribosomal peptide-bond formation; Synetos D et al.; The puromycin reaction, catalyzed by the ribosomal peptidyltransferase, has been carried out so as to make the definition of two distinct parameters of this reaction possible . These are (a) the final degree of the reaction which gives the proportion of peptidyl (P)-site binding of the donor and (b) the reactivity of the donor substrate expressed as an apparent rate constant (kobs) . This kobs varies with the concentration of puromycin; the maximal value (k3) of the kobs, at saturating concentrations of puromycin, gives the reactivity of the donor independently of the concentrations of both the donor and puromycin . k3 is also a measure of the activity of peptidyltransferase expressed as its catalytic rate constant (kcat) . If we assume that the puromycin-reactive donor is bound at the ribosomal P site, we observe the following, depending on the conditions of the experiment: the proportion of P-site binding of the donor substrates AcPhe-tRNA or fMet-tRNA can be the same and close to 100%, while there is a tenfold increase in the reactivity of the donor (k3 = 0.8 min-1 versus 8.3 min-1) . On the other hand there are conditions, under which the proportion of P-site binding increases from 30% to 100% while k3 remains low and equal to 0.8 min-1 . Using the puromycin reaction it was also found that an increase of Mg2+ from 10 mM to 20 mM reduces the reactivity of the donor and, hence, the activity of peptidyltransferase, provided that this change in Mg2+ occurs during the binding of the donor but not when it occurs during peptide bond formation per se . The fact that the donor substrate may exist in various states of reactivity in this cell-free system raises the possibility that the rate of peptide bond formation may not be uniform during protein synthesis.

Biopolymers, 1989 Sep, 28(9), 1515 - 26
Raman spectroscopy of supercoiled and nicked ColE1 plasmid; Christens-Barry WA et al.; Native supercoiled and nicked ColE1 DNA were examined using laser Raman spectroscopy . ColE1 contains 6646 base pairs (bp) and, when supercoiled, approximately 47 negative supercoils . An analytical buoyant density gradient centrifugation technique developed by Burke and Bauer was scaled to preparative quantities, and used to isolate the supercoiled plasmid fraction from its nicked counterpart . This procedure allowed enriched fractions of the supercoiled plasmid to be extracted without the use of the optical contaminant ethidium bromide . The intensities of several Raman bands were altered between the spectra of the two topological forms . Notably absent were any changes in bands arising from cytosine and guanine vibrations . The observed changes are interpreted in terms of the polymorphic structures which have been observed in many DNA structural studies . The results of this study suggest that accommodation of supercoiling takes place chiefly in A-T base pairs and backbone moieties, without substantial modification of G-C base-pair structure . Premelting effects may account for the observed changes, including a slight shift to lower frequency of a band known to be responsive to base-pair disruption . Heteronomous ribose sugar pucker is evident in both supercoiled and nicked plasmid species . No gross conformational transitions were detected for native supercoiled DNA, and consequently, subtle rearrangements appear sufficient to absorb the supercoiling deformations.

J Mol Endocrinol, 1989 Sep, 3(2), 105 - 12
Expression and partial purification of human pro-opiomelanocortin in Escherichia coli; Grewal TS et al.; A large portion of the human pro-opiomelanocortin (POMC) peptide corresponding to amino acid residues 59-241 has been cloned and expressed in Escherichia coli . A 1.0 kb DNA fragment encoding this peptide was cloned into the expression vectors pUC8 and pUR291 . Plasmid pJMBG51 (a pUC8 recombinant) was found to direct the expression of a 24 kDa peptide . The recombinant pUR291 (pJMBG52) was shown to produce a beta-galactosidase fusion protein of 140 kDa . Western blot analysis showed that both the 24 kDa and 140 kDa peptides are recognized by antibodies raised against POMC-derived peptides . The beta-galactosidase fusion protein has been partially purified from crude E . coli cell lysates using affinity chromatography on p-amino-benzyl-1-thio-beta-D-galactopyranoside agarose.

Arch Biochem Biophys, 1989 Sep, 273(2), 440 - 8
Isolation of a cDNA encoding functional Drosophila alcohol dehydrogenase in Escherichia coli and purification of the bacterially produced enzyme; Green MM et al.; Because of the severe limitations on growing large quantities of Drosophila affinidisjuncta in the laboratory, direct purification of alcohol dehydrogenase (ADH) from this species has proven impossible . As an alternative source of this enzyme, a cDNA encoding functional ADH was isolated from a newly constructed cDNA library made from larval poly(A)-containing RNA . The cDNA was recovered by virtue of its hybridization to a previously isolated genomic ADH gene . Nucleotide sequence analysis confirmed the identity of the newly isolated cDNA . When the cDNA was inserted in the proper orientation downstream of the lac promoter on the vector pUC8, the cDNA directed the synthesis of functional ADH by the bacterial host . The bacterially produced enzyme was purified to homogeneity and used to elicit polyclonal antibodies in rabbits . The purified ADH has identical apparent subunit molecular weight to that of authentic ADH in larval fly extracts as determined by immunoblotting . Further, comparisons of the kinetic parameters of the bacterially produced enzyme and ADH activity in larval fly extracts indicate similar substrate preferences, pH dependencies, and Km values for 2-propanol and NAD . These results show that expression of a cDNA in Escherichia coli is a valid strategy for isolation of an ADH that would otherwise be difficult or impossible to purify.

Arch Biochem Biophys, 1989 Sep, 273(2), 423 - 32
Characterization of a variant rat glutathione S-transferase by cDNA expression in Escherichia coli; Lai HC et al.; We have isolated a glutathione S-transferase Yb1 subunit cDNA from a lambda gt11 cDNA collection constructed from rat testis poly(A) RNA enriched for glutathione S-transferase mRNA activities . This Yb1 cDNA, designated pGTR201, is identical to our liver Yb1 cDNA clone pGTR200 except for a shorter 5'-untranslated sequence . Active glutathione S-transferase is expressed from this Yb1 cDNA driven by the tac promoter on the plasmid construct pGTR201-KK . The expressed glutathione S-transferase protein begins with the third codon (Met) of the cDNA, and is missing the N-terminal proline of rat liver glutathione S-transferase 3-3 . Therefore, our Escherichia coli expressed glutathione S-transferase protein represents a variant form of glutathione S-transferase 3-3 (Yb1Yb1), designated GST 3-3(-1) . The expressed Yb1 subunits are assembled into a dimer as purified from sonicated E . coli crude extracts . In the absence of dithiothreitol three active isomers can be resolved by ion-exchange chromatography . The pure protein has an extinction coefficient of 9.21 x 10(4) M-1 cm-1 at 280 nm or E0.1% 280 = 1.78 and a pI at 8.65 . It has a substrate specificity pattern similar to that of the authentic glutathione S-transferase 3-3 . The GST 3-3(-1) has a KM of 202 microM for reduced GSH and of 36 microM for 1-chloro-2,4-dinitrobenzene . The turnover number for this conjugation reaction is 57 s-1 . Results of kinetic studies of this reaction with GST 3-3(-1) are consistent with a sequential substrate binding mechanism . We conclude that the first amino acid proline of glutathione S-transferase 3-3 is not essential for enzyme activities.

Proc Natl Acad Sci U S A, 1989 Sep, 86(17), 6621 - 5
Cloning the mRNA encoding 1-aminocyclopropane-1-carboxylate synthase, the key enzyme for ethylene biosynthesis in plants; Sato T et al.; Ethylene is the plant hormone that controls several features of plant growth and development . The rate-limiting step in its synthesis is the formation of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) from S-adenosylmethionine (AdoMet), catalyzed by ACC synthase . We have isolated a complementary DNA sequence encoding ACC synthase from zucchini (Cucurbita) fruits . The biological activity of the clone was confirmed by the ability of the cloned sequence to direct ACC synthase activity in Escherichia coli and yeast . In vivo studies using the ACC cDNA as probe showed that the ACC synthase gene is induced by a diverse group of inducers, including wounding, Li+ ions, and the plant hormone auxin.

Proc Natl Acad Sci U S A, 1989 Sep, 86(17), 6577 - 81
Mutations in the Escherichia coli UvrB ATPase motif compromise excision repair capacity; Seeley TW et al.; The Escherichia coli UvrB protein possesses an amino acid sequence motif common to many ATPases . The role of this motif in UvrB has been investigated by site-directed mutagenesis . Three UvrB mutants, with amino acid replacements at lysine-45, failed to confer UV resistance when tested in the UV-sensitive strain N364 (delta uvrB), while five other mutants constructed near this region of UvrB confer wild-type levels of UV resistance . Because even the conservative substitution of arginine for lysine-45 in UvrB results in failure to confer UV resistance, we believe we have identified an amino acid side chain in UvrB essential to nucleotide excision repair in E . coli . The properties of two purified mutant UvrB proteins, lysine-45 to alanine (K45A) and asparagine-51 to alanine (N51A), were analyzed in vitro . While the K45A mutant is fully defective in incision of UV-irradiated DNA, K45A is capable of interaction with UvrA in forming an ATP-dependent nucleoprotein complex . The K45A mutant, however, fails to activate the characteristic increase in ATPase activity observed with the wild-type UvrB in the presence of UvrA and DNA . From these results we conclude that there is a second nucleotide-dependent step in incision following initial complex formation, which is defective in the K45A mutant . This experimental approach may prove of general applicability in the study of function and mechanism of other ATPase motif proteins.

Proc Natl Acad Sci U S A, 1989 Sep, 86(17), 6562 - 6
Stabilization of phage T4 lysozyme by engineered disulfide bonds; Matsumura M et al.; Four different disulfide bridges (linking positions 9-164, 21-142, 90-122, and 127-154) were introduced into a cysteine-free phage T4 lysozyme at sites suggested by theoretical calculations and computer modeling . The new cysteines spontaneously formed disulfide bonds on exposure to air in vitro . In all cases the oxidized (crosslinked) lysozyme was more stable than the corresponding reduced (noncrosslinked) enzyme toward thermal denaturation . Relative to wild-type lysozyme, the melting temperatures of the 9-164 and 21-142 disulfide mutants were increased by 6.4 degrees C and 11.0 degrees C, whereas the other two mutants were either less stable or equally stable . Measurement of the equilibrium constants for the reduction of the engineered disulfide bonds by dithiothreitol indicates that the less thermostable mutants tend to have a less favorable crosslink in the native structure . The two disulfide bridges that are most effective in increasing the stability of T4 lysozyme have, in common, a large loop size and a location that includes a flexible part of the molecule . The results suggest that stabilization due to the effect of the crosslink on the entropy of the unfolded polypeptide is offset by the strain energy associated with formation of the disulfide bond in the folded protein . The design of disulfide bridges is discussed in terms of protein flexibility.

Proc Natl Acad Sci U S A, 1989 Sep, 86(17), 6557 - 61
The noncovalent complex between DNA and the bifunctional intercalator ditercalinium is a substrate for the UvrABC endonuclease of Escherichia coli; Lambert B et al.; We have demonstrated that the noncovalent complex formed between DNA and an antitumor bifunctional intercalator, ditercalinium, is recognized in vitro as bulky covalent DNA lesions by the purified Escherichia coli UvrABC endonuclease . It was established that no covalent drug-DNA adduct was formed during the incubation of the drug with DNA or during subsequent incubation with the UvrAB proteins . The nucleoprotein-ditercalinium complexes appear different from those generated by repair of pyrimidine dimers . The UvrA protein is able to form a stable complex with ditercalinium-intercalated DNA in the presence of ATP, whereas both UvrA and UvrB proteins are required to form a stable complex with pyrimidine dimer-containing DNA . The apparent half-life of the UvrA- and UvrAB-ditercalinium-DNA complexes following removal of free ditercalinium is 5 min . However, if the free ditercalinium concentration is maintained to allow the intercalation of one molecule of ditercalinium per 3000 base pairs, the half-life of the UvrA- or UvrAB-ditercalinium-DNA complex is 50 min, comparable to that of the complex of UvrAB proteins formed with pyrimidine dimer-containing DNA . UvrABC endonuclease incises ditercalinium-intercalated DNA as efficiently as pyrimidine dimer-containing DNA . However, unlike repair of pyrimidine dimers, the incision reaction is strongly favored by the supercoiling of the DNA substrate . Because UvrA- or UvrAB-ditercalinium-DNA complexes can be formed with relaxed DNA without leading to a subsequent incision reaction, these apparently dead-end nucleoprotein complexes may become lesions in themselves resulting in the cytotoxicity of ditercalinium . Our results show that binding of excision repair proteins to a noncovalent DNA-ligand complex may lead to cell toxicity.

Proc Natl Acad Sci U S A, 1989 Sep, 86(17), 6503 - 7
Isolation and expression of the Pneumocystis carinii thymidylate synthase gene; Edman U et al.; The thymidylate synthase (TS) gene from Pneumocystis carinii has been isolated from complementary and genomic DNA libraries and expressed in Escherichia coli . The coding sequence of TS is 891 nucleotides, encoding a 297-amino acid protein of Mr 34,269 . The deduced amino acid sequence is similar to TS from other organisms and is most closely related to the enzyme from Saccharomyces cerevisiae with 65% identity . TS is found on a 330-kilobase-pair chromosome in P . carinii . While TS and dihydrofolate reductase reside on a single polypeptide chain in all protozoa studied to date, TS is not linked to dihydrofolate reductase in P . carinii . The TS gene shows the presence of four small intervening sequences, some of which interrupt the coding sequence in highly ordered structural regions of the protein . Heterologous expression of P . carinii TS in E . coli was accomplished by cloning the coding sequence into plasmid vectors under control of the lac and tac promoters . These constructs direct the synthesis of catalytically active enzyme to the extent of 2% of total soluble protein.

Proc Natl Acad Sci U S A, 1989 Sep, 86(17), 6469 - 73
Mismatch-specific 3'----5' exonuclease associated with the mitochondrial DNA polymerase from Drosophila embryos; Kaguni LS et al.; The mitochondrial DNA polymerase from Drosophila embryos lacks dNTP turnover activity . However, a potent 3'----5' exonuclease activity can be detected by a specific assay in which the exonuclease excises mispaired nucleotides at the 3' termini of primed synthetic and natural DNA templates . The excision of a mispaired nucleotide occurs at a significantly greater rate than excision of a correctly paired nucleotide and, under conditions of DNA synthesis, hydrolysis of a mispaired terminal nucleotide occurs prior to primer extension . The 3'----5' exonuclease copurifies quantitatively with DNA polymerase gamma and cosediments with the nearly homogeneous enzyme under native conditions . These results suggest that the 3'----5' exonuclease provides a proofreading function to enhance the fidelity of DNA synthesis during Drosophila mitochondrial DNA replication.

Mol Biochem Parasitol, 1989 Sep, 36(2), 151 - 9
Application of a recombinant Echinococcus multilocularis antigen in an enzyme-linked immunosorbent assay for immunodiagnosis of human alveolar echinococcosis; Muller N et al.; A highly antigenic polypeptide fragment of the recombinant Echinococcus multilocularis antigen II/3 was produced in Escherichia coli and purified for application in enzyme-linked immunosorbent assay (ELISA) . The antigen II/3-encoding 1.0 kb DNA sequence was reduced by sonication into smaller DNA fragments which were subsequently cloned into lambda gt11 . Three clones could be isolated from the sublibrary, all synthesizing a recombinant antigen as a stable beta-galactosidase fusion protein . In a further step, the 0.6-kb insert from one positive clone was subcloned into the plasmid pAR 3038, which directed efficient synthesis of the antigen fused to only 11 amino acids from the N-terminus of the phage T7 major capsid protein . The plasmid-encoded antigen (antigen II/3-10) was purified from a bacterial cell extract and then tested in an ELISA . Using sera from 88 patients with an E . multilocularis-infection, a high diagnostic sensitivity of 90% was demonstrated . Investigation of sera from 220 patients with various helminthic infections showed a specificity of 99%, suggesting the suitability of the antigen II/3-10 as an immunodiagnostic tool.

Mutat Res, 1989 Sep, 218(2), 105 - 9
Enhancement and inhibition of mutation by o-vanillin in Escherichia coli; Watanabe K et al.; 2-Hydroxy-3-methoxybenzaldehyde (omicron-vanillin), the antimutagenic effect of which has been reported on mutagenesis induced by 4-nitroquinoline 1-oxide (4NQO) in Escherichia coli WP2s, enhanced N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-induced mutagenesis in the same strain . A remarkable enhancement of mutagenesis provoked by N-methyl-N-nitrosourea (MNU) was also observed by the addition of omicron-vanillin . No enhancing effect was observed on mutagenesis induced by other mutagens such as methyl methanesulfonate (MMS), dimethylsulfate, N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG), N-ethyl-N-nitrosourea (ENU), ethyl methanesulfonate, diethylsulfate, 4NQO and furylfuramide (AF-2) . On the contrary, omicron-vanillin greatly suppressed AF-2- and 4NQO-induced mutagenesis and showed a slight suppressing effect against mutagenesis induced by MMS, ENNG and ENU . One possible explanation for the enhancing effect of omicron-vanillin on the mutagenesis induced by MNNG or MNU in E . coli WP2s may be inhibition of an inducible adaptive response . Among 7 derivatives of omicron-vanillin, 2-hydroxy-3-ethoxy-benzaldehyde, omicron-hydroxybenzaldehyde and m-methoxybenzaldehyde showed an enhancing effect on MNNG-induced mutagenesis.

J Bacteriol, 1989 Sep, 171(9), 5190 - 3
Both CheA and CheW are required for reconstitution of chemotactic signaling in Escherichia coli; Conley MP et al.; If cells of Escherichia coli deleted for genes that specify transducers and all known cytoplasmic chemotaxis proteins are reconstituted with CheA, CheW, and CheY, they spin their flagella alternately clockwise and counterclockwise . If the aspartate receptor also is present, clockwise rotation is suppressed upon addition of aspartate . If either CheA or CheW is absent, the fraction of time that the flagella spin clockwise is reduced and responses to aspartate do not occur.

J Bacteriol, 1989 Sep, 171(9), 5176 - 8
Molecular characterization of two cya mutations, cya-854 and cyaR1; Glaser P et al.; The cya-854 mutation of Escherichia coli K-12 (E . Brickman, L . Soll, and J . Beckwith, J . Bacteriol . 116:582-587, 1973) has been shown to consist of a 200-base-pair deletion generating a new translation start that permits synthesis of the adenylate cyclase carboxy terminus . Contrary to expectations (C . Guidi-Rontani, A . Danchin, and A . Ullmann, J . Bacteriol . 148:753-761, 1981), cyaR1 results from the insertion of an IS1 element into the sequence coding for the catalytic center of the protein.

J Bacteriol, 1989 Sep, 171(9), 5127 - 34
Involvement of ExbB and TonB in transport across the outer membrane of Escherichia coli: phenotypic complementation of exb mutants by overexpressed tonB and physical stabilization of TonB by ExbB; Fischer E et al.; The exb locus in Escherichia coli consists of two genes, termed exbB and exbD . Exb functions are related to TonB function in that most TonB-dependent processes are enhanced by Exb . Like tonB mutants, exb mutants were resistant to colicin M and albomycin but, in contrast to tonB mutants, showed only reduced sensitivity to colicins B and D . Overexpressed tonB on the multicopy vector pACYC177 largely restored the sensitivity of exb mutants to colicins B, D, and M but only marginally increased sensitivity to albomycin . Suppression of the btuB451 mutation in the structural gene for the vitamin B12 outer membrane receptor protein by a mutation in tonB occurred only in an exb+ strain . Degradation of the unstable overproduced TonB protein was prevented by overproduced ExbB protein . The ExbB protein also stabilized the ExbD protein . Pulse-chase experiments with radiolabeled ferrichrome revealed release of ferrichrome from exbB, tonB, and fhuC mutants, showing that ferrichrome had not crossed the cytoplasmic membrane . It is concluded that the ExbB and ExbD proteins contribute to the activity of TonB and, like TonB, are involved in receptor-dependent transport processes across the outer membrane.

J Bacteriol, 1989 Sep, 171(9), 4958 - 62
Regulation of the Escherichia coli glyA gene by the metR gene product and homocysteine; Plamann MD et al.; The methionine component of glyA gene regulation in Escherichia coli K-12 was investigated . The results indicate that the glyA gene is positively controlled by the metR gene product . Activation of glyA by the MetR protein requires homocysteine, an intermediate in methionine biosynthesis . The positive-acting metR regulatory system functions independently of a regulatory system shown previously to control glyA gene expression.

J Bacteriol, 1989 Sep, 171(9), 4888 - 99
MalI, a novel protein involved in regulation of the maltose system of Escherichia coli, is highly homologous to the repressor proteins GalR, CytR, and LacI; Reidl J et al.; The maltose regulon of Escherichia coli comprises several operons that are under common regulatory control of the MalT activator protein . Five mal genes, organized in two divergent operons, code for a binding-protein-dependent transport system specific for maltose and maltodextrins . MalK, one of the subunits of this transport system, not only is essential for transport but also plays a role in regulation . Mutations abolishing MalK function not only result in inability to transport maltose but also cause constitutive expression of the maltose regulon . For this constitutivity to be exerted, the function of an additional gene product, MalI, is necessary . Using the constitutive expression of a malK-lacZ fusion as a signal, we cloned the malI gene, expressed it in minicells, and determined its DNA sequence . The sequence predicted a protein of 34,729 molecular weight, in agreement with the apparent molecular weight of the protein (35,000) when expressed in minicells and analyzed by polyacrylamide gel electrophoresis and autoradiography . MalI exhibited high homology to the repressor proteins GalR, CytR, and LacI . When the amino acid sequences were appropriately aligned, MalI showed 28% identity to GalR, 21% to CytR, and 24% to LacI . Including conservative amino acid exchanges, these numbers increased to 69, 56, and 58%, respectively . The regions of high homology were clustered in particular at the N-terminal portion of the protein that includes the helix-turn-helix motif thought to be involved in DNA binding . The protein contained a short stretch of 30 amino acids that was surprisingly homologous to a sequence in MalT . The amino-terminal half of the protein exhibited significant homology with MalK . The transcriptional start of malI was determined by reverse transcriptase and by S1 nuclease mapping . We found a possible binding site for cyclic AMP receptor protein in the promoter region of malI as well as two perfect direct repeats of 14 base pairs with twofold symmetry indicating their possible role as operator sites . Upstream to malI we observed a divergent open reading frame that extended to the end of the sequenced DNA.

J Bacteriol, 1989 Sep, 171(9), 4767 - 77
Overlap between pdxA and ksgA in the complex pdxA-ksgA-apaG-apaH operon of Escherichia coli K-12; Roa BB et al.; We report that pdxA, which is required for de novo biosynthesis of pyridoxine (vitamin B6) and pyridoxal phosphate, belongs to an unusual, multifunctional operon . The pdxA gene was cloned in the same 3.5-kilobase BamHI-EcoRI restriction fragment that contains ksgA, which encodes the 16S rRNA modification enzyme m6(2)A methyltransferase, and apaH, which encodes diadenosine tetraphosphatase (ApppA hydrolase) . Previously, Blanchin-Roland et al . showed that ksgA and apaH form a complex operon (Mol . Gen . Genet . 205:515-522, 1986) . The pdxA gene was located on recombinant plasmids by subcloning, complementation, and insertion mutagenesis, and chromosomal insertions at five positions upstream from ksgA inactivated pdxA function . DNA sequence analysis and minicell translation experiments demonstrated that pdxA encoded a 35.1-kilodalton polypeptide and that the stop codon of pdxA overlapped the start codon of ksgA by 2 nucleotides . The translational start codon of pdxA was tentatively assigned based on polypeptide size and on the presence of a unique sequence that was also found near the translational start of PdxB . This conserved sequence may play a role in translational control of certain pyridoxine biosynthetic genes . RNase T2 mapping of chromosomal transcripts confirmed that pdxA and ksgA were members of the same complex operon, yet about half of ksgA transcripts arose in vivo under some culture conditions from an internal promoter mapped near the end of pdxA . Transcript analysis further suggested that pdxA is not the first gene in the operon . These structural features support the idea that pyridoxine-biosynthetic genes are members of complex operons, perhaps to interweave coenzyme biosynthesis genetically with other metabolic processes . The results are also considered in terms of ksgA expression.

J Bacteriol, 1989 Sep, 171(9), 4674 - 8
Identification and characterization of a cyanate permease in Escherichia coli K-12; Sung YC et al.; Escherichia coli contains an inducible enzyme, cyanase, that catalyzes the decomposition of cyanate into ammonia and bicarbonate . The gene encoding cyanase, cynS, was cloned and found to be on a DNA fragment that contained the lac operon . Characterization of a plasmid encoding cyanase indicated that a 26-kilodalton (kDa) protein of unknown function was also induced by cyanate (Y-C . Sung, D . Parsell, P.M . Anderson, and J.A . Fuchs, J . Bacteriol . 169:2639-2642, 1987) . The gene encoding the 26-kDa protein was located between cynS and its promoter, indicating the existence of a cyn operon . The 26-kDa protein was identified as a cyanate permease that transports exogenous cyanate by active transport . E . coli was shown to contain a cyanate transport system that is energy dependent and saturable by cyanate.

J Bacteriol, 1989 Sep, 171(9), 4640 - 7
Mutations that improve export of maltose-binding protein in SecB- cells of Escherichia coli; Collier DN et al.; It previously has been proposed that the Escherichia coli SecB protein promotes the export of the maltose-binding protein (MBP) from the cytoplasm by preventing the folding of the precursor MBP (preMBP) into a translocation-incompetent conformation . The export of wild-type MBP is only partially blocked in SecB- cells . In contrast, the export of MBP16-1, an MBP species with a defective signal peptide, is totally dependent on SecB; hence, SecB- cells that synthesize MBP16-1 are unable to utilize maltose as a sole carbon source . The selection of Mal+ revertants primarily yielded mutants with alterations in the MBP16-1 signal peptide that permitted SecB-independent MBP export to the periplasm to various extents . Although each of these alterations increased the overall hydrophobicity of the signal peptide, it was not possible to strictly equate changes in hydrophobicity with the degree of SecB-independent export . Somewhat unexpectedly, two mutants were obtained in which MBP export in SecB- cells was markedly superior to that of the wild-type MBP . Although wild-type MBP is not cotranslationally translocated in SecB- cells, the two mutant proteins designated MBP172 and MBP173 exhibited significant cotranslational export in the absence of SecB . Thus, the role of SecB was partially supplanted by a signal peptide that promoted more rapid movement of MBP through the export pathway . When preMBP included the MBP172 signal peptide as well as an alteration in the mature moiety that slows folding, the SecB requirement for maximal MBP export efficiency was almost totally eliminated . These results provide additional strong support for the proposed antifolding role of SecB in MBP export.

J Bacteriol, 1989 Sep, 171(9), 4633 - 9
Cell length, nucleoid separation, and cell division of rod-shaped and spherical cells of Escherichia coli; Donachie WD et al.; By comparing the dimensions and DNA contents of normal rod-shaped Escherichia coli with those of mutants that grow as spheres or ellipsoids, we have determined that two parameters remain unchanged: the DNA/mass ratio and the average cell length (diameter, for spherical cells) . In consequence, the average volumes and DNA contents of the spherical mutant cells are about four to six times greater than those of rod-shaped cells growing at a similar rate . In addition, it was found that cells of both rod and sphere forms had approximately the same number of nucleoids (as seen when the DNA was condensed after inhibition of protein synthesis) . The nucleoids of the spherical cells therefore consist of four to six completed chromosomes each (polytene nucleoids) . We suggest that the attainment of a minimum cell length is necessary for nucleoid separation after chromosome replication and that such a separation is itself a prerequisite for septum formation.

DNA, 1989 Sep, 8(7), 491 - 501
Expression of human and murine interleukin-5 in eukaryotic systems; Tavernier J et al.; A cDNA coding for murine interleukin-5 (IL-5) was isolated from the EL4.ExC5 cell line . With the exception of a single amino acid substitution at position 79 (Arg----His), it is identical to a published sequence . The coding sequence for human IL-5 was synthesized chemically, allowing the introduction of strategically located restriction enzyme cleavage sites . Both cDNAs were expressed in various eukaryotic systems . Deletion of the 3' untranslated region of the murine IL-5 gene led to a 5- to 10-fold increase in expression in Xenopus laevis oocytes and in NIH-3T3 cells . The highest production, however, was obtained in Sf9 cells using a baculovirus vector . Human IL-5 was obtained from transformed Saccharomyces cerevisiae as a secreted, mature form using an in-frame fusion to the leader sequence of alpha-mating type factor, and was purified to homogeneity . In all cases mentioned, IL-5 was found to be glycosylated, and its biological activity was dependent on a 40- to 50-kD homodimer configuration, linked together by disulfide bridges . Deglycosylation did not affect the biological activity . Recombinant human IL-5 is biologically active on some human B-CLL cells (proliferation in the presence of IL-2) and on murine BCl1 cells (proliferation) at a low specific activity (about 1-2 x 10(3) U/mg) and on human eosinophils (eosinophil peroxidase assay) at a high specific activity (at least 5 x 10(6) U/mg) . Recombinant murine IL-5 from Sf9 cells has a specific activity of 1-2 x 10(7) U/mg in the BCl1 proliferation assay . An additive effect is seen in the presence of murine granulocyte-macrophage colony-stimulating factor (GM-CSF) and a synergistic effect in the presence of murine IL-4.

J Infect Dis, 1989 Sep, 160(3), 452 - 9
Epithelial cell invasion: an overlooked property of enteropathogenic Escherichia coli (EPEC) associated with the EPEC adherence factor; Donnenberg MS et al.; In order to investigate the ability of enteropathogenic Escherichia coli (EPEC) to invade epithelial cells, 24 strains of diarrhea-causing E . coli were studied with a HEp-2 cell-gentamicin invasion assay . Invasive ability was expressed as the percentage of the inoculum surviving gentamicin after incubation of bacteria with HEp-2 cells . Geometric mean survival of EPEC strains possessing the EPEC adherence factor (EAF+ EPEC) was 5.177%, which was significantly greater than survival of enteroinvasive E . coli (EIEC) strains (1.871%) . EPEC strains lacking EAF (EAF-EPEC), enterotoxigenic E . coli (ETEC), and enterohemorrhagic E . coli (EHEC) were significantly less invasive (geometric mean survival, 0.032%, 0.013%, and 0.009%, respectively) . The variation in bacterial recovery was not due to differences in the number of HEp-2 cells remaining attached to the plates, as measured by the retention of crystal violet stain in parallel assays . Transmission electron microscopy confirmed the presence of many intracellular EAF+ EPEC and EIEC, whereas EAF- EPEC, EHEC, and ETEC were found primarily outside the cells . Epithelial cell invasion is an overlooked property of EAF+ EPEC of potential relevance in disease pathogenesis.

J Infect Dis, 1989 Sep, 160(3), 422 - 9
The effects of acute and chronic alcoholism on tumor necrosis factor and the inflammatory response; Nelson S et al.; Ethanol intoxication has been shown to suppress selected functions of the immune system, thereby compromising host defenses against bacterial infections . Because the macrophage secretory protein, tumor necrosis factor (TNF), plays a central role in the inflammatory cascade, the effect of acute and chronic alcoholism on lipopolysaccharide (LPS)-induced TNF activity was studied . Saline or ethanol was given intraperitoneally to normal or chronic alcoholic rats followed 30 min later by either intravenous or intratracheal LPS . Intravenous LPS caused a substantial increase in serum TNF at 90 min in both normal and chronic alcoholic rats . In marked contrast, peak serum TNF levels were significantly suppressed in normal and chronic alcoholic rats given an acute injection of ethanol . When LPS was instilled intratracheally into normal rats, high levels of TNF appeared in the bronchoalveolar lavage fluid . Similar levels of TNF were found in chronic alcoholic rats after intratracheal LPS . However, acute ethanol intoxication significantly inhibited LPS-induced TNF in bronchoalveolar lavage fluid . In a similar manner, acute ethanol intoxication, but not chronic alcohol consumption, markedly inhibited both systemic and intrapulmonary polymorphonuclear leukocyte aggregation in response to either intravenous or intratracheal LPS . Alcohol-induced inhibition of TNF is a potential mechanism of the antiinflammatory effects of ethanol.

Infect Immun, 1989 Sep, 57(9), 2878 - 85
Acylation of the 47-kilodalton major membrane immunogen of Treponema pallidum determines its hydrophobicity; Chamberlain NR et al.; The 47-kilodalton (kDa) major integral membrane immunogen of Treponema pallidum was recently found to be a proteolipid . Similar two-dimensional electrophoretic mobilities and common hydrophobic properties displayed by the native (T . pallidum) and recombinant (Escherichia coli) 47-kDa antigens suggested that the recombinant antigen also possesses covalently bound lipid . Both intact E . coli and E . coli minicells acylated the 47-kDa antigen; immunoprecipitation with a monoclonal antibody specific for the 47-kDa immunogen supported the contention that the acylated product of E . coli corresponds to the cloned T . pallidum antigen . Triton X-114 phase partitioning was used to compare the relative hydrophobicities of 47-kDa molecules synthesized by in vitro and in vivo protein translation systems . The products synthesized by T . pallidum, intact E . coli, or E . coli minicells were hydrophobic, while the protein synthesized in an E . coli cell-free translation system was hydrophilic . Processing experiments with E . coli suggested that the primary gene translation product of the protein is not synthesized in a precursor form, unlike other bacterial proteolipids . These results indicate that the hydrophobicity of the 47-kDa integral membrane protein is conferred substantially by the covalently attached lipid(s) . The biochemical similarities between the native and recombinant 47-kDa proteolipids will provide a foundation for future investigations into the structure and immunogenicity of this integral membrane protein of T . pallidum.

Infect Immun, 1989 Sep, 57(9), 2811 - 4
Hybridization of Escherichia coli producing Shiga-like toxin I, Shiga-like toxin II, and a variant of Shiga-like toxin II with synthetic oligonucleotide probes; Brown JE et al.; Synthetic oligonucleotides, constructed from the nucleotide sequences of genes coding for the A subunit of Shiga-like toxin (SLT) I and the B subunit of SLT-II, were used as probes at different degrees of stringency to identify Escherichia coli producing different types of SLTs . At 45 degrees C, the A-I oligonucleotide probe hybridized with E . coli producing SLT-I, SLT-II, and variant of SLT-II (SLT-IIv) . At 53 degrees C, only SLT-I-producing E . coli hybridized with this probe . At 45 degrees C, the B-II oligonucleotide probe hybridized with SLT-II- and SLT-IIv-producing E . coli . At 53 degrees C, this probe hybridized with only SLT-II-producing E . coli . The A-I and B-II oligonucleotide probes were subsequently tested for hybridization with 73 SLT-producing E . coli and 49 non-SLT-producing E . coli isolated in Asia and Canada . At 45 degrees C, the A-I oligomer had a sensitivity of 97% and a specificity of 100% in identifying SLT-producing E . coli . At 53 degrees C, the A-I oligonucleotide probe had a sensitivity of 92% and a specificity of 91% in identifying E . coli containing genes encoding SLT-I . At 45 degrees C, the B-II oligonucleotide had a 100% sensitivity and 97% specificity in identifying E . coli that hybridized with the SLT-II probe . Of 17 E . coli that hybridized only with the SLT-II probe, 10 did not hybridize with the B-II oligonucleotide at 53 degrees C . All 10 isolates were cytotoxic to Vero cells but not to HeLa cells, confirming that the B-II oligonucleotide probe used at 53 degrees C will differentiate isolates producing SLT-II and SLT-IIv.

Plasmid, 1989 Sep, 22(2), 91 - 8
Conserved DNA sequences in chlamydial plasmids; Hugall A et al.; Two 7.4-kb plasmids from Chlamydia psittaci have been cloned and characterized . These plasmids are quite distinct from the 6.2-kb C . psittaci and the C . trachomatis plasmids when compared by restriction endonuclease analysis . The plasmids show considerable cross-hybridization, with only a small region highly conserved and identified as a 4 X 22-bp tandemly repeated region . This sequence is identical in the two size categories of C . psittaci plasmids and differs from C . trachomatis plasmids by only 2 bp in the 22-bp motif . AT-rich clusters 5' to the repeat region which are present in C . trachomatis and Escherichia coli plasmids were absent from both classes of C . psittaci plasmids . Extensive regions are less highly conserved but show a sufficient degree of cross-hybridization to suggest that the plasmids are homologous.

Arzneimittelforschung, 1989 Sep, 39(9), 1085 - 9
Immunomodulation by the new synthetic thiazole derivative tiprotimod . 2nd communication: immunopharmacological activity; Schorlemmer HU et al.; The thiazole derivative {2-(3-carboxy-1-propylthio)-4-methyl-1,3-thiazole} acetic acid (tiprotimod, HBW 538) a new synthetic immunopotentiator of low molecular weight, has been tested in vivo and ex vivo in various experimental models . Its influence on parameters of macrophage functions, on DTH (delayed type hypersensitivity)-reaction and antibodies to sheep erythrocytes (SRBC), Tetanus toxoid and heatkilled E . coli bacteria in mice, and in the popliteal lymph node assay in rats was investigated . When mice were treated with the test substance i.v., i.p., or p.o . in a dose range from 1-100 mg/kg, a time and dose-dependent stimulation of macrophage activity was observed . The drug was able to enhance the DTH-response against SRBC and to stimulate the humoral immune response against Tetanus toxoid and heat-killed E . coli . In the popliteal lymph node assay, a murine graft-vs-host model, a stimulating effect of the substance was observed when it was administered at the same time of the grafts to rats . These results demonstrate that tiprotimod is a potent immunopotentiator for both humoral and cell mediated immune response in experimental animals.

Nippon Geka Gakkai Zasshi, 1989 Sep, 90(9), 1576 - 8
{Detection of cellular DNA strand breaks induced by antitumor drug and heat using in situ nick translation}; Maehara Y et al.; Cellular DNA strand break induced by an alkylating agent: Carboquone (CQ), and heat (43 degrees C) was detected in HeLa cells in vitro and mouse sarcoma-180 cells in vivo . The break sites in the DNA were translated artificially in the presence of Escherichia coli DNA polymerase I and {3H}-labeled dTTP and sites in the DNA were visualized by autoradiographic observation of grains in the nuclei . These breaks increased in a dose and time dependent manner, compared to findings in the control cells . Our findings show that the surviving response of cells decreases while the level of DNA strand breaks increases following exposure to CQ or heat . The nick translation method is a rapid in situ assay for determining drug and heat induced DNA damage of tumor cells, under in vitro and in vivo conditions and in a semi-quantitative manner.

Gene Anal Tech, 1989 Sep-Oct, 6(5), 97 - 100
An improved vector for the expression of proteins in all three translational reading frames; Seth A et al.; We have constructed a novel vector (pN-7) that is capable of producing large amounts of recombinant proteins in E . coli and requires minimal manipulation for the construction of recombinant expression vectors . This expression vector (pN-7) contains the tightly regulated lambda pL promoter, cII ribosome binding site, and initiator condon ATG . The pN-7 vector also contains cleavage sites for the restriction enzymes SmaI, EcoRV, and HpaI that provide blunt ends in all three reading frames . Thus after cleavage with the appropriate restriction enzyme, this novel vector can be directly ligated to the DNA fragment that contains the open reading frame without further manipulation.

Biochimie, 1989 Sep-Oct, 71(9-10), 1005 - 12
Regulation of transcription of the glnALG operon of Escherichia coli by protein phosphorylation; Magasanik B; The transcription of glnA, the structural gene for glutamine synthetase in enteric bacteria, is regulated by the phosphorylation and dephosphorylation of an effector protein, NRI . In its phosphorylated form the effector activates the initiation of transcription at promoters specific of sigma 54, rather than the abundant sigma 70 . The ability of NRI-phosphate to stimulate the formation of open promoter-sigma 54 RNA polymerase complexes is enhanced by specific binding sites, located in the case of glnA 100 and 130 base pairs upstream from the transcriptional start site . These sites can be moved more than 1000 base pairs upstream or downstream without losing their effectiveness . The phosphorylation and dephosphorylation of NRI-NRI-phosphate is catalyzed by the modulator protein NRII . Its activity is controlled by an intracellular signal, the ratio of glutamine to 2-ketoglutarate, which is generated by glutamine synthetase in response to the environmental stimulus, the availability or lack of ammonia . The signal is transduced to the modulator by means of 2 additional proteins: uridylytransferase and PII.

Mol Gen Genet, 1989 Sep, 218(3), 531 - 5
Analysis of expression of hybrid yeast genes containing ARS elements; Kipling D et al.; In an attempt to devise a new assay for ARS-binding proteins we have inserted the HO ARS between the upstream activation site and the TATA region of the yeast CYC1 promoter . A marked reduction in promoter activity is observed . Inactivation of the HO ARS element by point mutation does not restore promoter activity to its original level, although a modest activation is seen . We have also inserted the HO ARS into the intron of the yeast actin gene; although there is no apparent deleterious effect on transcription, the activity of the ARS is abolished in this new environment.

Mol Gen Genet, 1989 Sep, 218(3), 460 - 4
Cloning of the excC and excD genes involved in the release of periplasmic proteins by Escherichia coli K12; Lazzaroni JC et al.; Strains of Escherichia coli K12 carrying a tolA, tolB, lky or exc mutation located at min 16.5 on the genetic map released periplasmic proteins into the extracellular medium . Wild-type genes defined by these mutations have been cloned from E . coli genomic bank made with plasmid pBR328 . Subcloning experiments and complementation studies showed that lky and exc mutations were located either in the previously described tolA and tolB genes or in the newly characterized excC and excD genes . Using minicells, excC and excD gene products were identified as proteins with a molecular mass of 19 and 21 kDa, respectively.

J Clin Microbiol, 1989 Sep, 27(9), 2123 - 4
Mannose-resistant hemagglutination, enzyme-linked immunosorbent assay, and immune electron microscopy for detection of K99 fimbrial antigen in Escherichia coli from calves; Hernandez F et al.; Mannose-resistant hemagglutination (MRHA) was evaluated for identification of Escherichia coli with K99 fimbriae . The sensitivity and specificity of MRHA, relative to the enzyme-linked immunosorbent assay, were 21 and 79%, respectively . Disagreement between the tests may have been due in part to separation of pili from the cells, with resultant enzyme-linked immunosorbent assay-positive, MRHA-negative tests . MRHA was not useful as the sole means for the identification of E . coli with K99 fimbriae.

J Bacteriol, 1989 Sep, 171(9), 5202 - 5
Examination of enterotoxigenic Escherichia coli H10407 (colonization factor antigen I+) by scanning electron microscopy with conductive staining; Sherburne R et al.; We have used the scanning electron microscope to examine enterotoxigenic Escherichia coli H10407, which expresses colonization factor antigen I pili . The use of low accelerating voltages and conductive staining procedures allowed us to obtain images of colonization factor antigen I pili and other structural details which were obscured by conventional gold-coating techniques.

J Bacteriol, 1989 Sep, 171(9), 5169 - 72
DNA sequence of the Escherichia coli K-12 gamma-glutamyltranspeptidase gene, ggt; Suzuki H et al.; The DNA sequence of ggt, the gene that codes for gamma-glutamyltranspeptidase (EC 2.3.2.2) of Escherichia coli K-12, has been determined . The sequence contains a single open reading frame encoding the signal peptide and large and small subunits, in that order . This result suggests that E . coli gamma-glutamyltranspeptidase is processed posttranslationally.

Rev Cubana Med Trop, 1989 Sep-Dec, 41(3), 435 - 42
{Rotavirus infection in children with acute diarrhea in Habana City}; Esquivel Rivero M et al.; This paper shows the incidence of rotavirus and other pathogenic agents in 256 children under three years of age with a diagnosis of acute diarrhea . This study included the months of December 1983 through May 1984 . Rotavirus was found in 27.7% of patients, followed by enteropathogenic E . coli (17%) . No positive cases were found in the control group . The highest incidence of rotavirus infection coincided with the coldest months where the lowest volumes of rains were reported.

Mol Biol (Mosk), 1989 Sep-Oct, 23(5), 1391 - 9
{Cloning of tobacco DNA fragment with promoter properties in a transgenic plant}; Domanskii NN et al.; A selection system for isolating DNA sequences with transcription-promoting activity by their functioning in bacterial cells has been proposed . Tobacco nuclear DNA fragments were inserted in front of the promoterless neomycin 3'-phosphotransferase II (NPT-II) gene and promoter-like sequences were identified by their ability to restore NTP-II activity in E . coli cells . One of these recombinant plasmids was introduced in tobacco protoplasts by direct gene transfer and transformed calli were isolated by kanamycin selection . The NTP-II expression in regenerated transgenic plants were highest in root, slightly lower in stem and were practically absent in leaf . Sequence analysis of cloned segment showed the presence of conserved sequences essential for promoter activity in eukaryotic cells . A transcription start site was observed by S1 mapping . The size of protected fragments corresponds to the initiation of transcription 176 and 179 base pairs upstream the initiation codon in tobacco and 75 base pairs in E . coli.

Mol Biol (Mosk), 1989 Sep-Oct, 23(5), 1279 - 88
{Characteristic features of the structure of co-integrating plasmids and simple insertions formed during transposition of the IS1 element in transposon Tn9'}; Danilevich VN et al.; Earlier we have studied unstable dissociating IS1/Tn9'-mediated cointegrates between the plasmids pDK57 (pBR322::Tn9') and pRP3.1, a deletion derivative of RP1, and two types of such cointegrates containing three and four copies of IS1 were revealed . In the present paper we studied the structure of stable IS1/Tn9'-mediates cointegrates and simple insertions formed by interaction between the plasmids pDK57 and pRP3.1 in the E . coli recA- cells . It was shown, that the stable cointegrates were formed by insertion of pDK57 in different loci of pRP3.1 and these cointegrates contain three copies of IS1, i.e . one copy of IS1 and a copy of Tn9' at the junction of the two replicons . The cointegrates are formed predominantly due to the activity of the left copy of Tn9', which occupies a proximal position in regard to the promoter of the cat gene . It was found that the integration of pDK57 into the kan gene region of pRP3.1 leading to the formation of the KmS cointegrates occurs only in one of the two possible orientations . Meanwhile the insertions of the transposon Tn9' into the kan region of pRP3.1 leading to simple insertions occurs in the orientation opposite to the orientation of the transposon in the KmS cointegrates . It is proposed that simple insertions are not the products of direct transposition of Tn9', but they are formed from unstable cointegrates under the action of IS1-specific resolvase.

J Biochem (Tokyo), 1989 Sep, 106(3), 528 - 32
UDP-glucose pyrophosphorylase from potato tuber: purification and characterization; Nakano K et al.; UDP-glucose pyrophosphorylase from potato tuber was purified 243-fold to a nearly homogeneous state with a recovery of 30% . The purified enzyme utilized UDP-glucose, but not ADP-glucose, as the substrate, and was not activated by 3-phosphoglyceric acid . Product inhibition studies revealed the sequential binding of UDP-glucose and MgPPi and the sequential release of glucose-1-phosphate and MgUTP, in this order . Analyses of the effects of Mg2+ on the enzyme activity suggest that the MgPPi and MgUTP complexes are the actual substrates for the enzyme reaction, and that free UTP acts as an inhibitor . The enzyme exists probably as the monomer of an approximately 50-kDa polypeptide with a blocked amino terminus . For structural comparison, 29 peptides isolated from a tryptic digest of the S-carboxymethylated enzyme were sequenced . The results show that the potato tuber enzyme is homologous to UDP-glucose pyrophosphorylase from slime mold, but not to ADP-glucose pyrophosphorylase from Escherichia coli, and provide structural evidence that UDP-glucose and ADP-glucose pyrophosphorylase are two different protein entities.

Int J Pancreatol, 1989 Sep, 5(2), 203 - 11
Direct ESR measurement of free radicals in mouse pancreatic lesions; Nonaka A et al.; In this experiment, free radicals in the pancreas of endotoxemia and ethionine induced acute pancreatitis in mice were attempted to be detected directly by ESR spectroscopy, using 77 K freeze-trapping and 25 degrees C DMPO spin trapping techniques . In the 77 K freeze-trapping method, Mn (II) ion and R-00 . radical were detected in endotoxemia and ethionine induced pancreatic lesions . The heme-NO radical was observed at 6 and 24 h after isolation of the normal pancreas, and signal intensity was increased with time . This finding supports that ESR spectroscopy is a useful method for detecting the tissue degeneration process from ischemia to necrosis . Using the DMPO spin trapping technique (25 degrees C), 6-line was detected at 6 h after intraperitoneal administration of E . coli in the model of endotoxemia, and 3- and 6-lines and a signal suggestive of DMPO-OH adduct were noted at 12 and 24 h in ethionine pancreatitis . These findings suggest that impaired pancreatic tissues exist in a considerably oxidative environment and oxygen derived free radicals may be considered to play an important role in the development of pancreatic lesions.

Biochimie, 1989 Sep-Oct, 71(9-10), 1059 - 64
Studies of the phosphorylation of Escherichia coli isocitrate dehydrogenase . Recognition of the enzyme by isocitrate dehydrogenase kinase/phosphatase and effects of phosphorylation on its structure and properties; McKee JS et al.; Escherichia coli isocitrate dehydrogenase is completely inactivated by phosphorylation of a single serine residue per subunit . We have examined the conformations of the active and phosphorylated forms of the enzyme using circular dichroism spectroscopy . The results support the view that phosphorylation prevents the binding of NADP, probably by direct blocking of the coenzyme-binding site . Labelling studies suggest that an arginine residue at the coenzyme-binding site may be close to the phosphorylatable serine residue . The phosphorylation of isocitrate dehydrogenase is thus unusual in that it occurs at the active site of the enzyme . We therefore investigated the recognition of isocitrate dehydrogenase by isocitrate dehydrogenase kinase/phosphatase . The kinase activity of this enzyme can phosphorylate intact isocitrate dehydrogenase but not proteolytic fragments derived from it, nor a synthetic peptide corresponding to the sequence round the phosphorylation site.

Biochimie, 1989 Sep-Oct, 71(9-10), 1051 - 7
Isocitrate dehydrogenase kinase/phosphatase; Laporte DC et al.; In Escherichia coli, isocitrate dehydrogenase (IDH) is regulated by phosphorylation . This phosphorylation cycle is catalyzed by an unusual, bifunctional protein:IDH kinase/phosphatase . IDH kinase/phosphatase is expressed from a single gene, aceK, and both activities are catalyzed by the same polypeptide . The amino acid sequence of IDH kinase/phosphatase does not exhibit the characteristics which are typical of other protein kinases, although it does contain a consensus ATP binding site . The available evidence suggests that the IDH kinase and IDH phosphatase reactions occur at the same active site and that the IDH phosphatase reaction results from the back reaction of IDH kinase tightly coupled to ATP hydrolysis . The function of the IDH phosphorylation cycle is to control the flux of isocitrate through the glyoxylate bypass . This pathway is essential for growth on acetate because it prevents the quantitative loss of the acetate carbons as CO2 in the Krebs' cycle . IDH kinase/phosphatase monitors general metabolism by responding to the levels of a wide variety of metabolites, many of which activate IDH phosphatase and inhibit IDH kinase . The ability of IDH kinase/phosphatase to monitor general metabolism allows . the IDH phosphorylation cycle to compensate for substantial perturbations of the system, such as a 15-fold overproduction of IDH . The significance of the cellular level of IDH kinase/phosphatase has also been evaluated . The level of this protein is in great excess of that required for steady-state growth on acetate . In contrast, IDH kinase/phosphatase is, in some cases, rate-limiting for the dephosphorylation of IDH which results when preferred carbon sources are added to cultures growing on acetate.

Mol Gen Genet, 1989 Sep, 218(3), 409 - 18
asgB, a gene required early for developmental signalling, aggregation, and sporulation of Myxococcus xanthus; Mayo KA et al.; The asgB genetic locus of Myxococcus xanthus specifies a function which is required early in the developmental pathway leading to aggregation and sporulation in fruiting bodies . The developmental defect of asgB mutants can be compensated by extracellular complementation using either intact wild-type cells or cell-free supernatants conditioned by developing wild-type cells . A Tn5 insertion was isolated closely linked to asgB480 and facilitated the cloning of both the wild-type (asgB+) and the mutant (asgB480) alleles in Escherichia coli plasmid . Tandem duplications of the asgB locus were constructed in M . xanthus; the completely wild-type phenotype of asgB+/asgB480 partial diploids implies that the asgB480 allele is recessive . This finding, along with extracellular complementation by wild-type cells, is consistent with the hypothesis that the asgB+ locus is required to produce a substance with an intercellular signalling function . At least part of the asgB gene was found to lie within a 1.2 kb SmaI DNA fragment . This 1.2 kb fragment, as well as smaller fragments derived from it, were used as DNA probes in RNA/DNA hybrid analyses of transcription in the asgB region . Two small mRNA species were detected, one about 650 bp long, and the other about 500 bp; the two species of mRNAs apparently overlap . Both mRNAs are present in low, but approximately equal amounts, in vegetatively growing cells . This is consistent with the observation that asg mutants display a mutant vegetative phenotype (a change in colony color and spreading behavior) as well as defective development.

EMBO J, 1989 Sep, 8(9), 2755 - 60
Bordetella pertussis adenylate cyclase: purification and characterization of the toxic form of the enzyme; Rogel A et al.; Bordetella pertussis produces a calmodulin-sensitive adenylate cyclase (AC) which is an essential virulence factor in mammalian pertussis . Here we report the purification and characterization of the toxic form of the enzyme, which penetrates eukaryotic cells and generates high levels of intracellular cAMP . This form was purified from an extract of B.pertussis strain carrying a recombinant plasmid which over-produced both enzymatic and toxic activities of the enzyme . Western blot analysis of the extract using anti-B.pertussis AC antibodies detected only one protein of 200 kd . However, gel filtration of the extract resolved two peaks of enzymatic activity . The first peak of aggregated material contained greater than 70% of the total enzymatic activity, and the second peak contained the majority of the toxic activity . Purification of the enzyme from both peaks yielded proteins of 200 kd, with similar biochemical and immunological properties . Yet only the enzyme purified from the second peak could penetrate human lymphocyte and catalyse the formation of intracellular cAMP . B.pertussis AC gene expressed in Escherichia coli produced a calmodulin-dependent enzyme of 200 kd, which lacked lymphocyte penetration capacity . It is proposed that a post-translational modification that occurs in B.pertussis but not in E.coli confers upon the 200 kd protein of B.pertussis AC the toxic properties.

EMBO J, 1989 Sep, 8(9), 2745 - 54
A cinematographic view of Escherichia coli RNA polymerase translocation; Metzger W et al.; A series of RNA synthesizing transcription complexes, initiated at the T7 A1 promoter and halted at specific base positions ranging from +12 to +40, were analyzed by footprinting techniques; exonuclease III was used to determine the position of the bound RNA polymerase on the DNA and hydroxyl radicals were used to visualize the protein--DNA contact sites within the protected areas . In the binding (open) complex without RNA there are two DNA-domains, differing in their protection pattern . The first, extending from position +18 to -13, termed 'melting domain', is fully protected, whereas the second, extending from -14 to -55, termed 'recognition domain', shows only partial protection . At this domain, RNA polymerase is attached to one side of the DNA only, as indicated by the 10-bp periodicity of the protection pattern . Our data show that the formation of a mature RNA transcribing complex is characterized by dissociation of the RNA polymerase from the recognition domain, whereby the size of the melting domain remains constant . This process is accomplished if the nascent RNA has reached a length of 11 bases . As the RNA reaches a length of 20 bases, the size of the melting domain decreases from approximately 30 to 23 bp . Further RNA synthesis leaves the protection pattern essentially unchanged . These data demonstrate that the formation of a mature RNA transcribing complex can be described by at least two transitions.

Virus Genes, 1989 Sep, 3(1), 57 - 68
The influence of the herpes simplex virus-1 DNA template environment on the regulation of gene expression; Leary K et al.; To determine the role of the HSV-1 genome structure and environment on the regulation of gene expression, we constructed recombinant viruses containing a heterologous gene inserted into either the immediate early ICPO or late glycoprotein C (gC) genes of HSV-1 . The heterologous gene consisted of the SV40 early promoter (without enhancer sequences) linked to the coding sequences for the bacterial chloramphenicol acetyl transferase (CAT) . The expression of CAT was examined in Vero cells infected with either virus (named ICP0-CAT and Sph 6) . For both recombinants, expression of CAT was not dependent upon prior viral protein synthesis . The kinetics of expression of CAT-specific mRNA resembled that of the HSV-1 genes into which CAT was inserted . Primer extension analysis revealed that the SV40 promoter is recognized and used when placed in cis in two different HSV-1 genome locations, and Northern hybridization experiments confirmed that the heterologous gene was expressed in the absence of prior viral protein synthesis . Therefore, this gene was not regulated as strictly as an HSV-1 gene, but was influenced by the environment into which it was placed, presumably by factors that are present when the normal viral gene is on.

Virus Genes, 1989 Sep, 3(1), 29 - 44
Serological probes for some foot-and-mouth disease virus nonstructural proteins; Tesar M et al.; Foot-and-mouth disease virus (FMDV) O1 Kaufbeuren-specific cDNA fragments were subcloned into the E . coli expression vector pRIT.2T . Fusion proteins thus produced in bacteria were purified by affinity chromatography and inoculated into rabbits . Three sera thus obtained were found to be monospecific for FMDV proteins 3A, 3C, and 3D, respectively . Two others were prevalently directed against protein 2C, but in addition, either to protein 2B or to protein 3A . Five out of six mature nonstructural virus proteins can therefore be separately investigated in FMDV-infected cells, either by indirect immunofluorescence or by radioimmunoprecipitation . Immunofluorescence shows all investigated proteins to be located exclusively in the cytoplasm . One of them, protein 2C, transiently forms aggregates at the periphery of cells . Radioimmunoprecipitation confirmed current knowledge on maturation of FMDV proteins . It was further used to characterize postinfectional sera with regard to FMDV-specific antibodies . Cattle and guinea pig were found to have responded differently to FMDV nonstructural antigens . Furthermore, antigenicity of yet to be described FMDV polypeptides was observed in the guinea pig.

Immunology, 1989 Sep, 68(1), 96 - 101
Proliferative T-cell response to glycoprotein B of the human herpes viruses: the influence of MHC and sequence of infection on the pattern of cross-reactivity; Chan WL et al.; Oligopeptides of the highly conserved herpes virus glycoprotein B (gB) were expressed from DNA fragments of the EBV gB (BALF4) and HSV-2 gB open reading frames as fusion proteins with the lambda CII protein and beta-galactosidase (GZ), respectively, in Escherichia coli . After immunopurification using anti-gB or anti-GZ affinity columns, the fusion proteins were used in vitro to stimulate human peripheral blood lymphocytes (PBL) or murine lymph node cells that have been primed with EBV, HSV-1, HSV-2, VZV or HCMV (all human herpes viruses) to proliferate . Results obtained in BALB/c mice indicate that different herpes viruses induce different levels of T-cell response to each other and to gB, over a range of type-specific and cross-reactive T-cell epitopes . There is a lack of correlation of immunogenicity and antigenicity in the generation of T-cell responses between some of the viruses . Major T-cell epitopes are located at the C terminal half of the gB molecule . The T-cell response to gB in healthy individuals seropositive for various combinations of the five herpes viruses differed markedly from individual to individual, even when they are seropositive to the same set of herpes viruses . However, two individuals with high proliferative T-cell response to VZV and sharing HLA A2, B7, DR2 and DQw1 are also good responders for cross-reactive gB/fragments and for virus antigen of all the five herpes viruses . Therefore the data obtained demonstrated that the MHC and the immune interaction arising from cross-reactive T-cell response evoked by other herpes viruses may determine the pathogenesis of a herpes virus infection.

Gene, 1989 Sep 1, 81(1), 1 - 15
Physical and biological consequences of interactions between integration host factor (IHF) and coliphage lambda late p'R promoter and its mutants; Kur J et al.; The integration host factor (IHF) binds to a site (ihf) that overlaps the -35 region of the phage lambda late rightward promoter (p'R) . This interaction represses p'R-promoted transcription, both in vivo and in vitro . In vivo repression was observed when a plasmid carrying both p'R and the galK reporter gene was transfected into IHF+ or IHF- hosts . In vitro repression of transcription by IHF was observed only with linear, but not with supercoiled wild-type p'R templates . When binding to ihf, IHF imposes a strong bend on the DNA and protects this site from cleavage by neocarzinostatin, pancreatic DNase I, and hydroxyl radicals, as assessed by footprinting experiments . Both the functional and nonfunctional p'R mutants, in which the upstream part of the -35 region was replaced by an EcoRI linker, show modified behavior toward IHF . Some are more sensitive to IHF-mediated repression, even in the supercoiled form, while others have lost their affinity for IHF . We conclude that IHF binding depends not only on the consensus ihf sequence, but also on a suitable combination of the sequences of both ihf and neighboring regions, together with the DNA conformation, which includes both natural and imposed bends in DNA and the degree of supercoiling . Based on most of the present data, it is difficult to predict the relationship between the ihf sequence and IHF interaction, since two very different sequences (less than 50% homology) show strong IHF binding, whereas very similar sequences (80-87% homology) show a very different behavior . However, the hydroxylradical footprinting data show that three A + T-rich sequences are protected by IHF: the central sequence, which overlaps the -35 region of p'R, and two flanking sequences removed by one helix turn . All three sequences are located on the same face of the helix, and the amino acid side chains of IHF seem to occupy the narrow minor groove . A novel consensus sequence is proposed.

Am J Vet Res, 1989 Sep, 50(9), 1604 - 8
Modulation of the cellular immune responses to T-cell-dependent and T cell-independent antigens in lambs with induced bovine viral diarrhea virus infection; Lamontagne L et al.; Functional interaction between lymphoid cells and lymphotropic viruses is particularly evident for bovine viral diarrhea virus (BVDV) in cattle and its closely related virus, the border disease virus (BVDV) in sheep . The most important aspect of acute or chronic phases of BVDV or BDV infection was the host's increased susceptibility to secondary bacterial or viral infection . To study the ability of BVDV to alter the development of the cellular immune responses to concomitant inoculation with T cell-dependent and T cell-independent antigens, lambs were inoculated twice with rabbit RBC and Escherichia coli lipopolysacharide (LPS) and then were infected with a cytopathic strain of BVDV at postinoculation day 3 . Leukopenia characterized by lymphopenia developed after BVDV infection . Increased {3H}thymidine incorporation was observed in resting or lectin-stimulated blood mononuclear cells in the first weeks after inoculation in BVDV-infected lambs, but was followed by decreased {3H}thymidine incorporation after the second inoculation for up to 8 weeks after initial inoculation . In contrast, transient decrease of blastogenic responses, associated with toxic effect of LPS, was detected in inoculated noninfected lambs, but was followed by stimulation of cellular immune responses . Inoculated noninfected lambs had good in vitro cellular immune response to rabbit RBC and LPS antigens, whereas lymphocytes from BVDV-infected lambs could not mount lasting cellular immune responses to antigens or BVDV . Results suggest that BVDV infection in lambs modulates the ability of lymphocytes to respond to lectins or antigenic stimuli according to the time after infection.(ABSTRACT TRUNCATED AT 250 WORDS)

Mol Microbiol, 1989 Sep, 3(9), 1173 - 82
Molecular cloning and expression of a locus (mdoA) implicated in the biosynthesis of membrane-derived oligosaccharides in Escherichia coli; Lacroix JM et al.; Mutants of Escherichia coli defective in the mdoA locus are blocked at an early stage in the biosynthesis of membrane-derived oligosaccharides . The mdoA locus has now been cloned into multicopy plasmids . A 5 kb DNA fragment is necessary to complement mdoA mutations . Cells harbouring the mdoA+ plasmid produced three to four times more MDO than wild-type cells . MDO overproduction did not affect the degree of MDO substitution with sn-1-phosphoglycerol residues . The biosynthesis of MDO remained under osmotic control in overproducing strains.

Afr J Med Med Sci, 1989 Sep, 18(3), 163 - 8
Properties of lactase produced by enteropathogenic Escherichia coli from diarrhoeic children; Olusanya O et al.; The quantity of lactase produced by enteropathogenic Escherichia coli (EPEC) was significantly higher (P less than 0.01) than that produced by non-EPEC . The enzyme production was induced by lactose but repressed by glucose and galactose . The lactase from EPEC which was partially purified by ammonium sulphate precipitation and gel permeation chromatography had a molecular weight of 56 kD and apparent Km of approximately 2.78 mM for lactose . The lactase exhibited optimum activity at pH 7.0 at 40 degree C and was stimulated by Mg2+, Mn2+, Na+ and inhibited by Ba2+, Ca+, Cu2+, EDTA, iodo acetic acid (IAA) and Hg2+ and U2+ ions . The higher production of lactase by EPEC may be linked to its pathogenic role in childhood diarrhoea.

Proc Natl Acad Sci U S A, 1989 Sep, 86(18), 7195 - 9
Cloning of the cDNA and functional expression of the 47-kilodalton cytosolic component of human neutrophil respiratory burst oxidase; Volpp BD et al.; Neutrophil NADPH oxidase is a multicomponent enzyme that is activated to generate superoxide anion and is defective in the cells of patients with chronic granulomatous disease . It requires both membrane and cytosolic components, the latter including 47- and 67-kDa proteins recognized by the polyclonal antiserum B-1 . Immunoscreening of an induced HL-60 lambda ZAP cDNA library yielded seven cross-hybridizing cDNAs encoding the 47-kDa component . Fusion proteins of 22-50 kDa were recognized by B-1 . Antiserum against a fusion protein recognized a 47-kDa protein in normal neutrophils but not in those from patients with autosomal chronic granulomatous disease who lack the 47-kDa cytosolic oxidase component . In a cell-free NADPH oxidase system full-length and C-terminal fusion proteins augmented superoxide generation and reconstituted the cytosolic defect of a patient missing the 47-kDa protein . The cDNA hybridized with a 1.4-kilobase mRNA from induced HL-60 cells . The longest cDNA contained an open reading frame encoding a protein of 41,440 Da with a calculated pI of 10.4, an N-terminal glycine, sites favorable for phosphorylation, a nucleotide binding domain, and a region of homology to the src protein kinases, phospholipase C, and alpha-fodrin . These structural features are pertinent to proposed functional roles of the protein in the respiratory burst oxidase.

Eur J Biochem, 1989 Sep 1, 184(1), 197 - 203
Modulatory activity of 9-hydroxy- and 9-hydroperoxy-octadecadienoic acid towards reactive oxygen species from guinea-pig pulmonary macrophages; Engels F et al.; As guinea-pig pulmonary macrophages (PM) synthesize the linoleic acid metabolite 9-hydroxy-octadecadienoic acid (9-OH-Lin) under non-stimulated conditions in relatively large quantities, we investigated whether this product has an effect on the macrophage's own phagocytic cell function . 9-OH-Lin, and also its hydroperoxy precursor 9-hydroperoxy-octadecadienoic acid (9-OOH-Lin), influenced the generation of PM chemiluminescence, a measure of the production of reactive oxygen species . The generation of lucigenin-enhanced chemiluminescence by stimulated and non-stimulated PM was inhibited concentration-dependently . Inhibition was observed at concentrations as low as 10 nM . Since 9-OH-Lin and 9-OOH-Lin also inhibited the generation of chemiluminescence by a cell-free enzyme system, i.e . xanthine/xanthine oxidase, the inhibitory effects might represent a scavenging activity towards reactive oxygen species . 9-OH-Lin and 9-OOH-Lin did not influence other phagocytic cell functions, e.g . PM phagocytic capacity, the aggregatory response to the calcium ionophore A23187, or the release of lysosomal enzymes . The effects of 9-OH-Lin and 9-OOH-Lin could be ascribed to the hydroxy and hydroxyperoxy moiety, respectively, as evidenced by lack of effect of the native fatty acid linoleic acid . We conclude that the formation of 9-OH-Lin and 9-OOH-Lin by PM may represent a regulatory mechanism towards the cell's own activity by modulating reactive oxygen species.

Arch Biochem Biophys, 1989 Sep, 273(2), 597 - 601
Nick translation of lambda phage DNA with a deoxycytidine analog spin labeled in the 5 position; Strobel OK et al.; The synthesis and properties of a novel C(5)-spin-labeled 2'-deoxycytidine 5'-triphosphate which serves as a suitable substrate for the template-directed enzyme Escherichia coli DNA polymerase I are reported . The spin label is readily incorporated into lambda phage DNA by nick translation where it reports the characteristic local base motion for double- and single-stranded DNA as determined by electron spin resonance . The high-frequency deoxycytidine motion is similar to the previously reported thymidine motion in double-stranded lambda phage DNA.

Mutat Res, 1989 Sep, 214(1), 23 - 31
Characterization of hydroxyl free radical mediated damage to plasmid pBR322 DNA; Schneider JE et al.; We have investigated hydroxyl free radical mediated damage to pBR322 DNA produced by ascorbate/iron and oxygen in a phosphate-buffered in vitro system . An observed lag phase in DNA nicking suggests a multi-target model of hydroxyl free radical attack on DNA . In the present report we further examine the model system and show that there is a "heat labile" component of the ascorbate/iron system which can be completely restored by the readdition of ascorbate . These observations have allowed us to rule out the possibility that intermediates build up in the reaction and act independently of ascorbate to increase the reaction rate . We have investigated the initial rate of OH production with two OH trapping agents, salicylate and deoxyguanosine, and find that the lag in DNA nicking is not due to a corresponding lag in the production of OH as assessed by formation of the products, dihydroxybenzoic acids and 8-hydroxydeoxyguanosine, respectively . We have found that the energy of activation for DNA supercoiled nicking is 13.9 kcal/mole and for OH trapping by salicylate is 21.1 kcal/nmole . These two activation energies are sufficiently different to suggest that the rate-limiting steps of these two reactions are different . Investigation of the rate of oxygen consumption during the ascorbate/iron-mediated DNA damage showed that oxygen was not a limiting component at any point in the reaction . The addition of catalase slowed down oxygen consumption by 31% and this data taken together with our previous observations on the model implicate hydrogen peroxide as a key intermediate in DNA damage caused by hydroxyl free radical.

J Bacteriol, 1989 Sep, 171(9), 5212 - 4
Orientation of IS50 transposase gene and IS50 transposition; Makris JC et al.; Reversal of transposase gene orientation with respect to the nonidentical ends of IS50 strongly decreased IS50 transposition in both Dam- and Dam+ hosts . In either orientation, IS50 transposase expression was unaffected . These effects were independent of the surrounding DNA context . This shows that the efficiency of IS50 transposition is dependent on transposase gene orientation . The transposition frequencies of transposons utilizing inverted IS50 inside ends (IE), IE-IE transposons, were lower than either outside end (OE)-IE or OE-OE transposons.

J Bacteriol, 1989 Sep, 171(9), 5183 - 6
Levels of DNA topoisomerases, single-stranded-DNA-binding protein, and DNA polymerase I in rho+ and rho-15 strains of Escherichia coli; Arnold GF et al.; The Escherichia coli rho-15 mutant, which is highly defective in transcription termination, was examined to see whether its reduced DNA superhelicity could be explained by altered expression of proteins that may affect DNA structure . Levels of DNA gyrase and topoisomerase I were normal; levels of single-stranded-DNA-binding protein, DNA polymerase I, and a protein tentatively identified as Lon were significantly altered.

J Bacteriol, 1989 Sep, 171(9), 4996 - 5004
Analysis of the transcriptional unit encoding the genes for rubredoxin (rub) and a putative rubredoxin oxidoreductase (rbo) in Desulfovibrio vulgaris Hildenborough; Brumlik MJ et al.; The nucleotide sequence of a 2.0-kilobase-pair EcoRI restriction fragment upstream from the gene (rub, 162 base pairs) encoding rubredoxin from Desulfovibrio vulgaris Hildenborough indicates that it is part of a larger transcriptional unit, containing an additional 378-base-pair open reading frame which terminates 16 nucleotides from the translational start of the rub gene and could encode a polypeptide of 14 kilodaltons (kDa) . Northern (RNA) blotting of RNA isolated from both D . vulgaris Hildenborough and Escherichia coli TG2 transformed with plasmid pJK29, which contains both genes on a 1.1-kilobase-pair SalI insert, confirms that the genes for this 14-kDa polypeptide and rubredoxin are present on a single transcript of 680 nucleotides . Strong evidence that the 14-kDa polypeptide is also a redox protein is provided by the fact that its NH2 terminus is homologous to desulforedoxin, which has been isolated from D . gigas as a small dimeric redox protein (36 amino acids per monomer), coordinating two iron atoms . Since rubredoxin is a potential redox partner for the 14-kDa protein, it has been tentatively named rubredoxin oxidoreductase, produced by the rbo gene . Southern blotting indicates that the rbo-rub operon is present in several species and strains of sulfate-reducing bacteria.

J Bacteriol, 1989 Sep, 171(9), 4914 - 22
Genes downstream from pucB and pucA are essential for formation of the B800-850 complex of Rhodobacter capsulatus; Tichy HV et al.; The formation of the light-harvesting complex B800-850 (LH-II) of Rhodobacter capsulatus requires, in addition to the synthesis of the polypeptides alpha and beta (the gene products of pucA and pucB), the synthesis of bacteriochlorophyll and carotenoids and the expression of at least one gene localized downstream from the pucBA operon . This was concluded from the observation that a Tn5 insertion downstream from pucBA inhibited the formation of the LH-II complex and the formation of the pucBA mRNA . The Tn5 insertion point was mapped and found to be over 500 base pairs (bp) downstream from the end of the pucA gene, suggesting the presence of additional puc genes . A region of about 3,000 bp including the pucB and pucA genes and DNA downstream from pucA was sequenced and found to contain three open reading frames (ORFs C, D, and E) . The polypeptide deduced from the first ORF (C) contains 403 amino acids with strongly hydrophobic stretches and one large and three small hydrophilic domains carrying many charged residues . The other two ORFs contain 113 (D) and 118 (E) codons . The amino acid sequences of the N terminus and two tryptic peptides of an alkaline-soluble Mr-14,000 subunit of the isolated LH-II complex were identical with the deduced amino acid sequence of ORF E.

J Bacteriol, 1989 Sep, 171(9), 4807 - 13
Method for selection of transposable DNA and characterization of a new insertion sequence, IS493, from Streptomyces lividans; Solenberg PJ et al.; A method to select for transposable elements from Streptomyces spp . by using insertional inactivation of a repressor gene that functions in Escherichia coli was developed . Plasmid pCZA126, which can replicate in Streptomyces spp . or E . coli, contains a gene coding for the lambda cI857 repressor and a gene, under repressor control, coding for apramycin resistance . E . coli cells containing the plasmid are apramycin sensitive but become apramycin resistant if the cI857 repressor gene is disrupted . Plasmids propagated in Streptomyces spp . can be screened for transposable elements that have disrupted the cI857 gene by transforming E . coli cells to apramycin resistance . This method was used to isolate a new 1.6-kilobase insertion sequence, IS493, from Streptomyces lividans CT2 . IS493 duplicated host DNA at the target site, had inverted repeats at its ends, and contained two tandem open reading frames on each strand . IS493 was present in three copies in the same genomic locations in several S . lividans strains . Two of the copies appeared to be present in regions of similar DNA context that extended at least 11.5 kilobases . Several other Streptomyces spp . did not appear to contain copies of IS493.

J Bacteriol, 1989 Sep, 171(9), 4617 - 22
New method for generating deletions and gene replacements in Escherichia coli; Hamilton CM et al.; We describe a method for generating gene replacements and deletions in Escherichia coli . The technique is simple and rapid and can be applied to most genes, even those that are essential . What makes this method unique and particularly effective is the use of a temperature-sensitive pSC101 replicon to facilitate the gene replacement . The method proceeds by homologous recombination between a gene on the chromosome and homologous sequences carried on a plasmid temperature sensitive for DNA replication . Thus, after transformation of the plasmid into an appropriate host, it is possible to select for integration of the plasmid into the chromosome at 44 degrees C . Subsequent growth of these cointegrates at 30 degrees C leads to a second recombination event, resulting in their resolution . Depending on where the second recombination event takes place, the chromosome will either have undergone a gene replacement or retain the original copy of the gene . The procedure can also be used to effect the transfer of an allele from a plasmid to the chromosome or to rescue a chromosomal allele onto a plasmid . Since the resolved plasmid can be maintained by selection, this technique can be used to generate deletions of essential genes.

J Virol, 1989 Sep, 63(9), 3769 - 76
Herpes simplex virus ribonucleotide reductase: expression in Escherichia coli and purification to homogeneity of a tyrosyl free radical-containing, enzymatically active form of the 38-kilodalton subunit; Ingemarson R et al.; Infection of mammalian cells with herpes simplex virus (HSV) induces a virus-encoded ribonucleotide reductase which is different from the cellular enzyme . This essential viral enzyme consists of two nonidentical subunits of 140 and 38 kilodaltons (kDa) which have not previously been purified to homogeneity . The small subunit of ribonucleotide reductases from other species contains a tyrosyl free radical essential for activity . We have cloned the gene for the small subunit of HSV-1 ribonucleotide reductase into a tac expression plasmid vector . After transfection of Escherichia coli, expression of the 38-kDa protein was detected in immunoblots with a specific monoclonal antibody . About 30 micrograms of protein was produced per liter of bacterial culture . The 38-kDa protein was purified to homogeneity in an almost quantitative yield by immunoaffinity chromatography . It contained a tyrosyl free radical which gave a specific electron paramagnetic resonance spectrum identical to that we have observed in HSV-infected mammalian cells and clearly different from that produced by the E . coli and mammalian ribonucleotide reductases . The recombinant 38-kDa subunit had full activity when assayed in the presence of HSV-infected cell extracts deficient in the native 38-kDa subunit.

Prikl Biokhim Mikrobiol, 1989 Sep-Oct, 25(5), 688 - 98
{The effect of cationic surface-active agents on the Escherichia coli ATPase}; Cherniavskaia MA et al.; The authors studied the effect of cationic surfactants (CS), such as alkyl(C8H17-C18H37)dimethylbenzylammonium (I), alkyl(C8H17-C16H33)benzyltrimethylammonium (II), alkyl(C8H17-C16H33)di-beta-hydroxyethylbenzylammonium (III) chlorides and chlorhydrate of glycine decyl ester (IV) on the ATPase activity of E . coli 1257 cell, spheroplasts, and isolated membranes . Changes in the ATPase activity of the E . coli cells and spheroplasts were found to depends on the concentration and the structure of the cationic surfactants . The removal of the cell wall increased the destroying effect of CS on the cytoplasmic membranes and enhanced the ATPase inhibition . The compounds with 16 and 18 carbon atom radical had the highest inhibitory effect . The action of cationic surfactants on the membrane is accompanied by changes in the protein and phospholipid composition and by significant solubilization of ATPase with pronounced inactivation of the enzyme . The kinetics of inhibition of E . coli membrane ATPase was studied to the presence of the homological series I and IV . The cationic surfactants under study inhibited the ATP hydrolysis catalysed by E . coli ATPase by a mixed type mechanism . Ki = 58.21.10(-4) M for IC10H21; 10.67.10(-4) M for IC12H25; 0.58.10(-4) M for IC16H33; 0.16.10(-4) M for IC18H37, and 5.93.10(-4) M for IV.

EMBO J, 1989 Sep, 8(9), 2703 - 9
Three pure chaperone proteins of Escherichia coli--SecB, trigger factor and GroEL--form soluble complexes with precursor proteins in vitro; Lecker S et al.; Diverse studies of three cytoplasmic proteins of Escherichia coli--SecB, trigger factor and GroEL--have suggested that they can maintain precursor proteins in a conformation which is competent for membrane translocation . These proteins have been termed 'chaperones' . Using purified chaperone proteins and precursor protein substrates, we find that each of these chaperones can stabilize proOmpA for translocation and for the translocation-ATPase . These chaperones bind to proOmpA to form isolable complexes . SecB and GroEL will also form complexes with another exported protein, prePhoE . In contrast, these chaperones do not form stable complexes with a variety of soluble proteins such as SecA protein, bovine serum albumin, ovalbumin or ribonuclease A . While chaperones may transiently interact with soluble proteins to catalyze their folding, the stable interaction between chaperones and presecretory proteins, maintaining an open conformation which is essential for translocation, may commit these proteins to the secretion pathway.

Genetics, 1989 Sep, 123(1), 5 - 17
Distribution of Chi-stimulated recombinational exchanges and heteroduplex endpoints in phage lambda; Cheng KC et al.; The recombination hotspot Chi, 5' G-C-T-G-G-T-G-G 3', stimulates the RecBCD recombination pathway of Escherichia coli . We have determined, with precision greater than previously reported, the distribution of Chi-stimulated exchanges around a Chi site in phage lambda . Crosses of lambda phages with single base-pair mutations surrounding a Chi site were conducted in and analyzed on mismatch correction-impaired hosts to preserve heteroduplex mismatches for analysis . Among phages recombinant for flanking markers, Chi stimulated exchanges most intensely in the intervals immediately adjacent to the Chi site, both to its right and to its left . Stimulation fell off abruptly to the right but gradually to the left (with respect to the orientation of the Chi sequence written above) . We have also determined that Chi stimulated the formation of heteroduplex DNA, which frequently had one endpoint to the right of Chi and the other endpoint to the left . These data support a model of Chi-stimulated recombination in which RecBCD enzyme cuts DNA immediately to the right of Chi and unwinds DNA to the left of Chi; segments of unwound single-stranded DNA are sometimes, but not always, degraded before synapsis with homologous DNA.

J Bacteriol, 1989 Sep, 171(9), 5017 - 24
Temperature-sensitive lethal mutant of era, a G protein in Escherichia coli; Inada T et al.; The era gene of Escherichia coli encodes a GTP-binding protein which has similarities to elongation factor Tu and the Saccharomyces cerevisiae RAS protein . To investigate its function, mutations affecting era were isolated . A mini-Tn10 insertion, which truncated 22 amino acids from the COOH end of Era, did not affect cell growth . By using this mini-Tn10 insert as a coselectable marker, a temperature-sensitive lethal era mutant was isolated by localized mutagenesis using P1 phage transduction . A single-base G to A change was found at position 23, causing a tyrosine residue to be substituted for the cysteine residue at position 8 (era-770), in addition to the COOH-terminal mini-Tn10 disruption . Both alterations were necessary for the temperature-sensitive phenotype . Purified Era-770 mutant protein exhibited reduced binding to GTP compared with that of the wild-type Era protein.

J Bacteriol, 1989 Sep, 171(9), 4609 - 16
Genetic studies on the inability of beta-galactosidase to be translocated across the Escherichia coli cytoplasmic membrane; Lee C et al.; When a signal sequence is attached to beta-galactosidase, the normally cytoplasmic protein is unable to fully traverse the cytoplasmic membrane . We used a genetic approach to study those features of beta-galactosidase responsible for the block in translocation . By using both in vivo and in vitro techniques, fragments of beta-galactosidase were interposed between a signal sequence and alkaline phosphatase . The alkaline phosphatase acts as a sensor for any blocking effects of beta-galactosidase on export . From these studies, we show that multiple regions of beta-galactosidase contribute to its failure to be translocated . These results are most easily interpreted if the folding of beta-galactosidase or of domains of it is responsible for the block in export . In addition, in certain constructs, positively charged amino acids directly following the signal sequence interfered with export.

Indian J Exp Biol, 1989 Sep, 27(9), 833 - 4
Interaction of EDTA with tributyltin induced cellular toxicity; Singh K et al.; Tributyltin inhibited the growth of cells in concentration dependent manner . The inhibitory effect of tributyltin on E . coli was enhanced in the presence of metal ion chelator, EDTA . This was due to EDTA induced increase of permeability of bacterial cell envelope to tributyltin . In case of EDTA treated cells the rate of uptake of tributyltin was more pronounced as compared to control.

Pharmazie, 1989 Sep, 44(9), 608 - 11
{Synthesis and in vivo antitumor effect of N,N-di(2-chlorethyl) hydrazides of natural alpha-aminocarboxylic acids}; Zakhariev S et al.; N,N-Di(2-chlorethyl)hydrazides of the following alpha-amino-carboxylic acids were synthesized: glycine, valine, norvaline, lysine, phenylalanine, tyrosine, cystine, homocystine, aspartic and glutamic acid as well as the N,N-diethylhydrazides of glycine, phenylalanine and cystine . The N,N-di-(2-chlorethyl)hydrazides have a pronounced effect on solid tumours (tumour growth inhibition by 30-100%), whereas their inhibition activity with ascite tumours is negligible . N,N-diethylhydrazides show analogous but less expressed biological effect.

Genetika, 1989 Sep, 25(9), 1551 - 8
{Induction of SOS function in Escherichia coli after exposure to various types of chemical mutagens}; Dzhedzhelava DA et al.; On the basis of the SOS-chromotest which was developed by the authors earlier, the minimum time required for expression of the SOS-response is shown to be 15 min, the maximum being recorded 2 h later . An assay of induction of the SOS-response to the mutagens which act on DNA via various mechanisms (e . g., UV-irradiation, mitomycin C, nalidixic acid, nitroso-methylurea and acridine orange) revealed that all the mutagens under study caused induction of the SOS-response . Analysis of efficiency of SOS induction under the action of a mixture of formaldehyde with various amino acids has shown that arginine, asparagine and, especially, cysteine decrease the level of the SOS-response induction . This fact suggests an opportunity for using the SOS-chromotest not only to identify the mutagens and cancerogens, but also to screen the agents for their antimutagenic activity . The study of the SOS-response in the cells which are deficient in genes for repair and recombination has shown that induction is significantly suppressed in the mutants recA, lexA and recF; it is not modified in sbcB mutants and is significantly increased in the mutant lines recBC, uvrB and, particularly, in the double mutant recBCsbcB.

Biochimie, 1989 Sep-Oct, 71(9-10), 1095 - 100
Phosphorylation of Escherichia coli enolase; Dannelly HK et al.; In vivo labeling of Escherichia coli JA200 pLC 11-8 resulted in 32P incorporation into enolase as demonstrated by immunoaffinity chromatography and electrophoresis followed by autoradiography . Complete acid hydrolysis, followed by thin layer chromatography was employed for determination of the phosphoamino acid residue . Comparison with phosphoamino acid standards resulted in the identification of a labeled residue corresponding to phosphoserine . In vitro labeling of cell extracts from glucose and acetate grown cells resulted in differential labeling of enolase . When specific radioactivities of in vivo labeled enolase were compared, 7 times more label was incorporated at late log phase in glucose grown cells than in late log acetate grown cells . At stationary phase, only 2.5 times more label was incorporated into glucose compared to acetate . When 32P-labeled enolase from glucose grown cells was subjected to treatment with potato acid phosphatase, dephosphorylation of the enzyme could be observed . Monitoring enzyme activity during the acid phosphatase treatment revealed a 70% decrease for the forward enzyme reaction, and a 3-fold increase, followed by a gradual decrease to almost zero, for the reverse enzyme reaction . Complete reversal of the changes in activity was possible by adding an aliquot of partially purified enolase kinase plus ATP.

Biochimie, 1989 Sep-Oct, 71(9-10), 1079 - 87
Phosphorylation of glutaminyl-tRNA synthetase and threonyl-tRNA synthetase by the gene products of dnaK and dnaJ in Escherichia coli K-12 cells; Itikawa H et al.; In Escherichia coli K-12, the heat shock protein DnaK and DnaJ participate in phosphorylation of both glutaminyl-tRNA synthetase and threonyl-tRNA synthetase since when cellular proteins extracted from the dnaK7(Ts), dnaK756(Ts) and dnaJ259(Ts) mutant cells labeled with 32Pi at 42 degrees C were analyzed by two-dimensional gel electrophoresis, no phosphorylation of glutaminyl-tRNA synthetase and threonyl-tRNA synthetase was observed while phosphorylation of both aminoacyl-tRNA synthetases was detected in the samples extracted from wild-type cells.

Biochimie, 1989 Sep-Oct, 71(9-10), 1071 - 7
Biochemical properties of the Escherichia coli dnaK heat shock protein and its mutant derivatives; Cegielska A et al.; The dnaK protein of Escherichia coli has been shown to possess both autophosphorylating and 5'-nucleotidase activities . The dnaK protein has been shown to bind avidly to ATP, but hydrolyzing it slowly . In vitro autophosphorylation occurs at a threonine residue when either ATP or GTP are used as phosphate donors . The extent of autophosphorylation is low; only a few percent of the molecules are phosphorylated . This activity is stimulated at least tenfold in the presence of Ca2+ ions with either ATP or GTP as the donor . The autophosphorylating activity of the mutant dnaK756 protein in the presence or absence of Ca2+ is reduced compared to that of the wild type.

Biochimie, 1989 Sep-Oct, 71(9-10), 1065 - 70
Phosphorylation of isocitrate lyase in Escherichia coli; Robertson EF et al.; Isocitrate lyase from Escherichia coli becomes phosphorylated in vitro by an endogenous kinase when partially purified extracts are incubated with {gamma-32P}ATP . Treatment of isocitrate lyase with histidine modifying reagents, and alkaline hydrolysis of in vitro phosphorylated enzyme indicated the presence of a phosphohistidine residue . Phosphorylation of isocitrate lyase can also occur in vivo, which indicates a possible regulatory significance of this modification . In addition to phosphorylation, isocitrate lyase is capable of incorporating label from both {alpha-32P}ATP and {14C}ATP suggesting that more than one type of covalent modification occurs on this enzyme . This report reviews the studies which have demonstrated the phosphorylation and modification of isocitrate lyase from Escherichia coli.

Biochimie, 1989 Sep-Oct, 71(9-10), 1043 - 9
Utilization of acetate in Escherichia coli: structural organization and differential expression of the ace operon; Cortay JC et al.; Growth of Escherichia coli on acetate as the sole source of carbon and energy requires operation of the glyoxylate bypass in connection with the expression of the polycistronic ace operon . The structural organization of this operon is presented, including the 3 structural genes coding respectively for malate synthase (aceB), isocitrate lyase (aceA) and isocitrate dehydrogenase kinase/phosphatase (aceK), and the surrounding genes iclR and metA . In addition, the differential expression of genes aceB, aceA, and aceK has been tested both in vivo in a minicell system and in vitro in a plasmid-directed transcription-translation coupled system . Moreover, the codon usage and adaptation to transfer RNA frequencies during translation of the corresponding messenger RNAs have been measured.

Kosm Biol Aviakosm Med, 1989 Sep-Oct, 23(5), 26 - 32
{Biological experiments on "Kosmos-1887"}; Alpatov AM et al.; In the 13-ray space flight on Kosmos-1887 various experiments in the field of cell biology, genetics, biorhythm, developmental biology and regeneration were performed using bacteria, protozoa, plants, worms, insects, fish and amphibia . Paramecia showed enhanced cell proliferation, spheroidization and diminished protein content . Experiments on fruit-flies, newt oocytes and primate lymphocytes confirmed involvement of the cell genetic apparatus in responses to microgravity . Beetles exhibited a reduction of the length of the spontaneous period of freely running circadian rhythms . Carausius morosus developed latent changes in early embryogenesis which manifested at later stages of ontogenesis . Exposure to microgravity did not prevent recovery of injured tissues; moreover their regeneration may be accelerated after recovery . Biology research programs in future biosatellite flights are discussed.

Mol Gen Genet, 1989 Sep, 218(3), 481 - 6
Autoregulation of the ccd operon in the F plasmid; de Feyter R et al.; Mini-F sequences, including the promoter and portions of the ccd region, were inserted upstream of lacZ in promoterless lacZ vectors, and beta-galactosidase specific activities were measured . The results showed that the H (ccdA), G (ccdB) and D genes, together with a promoter, comprise an operon . Ccd operon expression was shown to be regulated at the level of transcription by the G gene product, probably in concert with the H gene product . Thus expression is autoregulated . Expression of the D gene was largely dependent on the ccd promoter, although low levels of transcription from another promoter within the ccd coding region were detected.

Gene, 1989 Sep 1, 81(1), 129 - 37
Mature apolipoprotein AI and its precursor proApoAI: influence of the sequence at the 5' end of the gene on the efficiency of expression in Escherichia coli; Isacchi A et al.; Apolipoprotein AI (ApoAI) plays a central role in the regulation of lipid metabolism . Initial attempts to express human apoAI cDNA in Escherichia coli did not yield detectable levels of the mature protein . By analyzing the efficiency of expression of apoAI-lacZ gene fusions, we have been able to show that the sequence at the 5' end of the ApoAI-coding region is a critical parameter . Indeed, silent changes in the codons for the first 8 residues of ApoAI, which did not alter the amino acid sequence, affected expression dramatically . Analysis of the corresponding mRNA steady-state levels suggested a role for differential mRNA stability in the control of apoAI expression in this system . Among all the possible alternative sequences, we have identified an optimal sequence which, when reinserted in the original expression plasmid, yields high level production of mature ApoAI . This procedure has been extended to the production of the natural variant ApoAI-Milano and the precursor proApoAI . Availability of these recombinant molecules would allow the investigation of their structural and biological features . In addition, the methodology used to optimize ApoAI expression is of general interest in assuring high expression of heterologous proteins in E . coli.

Mol Microbiol, 1989 Sep, 3(9), 1221 - 9
Molecular and genetical analysis of the fructose-glucose transport system in the cyanobacterium Synechocystis PCC6803; Zhang CC et al.; Complementation for glucose transport capacity of deficient mutants from Synechocystis PCC6803 allowed the cloning of the corresponding gene, glcP . The protein predicted from one open reading frame (ORF) in the DNA sequence was 468 residues long . It showed 46-60% amino acid sequence homology and similarity in size and predicted structure (including twelve probable membrane-spanning regions) with a group of non-phosphorylating sugar transporters from mammals, yeasts and Escherichia coli . A second ORF, 64 base pairs downstream from glcP, was detected . Its function, dispensable under auto- and heterotrophic conditions, could not be determined . Genetic analysis of mutants confirmed that the resistance to fructose, acquired simultaneously with the deficiency in glucose transport, resulted from mutations in the glcP gene, whose approximate location could be determined.

Prostaglandins, 1989 Sep, 38(3), 319 - 33
Relationship between PAF-acether and thromboxane A2 biosynthesis in endotoxin-induced intestinal damage in the rat; Boughton-Smith NK et al.; PAF-receptor antagonists are known to inhibit gastrointestinal damage induced by endotoxin . In the present study, the interaction between the biosynthesis of PAF and thromboxane (TX) A2, as putative mediators of the acute intestinal damage induced by endotoxin, has been investigated in the anaesthetised rat . Bolus intravenous administration of lipopolysaccharide from E . coli (5-50 mg/kg) induced dose-related jejunal damage, assessed using both macroscopic and histological techniques . This damage was accompanied by significant increases in the jejunal formation of PAF determined by bioassay, and of TXB2, determined by radioimmunoassay . Pretreatment with the structurally-unrelated thromboxane synthase inhibitors, 1-benzyl imidazole (10-50 mg/kg) or OKY 1581 (25 mg/kg) substantially reduced both jejunal damage and TXB2 formation, but did not inhibit PAF formation . Likewise, pretreatment with indomethacin (5 mg/kg) or BW 755C (50 mg/kg) reduced jejunal damage and TXB2 formation but did not affect PAF formation . Pretreatment (2h) with dexamethasone (4 mg/kg) reduced jejunal damage and the formation of both TXB2 and PAF . Intravenous infusion of PAF (100 ng/kg/min for 10 min) induced jejunal damage and significantly increased the formation of TXB2, whereas non-specific jejunal damage induced by oral administration of ethanol did not augment PAF formation . The present findings that inhibition of jejunal thromboxane formation is associated with a substantial reduction in jejunal damage, with no corresponding inhibition in PAF formation, therefore suggests a complex interaction or sequential release of these tissue destructive mediators underlying the intestinal damage induced by endotoxin.

Eur J Biochem, 1989 Sep 1, 184(1), 63 - 8
Methyl-coenzyme-M reductase from Methanobacterium thermoautotrophicum (strain Marburg) . Purity, activity and novel inhibitors; Ellermann J et al.; Methyl-coenzyme-M reductase from Methanobacterium thermoautotrophicum (strain Marburg) was purified to a stage where, besides the alpha, beta and gamma subunits, no additional polypeptides were detectable in the preparation . Under appropriate conditions the enzyme was found to catalyze the reduction of methyl-CoM with 7-mercaptoheptanoylthreonine phosphate (H-S-HTP) to CH4 at a specific rate of 2.5 mumol.min-1.mg protein-1 . This finding contradicts a recent report that methyl-CoM reductase is only active when some contaminating proteins are present . The two polypeptides encoded by the open reading frames ORF1 and ORF2 of the methyl-CoM reductase transcription unit did not co-purify with the alpha, beta and gamma subunits . They were neither required nor did they stimulate the activity under the assay conditions . 3-Bromopropanesulfonate (apparent Ki = 0.05 microM) and 2-azidoethanesulfonate (apparent Ki = 1 microM) were found to be two new competitive inhibitors of methyl-CoM reductase . Both inhibitors were considerably more effective than the "classical" 2-bromoethanesulfonate (apparent Ki = 4 microM).

Am J Clin Nutr, 1989 Sep, 50(3), 524 - 7
Dietary iron and recovery from peritonitis in guinea pigs; Peck MD et al.; Ninety female Hartley guinea pigs underwent gastrostomy placement . One week later they underwent implantation of an osmotic pump, which allowed constant delivery of bacteria into the peritoneal cavity . Three days after pump implantation the animals were begun on enteral diets differing only in iron content (the None {no Fe}, Low {1 X RDA}, and High {10 X RDA} groups) . When survivors were killed no differences were found in body, carcass, or organ weights among the three groups . Serum Fe and percent Fe-binding sites occupied were significantly lower in the None group although total Fe-binding capacity was similar . Mortality was not statistically different (p = 0.29): 18/32 in the None group (56%), 14/24 in the Low group (58%), and 25/34 in the High group (73%) . We conclude that although deprivation of dietary sources of Fe does affect available circulating Fe, diet-induced hypoferremia does not alter mortality rates from bacterial peritonitis in the guinea pig.

Proc Natl Acad Sci U S A, 1989 Sep, 86(17), 6538 - 42
Selective expression of a protein-tyrosine kinase, p56lyn, in hematopoietic cells and association with production of human T-cell lymphotropic virus type I; Yamanashi Y et al.; This paper reports the identification of the lyn gene product, a member of the src-related family of protein-tyrosine kinases, and its expression in hematopoietic cells . A lyn-specific sequence (Arg-25 to Ala-119 of the protein) was expressed in Escherichia coli as a fusion protein with beta-galactosidase . Antiserum raised against the fusion protein immunoprecipitated a 56-kDa protein from human B lymphocytes . Incubation of the immunoprecipitate with {gamma-32P}ATP resulted in the phosphorylation of this protein at tyrosine residues . Immunohistological and immunoblotting analyses showed that the lyn gene product was expressed in lymphatic tissues (spleen and tonsil) and in adult lung, which contains many macrophages . Furthermore, both the transcripts and the protein products of the lyn gene accumulated in macrophages/monocytes, platelets, and B lymphocytes but were not expressed appreciably in granulocytes, erythrocytes, or T lymphocytes, suggesting that lyn gene products function primarily in certain differentiated cells of lymphoid and myeloid lineages.

J Nucl Med, 1989 Sep, 30(9), 1538 - 45
Autoradiographic method for quantitation of radiolabeled proteins in tissues using indium-111; Morrell EM et al.; A quantitative autoradiographic method was developed to measure 111In-labeled proteins in extravascular tissues with a spatial resolution sufficient to associate these proteins with tissue morphology . A linear relationship between measured grain density and isotope concentration was demonstrated with uniformly-labeled standard sources of epoxy-embedded gelatin containing {111In}albumin; half-distance of spatial resolution was 0.6 micron . The technique was illustrated by measuring 24-hr accumulation of diethylenetriaminepentaacetic acid-coupled 111In-labeled human polyclonal IgG and human serum albumin (HSA) in a thigh infection model in the rat . Gamma camera images localized the infection and showed target-to-background ratios of 2.5 +/- 0.3 for IgG and 1.4 +/- 0.02 for human serum albumin (mean +/- s.d., n = 3) . Using quantitative autoradiography, significantly higher average tissue concentrations were found in the infected thighs at 4 to 4.5% of the initial plasma concentrations as compared to 0.2 to 0.3% of initial plasma concentrations in the noninfected thigh (p less than 0.05); these radiolabeled proteins were not inflammatory cell associated and localized primarily within the edematous interstitial spaces of the infection.

J Bacteriol, 1989 Sep, 171(9), 5095 - 102
L-serine degradation in Escherichia coli K-12: cloning and sequencing of the sdaA gene; Su HS et al.; A new mutant of Escherichia coli K-12 unable to grow with L-serine, glycine, and L-leucine has been isolated by lambda plac Mu insertion and shown to be deficient in L-serine deaminase activity . The corresponding gene, sdaA, has been cloned from a prototrophic strain, and the clone has been characterized and sequenced . The evidence is consistent with the hypothesis that sdaA is the structural gene for L-serine deaminase . However, other possibilities are also considered . No significant homology with previously reported DNA or protein sequences was detected.

J Bacteriol, 1989 Sep, 171(9), 4569 - 76
The hydrophobic domain of cytochrome b5 is capable of anchoring beta-galactosidase in Escherichia coli membranes; George SK et al.; Cytochrome b5 is inserted posttranslationally into membranes in vivo and spontaneously into liposomes in vitro by a short carboxyl-terminal hydrophobic membrane-anchoring sequence . DNA corresponding to this hydrophobic sequence has been synthesized, and two gene fusions with the Escherichia coli enzyme beta-galactosidase have been constructed by locating the hydrophobic domain in one case at the EcoRI site near the C terminus and in the other at the normal C terminus of the enzyme . The latter fusion protein was enzymatically active, having approximately 50% of the specific activity of beta-galactosidase, and cells expressing this protein grew normally with lactose as the sole carbon source . Both fusion proteins were localized to the E . coli inner membrane, converting beta-galactosidase from a cytoplasmic enzyme to a membrane-associated enzyme . The hydrophobic domain of cytochrome b5 therefore contains the information required to target polypeptides containing this domain to the membrane . Use of the cytochrome b5 hydrophobic peptide, either alone or in conjunction with other localizing sequences such as signal sequences, provides a general procedure for associating proteins with membranes . Polypeptides bearing this hydrophobic peptide may have considerable use as pharmaceuticals when associated with liposomes or cellular membranes.

DNA, 1989 Sep, 8(7), 535 - 41
Efficient DNA-mediated gene transfer into primary cultures of adult rat hepatocytes; Pasco DS et al.; An efficient, simple, and reproducible DNA-mediated gene transfer procedure has been developed for primary cultures of adult rat hepatocytes . Calcium phosphate-DNA precipitate is formed in complete culture medium during 5 hr incubation with cells . Unabsorbed precipitate is then washed out, and 40 hr later gene expression is measured . Under optimal conditions, up to 20-25% of cells in cultures transfected with the beta-galactosidase (lacZ) gene stain positively for this activity, and cells transfected with the chloramphenicol acetyl transferase (CAT) gene, fused to a strong promoter, express CAT activities of 10-14 nmoles/min per mg protein . Five conditions were optimized based on transfection efficiency, CAT expression, and cell viability . (i) Medium composition: the presence of protein, such as fetal bovine serum or bovine serum albumin, in the medium was essential . (ii) Cell substratum: tissue culture plastic was superior to calf skin collagen and Matrigel . (iii) Cell density: 0.5-1.0 X 10(6) cells/60-mm dish were superior to higher densities . (iv) Duration of exposure to calcium phosphate-DNA: 5-8 hr was better than shorter or longer times . (v) Length of time hepatocytes were maintained in culture before initiating transfection: 2-3 days was superior to earlier times . This procedure was successful with reporter genes linked to three different eukaryotic promoters . These included a chimeric promoter containing the polycyclic aromatic hydrocarbon-responsive enhancer of the cytochrome P450c gene (CYP1A1), which was shown to confer upon the CAT gene responsiveness to polycyclic aromatic hydrocarbons comparable to that of the native P450c gene . This transfection procedure should be of considerable use for the study of liver-specific gene expression in primary hepatocyte cultures.

Asia Oceania J Obstet Gynaecol, 1989 Sep, 15(3), 307 - 12
An experimental study on the effects of elastase, bleomycin and infection on the growth and tensile strength of elastic tissue in rabbit fetal membranes; Chimura T et al.; The effects of bleomycin, elastase and infection on the tensile strength of rabbit fetal membranes and the structure of the elastic tissue of rabbit amnion were studied . The experimental subjects were rabbits to which bleomycin (total dose, 20 mg) or elastase (total dose, 60 mg) was administered from 18 days of pregnancy . In addition to the fetal membranes from these treated animals, fetal membranes with amniotic infection induced by injection of Escherichia coli into the amniotic fluid (AF) of rabbits at 22 or 23 days of pregnancy were examined, and those from rabbits administered physiological saline solution were used as controls . The results obtained were as follows . Determination of tensile strength of the fetal membranes revealed that the infected membranes had minimum rupture pressure (18.67 +/- 6.30 mm Hg), followed by the elastase administration group (46.67 +/- 3.51 mm Hg) . The value in the bleomycin administration group (99.67 +/- 12.89 mm Hg) was significantly higher than that in the control group (61.80 +/- 6.30 mm Hg) . Histological changes in the fibroblast layer of these fetal membranes included elastic fibrosis due to excessive collagen production, which was mostly manifested as fibrosis densa, in the bleomycin administration group . In the elastase administration group, the elastic fibers were eliminated or became minute, as in the infection groups . The above results showed the adverse effects of elastase and bleomycin and the destructive effect of infection on the growth and properties of elastic tissue in the amnion, suggesting their pathophysiological importance.

Neurol Med Chir (Tokyo), 1989 Sep, 29(9), 861 - 3
Neonatal brain abscess with intracystic hemorrhage--case report; Nakagawa Y et al.; The authors describe the case of an 11-day-old boy with high fever and signs of meningeal irritation in whom computed tomography demonstrated a large brain abscess with intracystic hemorrhage in the right temporal lobe . Cerebrospinal fluid analysis indicated purulent meningitis caused by Escherichia coli . After aspiration of the abscess contents, the entire ventricular system gradually enlarged . Despite repeated ventricular drainage and ventriculoperitoneal shunting, the lateral horn of the left lateral ventricle remained dilated . The isolation of the lateral ventricle may have resulted from septation due to the inflammatory reaction . This fluid was also shunted to the peritoneal cavity.

Anal Biochem, 1989 Sep, 181(2), 360 - 70
Nucleic acid hybridization assays employing dA-tailed capture probes . II . Advanced multiple capture methods; Hunsaker WR et al.; A fourth capture is added to the reversible target capture procedure of the preceding paper . This results in an improved radioisotopic detection limit of 7.3 x 10(-21) mol of target . In addition, the standard triple capture method is converted into a nonradioactive format with a detection limit of under 1 amol of target . The principal advantage of nonradioactive detection is that the entire assay can be performed in about 1 h . Nucleic acids are released from cells in the presence of the ('capture probe') which contains a 3'-poly(dA) sequence and the ('labeled probe') which contains a detectable nonradioactive moiety such as biotin . After a brief hybridization in solution, the target is captured on oligo(dT) magnetic particles . The target is further purified from sample impurities and excess labeled probe by recapture either once or twice more on fresh magnetic particles . The highly purified target is then concentrated to 200 nl by recapture onto a poly(dT) nitrocellulose filter and rapidly detected with streptavidin-alkaline phosphatase using bromochloroindolyl phosphate and nitroblue tetrazolium . Using this procedure, as little as 0.25 amol of a target plasmid has been detected nonradioactively in crude samples in just 1 h without prior purification of the DNA and RNA . Finally, a new procedure called background capture is introduced to complement the background-reducing power of RTC.

Int J Parasitol, 1989 Sep, 19(6), 631 - 8
Characterization of cloned antigens of Taenia ovis; Howell MJ et al.; Fusion proteins derived from recombinant clones of Escherichia coli carrying the pEX series of expression vectors with cDNA inserts of the tapeworm, Taenia ovis, were characterized by their antigenicity when administered to mice and sheep . Antisera derived from these hosts were shown to react with a number of T . ovis adult worm and oncospheral antigens in Western blots, and to bind to oncospheres and worm tissue in fluorescent antibody tests . Epitopes produced by two clones (designated T07 and T08) are found on several antigenic polypeptides of T . ovis and a related species T . hydatigena . Those produced by a third clone (T03) are specific to T . ovis . The immunological results were confirmed by Northern blotting of T . ovis and T . hydatigena RNA: RNA transcripts corresponding to T07 and T08 are found in both T . ovis and T . hydatigena, whereas RNA corresponding to T03 is found in T . ovis only . Cross reacting epitopes in fusion proteins from T07 and T08 appear to have different amino acid sequences, and T . ovis antigenic epitopes are shared by a number of polypeptides in and between adult worms and oncospheres.

Biophys J, 1989 Sep, 56(3), 631 - 6
Modified reconstitution method used in patch-clamp studies of Escherichia coli ion channels; Delcour AH et al.; We have modified the procedure of Criado and Keller (1987) to study ion channels of Escherichia coli reconstituted in liposomes . The modifications include (a) excluding the use of any detergent and (b) inducing blisters from liposomes with Mg2+ . These blisters, which appear to be unilamellar, are stable for hours . They could be repeatedly sampled with different patch-clamp pipettes each achieving seal resistance greater than 10 GOhms . Activities of three types of ion channels are often observed by use of this method, including two voltage-sensitive cation channels of different conductances . Even the mechanosensitive channel, previously recorded from live E . coli cells (Martinac et al., 1987), was also detected in these blisters . Apparently the channel protein and any accessory structures, postulated to be needed for mechanotransduction, can be reconstituted together by this method.

Proc Natl Acad Sci U S A, 1989 Sep, 86(18), 7208 - 12
Extensive diversity of branched-RNA-linked multicopy single-stranded DNAs in clinical strains of Escherichia coli; Sun J et al.; Recently it was shown that a clinical strain of Escherichia coli contains a reverse transcriptase that is essential for the synthesis of a branched-RNA-linked multicopy single-stranded DNA (msDNA) . We now have examined 113 independent clinical isolates of E . coli for the existence of msDNA and found that 7 strains contained msDNA . Four of them were further analyzed by hybridization analysis, which indicated that three of the msDNAs were different, having little sequence homology . When the reverse transcriptase gene associated with one of these msDNAs was used as a probe, it did not hybridize with chromosomal DNA from the other strains containing msDNA . These results indicate that some clinical E . coli strains carry their own unique msDNA-synthesizing systems; msDNAs produced by these systems have little, if any, sequence homology in their RNA and DNA molecules and the reverse transcriptases required for the production of msDNA also have little sequence similarity . Such extensive diversity of the msDNA-synthesizing systems supports the notion that they were acquired by the E . coli genome late during the evolution of E . coli.

Proc Natl Acad Sci U S A, 1989 Sep, 86(18), 6925 - 9
Human immunodeficiency virus 1 tat protein binds trans-activation-responsive region (TAR) RNA in vitro; Dingwall C et al.; tat, the trans-activator protein for human immunodeficiency virus 1 (HIV-1), has been expressed in Escherichia coli from synthetic genes . Purified tat binds specifically to HIV-1 trans-activation-responsive region (TAR) RNA in gel-retardation, filter-binding, and immunoprecipitation assays . tat does not bind detectably to antisense TAR RNA sequences, cellular mRNA sequences, variant TAR RNA sequences with altered stem-loop structures, or TAR DNA.

Arch Biochem Biophys, 1989 Sep, 273(2), 347 - 58
Recombinant HIV-1 reverse transcriptase: purification, primary structure, and polymerase/ribonuclease H activities; Mizrahi V et al.; Recombinant HIV-1 reverse transcriptase (RT) was stably overproduced as a soluble protein in Escherichia coli using a double-plasmid expression system in which an RT precursor protein was expressed and processed in vivo by HIV-1 protease produced in trans . The RT thus produced consisted of an equimolar mixture of two polypeptides, p66 and p51, which were copurified to greater than 90% homogeneity and were found to share a common NH2 terminus as judged by sequence analysis of the polypeptide mixture . The observed sequence confirmed correct in vivo cleavage by protease at the protease-RT polyprotein junction to yield an NH2 terminus identical to that of genuine viral RT (M . M . Lightfoote et al . (1986) J . Virol . 60, 771-775; F . diMarzo Veronese et al . (1986) Science 231, 1289-1291) . The bacterially expressed RT had a specific activity similar to that of viral RT and inhibition studies with phosphonoformate confirmed that it was indistinguishable from the viral enzyme with respect to sensitivity to this inhibitor . Polymerase activated gel analysis of the mixture indicated that p66 was associated with a higher level of RT activity than p51 . RNase H activated gel analysis suggested that the purified preparation of recombinant RT was free of endogenous E . coli RNase H, and that the RNase H activity of RT was exclusively associated with the p66 polypeptide, supporting the hypothesis that the RNase H domain is located in the COOH-terminal region of the molecule.

J Bacteriol, 1989 Sep, 171(9), 5056 - 64
Role of countertranscript RNA in the copy number control system of an IncB miniplasmid; Praszkier J et al.; Transcriptional mapping studies of the IncB minireplicon pMU720 demonstrated the existence of a long RNA molecule, RNA II, whose 5' portion is complementary to the product of the incompatibility gene RNA I . By using gene fusion and transcriptional fusion plasmids, it was shown that RNA I regulated the expression of the RNA II gene product and that it did so primarily at the level of translation . The target of RNA I was mapped to lie within a 216-base region of RNA II containing the sequence complementary to RNA I . Introduction of the target for RNA I in trans increased the copy number of an IncB minireplicon, indicating that RNA I and RNA II form the basis of the copy number control system of IncB plasmids.

Biochim Biophys Acta, 1989 Aug 31, 997(3), 242 - 7
Calcium regulates thioredoxin reductase in human metastatic melanoma; Schallreuter KU et al.; Thioredoxin reductase has been purified from human metastatic melanotic melanoma and amelanotic melanoma tissues . Enzyme from the melanotic melanoma tissue contains bound calcium showing classical sigmoidal allosteric kinetics, whereas enzyme from the amelanotic melanoma yielded normal Michaelis-Menten saturation with substrate . Calcium inhibition can be partially reversed by oxidized thioredoxin . 45Ca has been used to label the amelanotic melanoma enzyme in order to determine the number of calcium-binding sites . These isotope experiments yielded only one calcium-binding site per enzyme molecule . Enzyme labeled with 45Ca was dialyzed for 24 h without loss of radioactivity, but the addition of oxidized thioredoxin to this labeled enzyme caused 60% calcium exchange in 24 h . Comparative studies with Escherichia coli thioredoxin reductase showed similar calcium inhibition as well as partial reactivation with oxidized thioredoxin . The enzyme from E . coli previously sequenced by others, showed considerable homology with the first EF-hands calcium-binding site of calmodulin . Detailed calcium-binding studies indicated that 10(-5) M of this fast exchange ion was sufficient to cause allosteric regulation in 10 min . This strong calcium-binding property could explain the allosteric nature of the thioredoxin reductase purified from human metastatic melanotic melanoma and its role in the regulation of melanin biosynthesis.

Nature, 1989 Aug 31, 340(6236), 730 - 2
Three-dimensional structure of Escherichia coli RNA polymerase holoenzyme determined by electron crystallography; Darst SA et al.; During transcription in E . coli, the DNA-dependent RNA polymerase locates specific promoter sequences in the DNA template, melts a small region containing the transcription start site, initiates RNA synthesis, processively elongates the transcript, and finally terminates and releases the RNA product . Each step is regulated by interactions between the polymerase, the DNA, the nascent RNA, and a variety of regulatory proteins and ligands . The E . coli enzyme contains a catalytic core of two alpha-subunits, one beta- and one beta'-subunit, with relative molecular masses (Mr) of 36,512, 150,619 and 155,162, respectively . The holoenzyme has an additional regulatory subunit, normally sigma, of Mr 70,236 . Preparations may also contain the omega-subunit (Mr approximately 10,000), which can be removed without affecting any known properties of the enzyme . Because the amino-acid sequences of the beta- and beta'-subunits are homologous to those of the largest subunits of the yeast, Drosophila and murine RNA polymerases, it seems likely that essential features of the three-dimensional structure and catalytic mechanism of RNA polymerase are also conserved across species . Crystals of RNA polymerase suitable for X-ray analysis have not yet been obtained, but two-dimensional crystals of E . coli RNA polymerase holoenzyme can be grown on positively charged lipid layers . Electron microscopy of these crystals in negative stain shows the enzyme in projection as an irregularly shaped complex approximately 100 x 100 x 160 A in size . We have now determined the three-dimensional structure by electron microscopy of negatively stained, two-dimensional crystals tilted at various angles to the incident electron beam . We find a structure in RNA polymerase similar to the active-site cleft of DNA polymerase I . In the light of functional similarities between these two enzymes, together with other evidence, this probably identifies the active-site region of RNA polymerase.

Biochem Biophys Res Commun, 1989 Aug 30, 163(1), 64 - 71
Chemical phosphorylation of proteins by zinc-ATP; Jarvis BW et al.; During an examination of in vitro phosphorylation of the adipocyte lipid-binding protein (ALBP) by the insulin receptor, we detected insulin receptor-independent, chemical phosphorylation of ALBP . This activity was present in ALBP purified to homogeneity from murine 3T3-L1 cells and in recombinant murine ALBP purified from expressing E . coli cultures . Phosphoamino acid analysis revealed that chemical phosphorylation of ALBP occurred primarily on Ser residues . The phosphorylation activity occurred in the alkaline pH range from 8 to 11 and exhibited a broad temperature dependence . The reaction rate was linearly dependent upon the ATP concentration and exhibited a biphasic kinetic profile . Eight of twelve other proteins tested also underwent chemical phosphorylation . Zn+2, Mg+2, or Mn+2 promoted optimal phosphorylation of different proteins . We conclude that many proteins are capable of undergoing chemical phosphorylation.

Biochem Biophys Res Commun, 1989 Aug 30, 163(1), 79 - 83
The effect of homocysteine on MetR regulation of metE, metR and metH expression in vitro; Cai XY et al.; An Escherichia coli S-30 DNA directed protein synthesis system was used to study the effect of homocysteine on the in vitro expression of the metE, metH and metR genes . In the presence of purified MetR protein, which is known to regulate the expression of these genes, homocysteine activates metE expression and inhibits both metR and metH expression . These findings support the recent in vivo results of Urbanowski, M.L . and Stauffer, G.V . (1989), J . Bacteriol . 171, 3277-3281.

Biochem Biophys Res Commun, 1989 Aug 30, 163(1), 438 - 43
Transient shut off of Escherichia coli heat shock protein synthesis upon temperature shift down; Taura T et al.; A moderate downward shift in growth temperature (37 to 30 degrees C in strain B/r and 37 to 24 degrees C in strain K-12) was found to depress markedly the synthesis of major heat shock proteins GroEL and DnaK in E . coli . The depression was transient and cancelled gradually to a new steady state level, taking 60-80 min . The synthesis of beta-galactosidase directed by transcription initiated at the groE promoter behaved similarly, suggesting that this regulation, termed "reverse heat shock response", occurs at the transcriptional level.

Biochem Biophys Res Commun, 1989 Aug 30, 163(1), 131 - 6
Expression of rat protein phosphatase 2C (IA) in Escherichia coli; Tamura S et al.; A cDNA containing the entire coding sequence of rat type 2C (IA) protein phosphatase was expressed in Escherichia coli . An extract of bacterial cells harboring the recombinant plasmid contained a major (Mr = 41,000 - 43,000) and a minor (Mr = 30,000) protein band; both of these reacted with an anti-type 2C protein phosphatase serum . The size of the major protein band agrees well with that of the 2C phosphatase conceptualized from the cognate cDNA . A Mg2+-dependent protein phosphatase activity was detected in extracts containing the recombinant protein, but not in host cell extracts . Based on these results, it is concluded that the isolated cDNA clone encodes a functional type 2C protein phosphatase.

FEBS Lett, 1989 Aug 28, 254(1-2), 69 - 73
Calmodulin-independent bovine brain adenylate cyclase . Amino acid sequence and nucleotide sequence of the corresponding cDNA; Lipkin VM et al.; An individual catalytic component of calmodulin-independent adenylate cyclase has been isolated from bovine brain cortex . Affinity chromatography on an immunosorbent was used . The amino acid sequence of adenylate cyclase as well as the corresponding nucleotide sequence of the cDNA has been determined . cDNA of adenylate cyclase encodes a protein consisting of 834 amino acid residues and the signal peptide (19 amino acid residues) . A series of adenylate cyclase isoforms has been found . A homology between adenylate cyclases from bovine brain, E . coli and Bordetella pertussis has been revealed.

J Biol Chem, 1989 Aug 25, 264(24), 14455 - 62
Quantification of aminofluorene adduct formation and repair in defined DNA sequences in mammalian cells using the UVRABC nuclease; Tang MS et al.; Using the UVRABC nuclease as a reagent coupled with DNA restriction and hybridization analysis we have developed a method to quantify N-acetoxy-2-acetylaminofluorene (NAAAF)-induced DNA damage in the coding and noncoding sequences of the dihydrofolate reductase (DHFR) gene in Chinese hamster ovary (CHO) cells . High performance liquid chromatography analysis shows that the only DNA adduct formed in NAAAF-treated CHO cells is N-(deoxyguanosine-C8-yl)-2-aminofluorene (dG-C8-AF) . DNA sequencing analysis demonstrates that the UVRABC nuclease incises at all potential sites in which dG-C8-AF adduct may form in linear DNA fragments . We have found that the formation and removal of dG-C8-AF adducts in the coding and 3' downstream noncoding sequences of the DHFR domain are similar in cells treated with 10 microM NAAAF (3.1 adducts/14 kilobases); DNA adduct removal attains 70% for both sequences within 24 h . This result contrasts with that obtained for the repair of cyclobutane dipyrimidines in the DHFR gene, in which the repair efficiency is much higher in the coding region than in the 3' downstream noncoding region . Our results suggest that in CHO cells the repair pathway for aminofluorene DNA adducts is not the same as that for cyclobutane dipyrimidines . This new technique has the potential to detect a variety of chemical carcinogen induced DNA adducts at the gene level in cultured cells and in DNA isolated from animal tissues.

Nucleic Acids Res, 1989 Aug 25, 17(16), 6545 - 51
A simple method for site-directed mutagenesis using the polymerase chain reaction; Hemsley A et al.; We have developed a general and simple method for directing specific sequence changes in a plasmid using primed amplification by the polymerase chain reaction (PCR) . The method is based on the amplification of the entire plasmid using primers that include the desired changes . The method is rapid, simple in its execution, and requires only minute amounts of plasmid template DNA . It is significant that there are no special requirements for appropriately placed restriction sites in the sequence to be manipulated . In our system the yield of transformants was high and the fraction of them harboring plasmids with only the desired change was consistently about 80% . The generality of the method should make it useful for the direct alteration of most cloned genes . The only limitation may be the total length of the plasmid to be manipulated . During the study we found that the Taq DNA polymerase used for PCR adds on a single extra base (usually an A) at the end of a large fraction of the newly synthesized chains . These had to be removed by the Klenow fragment of DNA polymerase to insure restoration of the gene sequence.

J Biol Chem, 1989 Aug 25, 264(24), 14478 - 85
Signal peptide subsegments are not always functionally interchangeable . M13 procoat hydrophobic core fails to transport alkaline phosphatase in Escherichia coli; Laforet GA et al.; Bacterial signal peptides display little amino acid sequence homology despite their shared role in mediating protein transport . This heterogeneity may exist to permit the establishment of signal peptide conformations that are appropriate for transport of particular proteins . In this paper we explore how signal peptides are composed of structural units that may interact with each other and with the mature protein to effect transport . Using a new application of cassette mutagenesis, we have replaced the hydrophobic core of the Escherichia coli alkaline phosphatase signal peptide with cores from the signals of maltose-binding protein, OmpA, and M13 major coat protein . The core regions from maltose-binding protein and OmpA effectively replaced the alkaline phosphatase core; the resultant hybrid signals performed as well as wild type in periplasmic transport and processing of alkaline phosphatase . However, the core region from M13 major coat protein generated a transport-incompetent hybrid signal peptide . Elimination of a proline-containing portion of the M13 major coat protein core did not improve transport effectiveness . However, restoration of the procoat cleavage region and the negatively charged amino terminus of the mature protein did ameliorate the transport defect . These results suggest that at least in the case of these procoat-derived signal peptide mutants, there is a requirement for complementarity among the hydrophobic core, cleavage region, and part of the mature protein in order for efficient protein transport to occur.

J Biol Chem, 1989 Aug 25, 264(24), 14396 - 402
Down-regulation and phosphorylation of glucocorticoid receptors in cultured cells . Investigations with a monospecific antiserum against a bacterially expressed receptor fragment; Hoeck W et al.; The expression of the glucocorticoid receptor (GR) gene and the phosphorylation of the GR protein has been studied as a function of time after hormone addition to NIH 3T3 cells . We detected a ligand-induced decrease of GR gene expression at both the level of RNA and protein . GR mRNA declined to 25% of the control within 3 h of dexamethasone treatment and remained at this level for at least 24 h . GR protein was analyzed with a monospecific antiserum directed against the DNA- and hormone-binding domains of the rat GR synthesized in Escherichia coli . Pulse-chase experiments revealed a decrease in GR half-life from 8 h in the absence of hormone to 3 h following hormone treatment . Both effects resulted in a reduction of total GR protein to 20% as determined by quantitative immunoblotting . The level of the GR protein returned to that of untreated cells within 24 h after withdrawal of hormone . Furthermore, dexamethasone treatment led to a 3-4-fold increase in GR phosphorylation within 60 min . The glucocorticoid antagonist 17 beta-hydroxy-11 beta-(4-dimethylamino-phenyl)-17 alpha-(1-propynyl)-oestra-4,9-dien-3-one (RU 486) did not change the phosphorylation state of the receptor, but a down-regulation of the GR was still observed . These results suggest that ligand-dependent receptor phosphorylation is not involved in down-regulation of the GR but may be important in the transcriptional activation of hormone responsive genes.

J Biol Chem, 1989 Aug 25, 264(24), 14129 - 35
psbG is not a photosystem two gene but may be an ndh gene; Nixon PJ et al.; A gene of the chloroplast genome has been designated the psbG gene on the basis that in maize the gene product is a 24-kDa polypeptide of photosystem two (PS2) (Steinmetz, A . A., Castroviejo, M., Sayre, R . T., and Bogorad, L . (1986) J . Biol . Chem . 261, 2485-2488) . We have located and sequenced the equivalent gene in wheat (Triticum aestivum) and have raised specific antibodies to the gene product following its expression in Escherichia coli as a beta-galactosidase fusion protein . Using these antibodies, we have investigated the location of the gene product in various thylakoid membrane fractions of pea (Pisum sativum) . The gene product of apparent molecular mass 27-28 kDa is severely depleted in PS2-enriched membrane preparations and its distribution between stromal and granal regions of the membrane is distinct to that of the psbC gene product which is known to be a core polypeptide of PS2 . We therefore conclude that psbG does not code for a component of PS2 but instead suggest that it is present in a novel protein complex of the thylakoid membrane . On the basis of 1) the conserved overlap between psbG and ndhC, a chloroplast gene which shows significant homology to a mitochondrial gene that codes for a subunit of the NADH-ubiquinone oxidoreductase of mitochondria, and 2) sequence similarity between the psbG gene product and the ndh gene product of E . coli, which codes for a respiratory NADH dehydrogenase, we propose that this ill-defined complex functions as a NADH or NADPH-plastoquinone oxidoreductase.

J Biol Chem, 1989 Aug 25, 264(24), 14090 - 4
Evidence for the physiological importance of the phosphotransfer between the two regulatory components, EnvZ and OmpR, in osmoregulation in Escherichia coli; Aiba H et al.; Previously, the transfer of the phosphoryl group between the EnvZ and OmpR proteins, which are involved in activation of the ompF and ompC genes in response to the medium osmolarity, has been demonstrated in vitro . In this study, we characterized mutant EnvZ and OmpR proteins in terms of their in vitro phosphorylation and dephosphorylation . The proteins isolated from the mutants, envZ11 and ompR3, were found to be defective in seemingly the same aspect, i.e . OmpR dephosphorylation . The protein isolated from the ompR77 mutant, which is a suppressor mutant specific for envZ11, was found to be defective in another aspect, i.e . OmpR phosphorylation . These results imply that the phosphotransfer reactions observed in vitro play roles in the mechanism underlying the osmoregulatory expression of the ompF and ompC genes in vivo . We provide evidence that the EnvZ protein is involved not only in OmpR phosphorylation but also in OmpR dephosphorylation.

J Biol Chem, 1989 Aug 25, 264(24), 14065 - 70
A single amino acid substitution in B subunit of Escherichia coli enterotoxin affects its oligomer formation; Iida T et al.; We isolated a mutant strain of enterotoxigenic Escherichia coli by nitrosoguanidine mutagenesis, which produces an immunologically altered B subunit of heat-labile enterotoxin . This mutant B subunit was detected as a monomer on sodium dodecyl sulfate-polyacrylamide gel electrophoresis even without prior heating, suggesting a problem in oligomer formation . Furthermore, this mutant B subunit could not form holotoxin with the native A subunit, and the affinity to GM1-ganglioside receptor was 10-fold lower than that of the native B subunit . The amino acid sequence analysis of this mutant B subunit revealed only one amino acid substitution compared with the native B subunit, at the 64th position from the N terminus (valine instead of alanine) . These data suggest that the alanine at position 64 from the N terminus is important for the native B subunit to form an oligomer structure and express its functions.

J Biol Chem, 1989 Aug 25, 264(24), 13967 - 70
Selective inhibition of protein disulfide isomerase by estrogens; Tsibris JC et al.; Protein disulfide isomerase (PDI) is a multifunctional microsomal enzyme that participates in the formation of protein disulfide bonds . PDI catalyzes the reduction of protein disulfide bonds in the presence of excess reduced glutathione and has been implicated in the reductive degradation of insulin; E . coli thioredoxin is homologous to two regions in PDI and can also degrade insulin . PDI activity, measured by 125I-insulin degradation or reactivation of randomly oxidized RNase in the presence of reduced glutathione, is non-competitively inhibited by estrogens; half-maximal inhibition was observed at approximately 100 nM estrogen . Other steroid hormones at 1 microM had little or no effect . PDI segment 120-163 (which corresponds to exon 3 of the PDI gene) and 182-230 have significant similarity with estrogen receptor segments 350-392 and 304-349, respectively, located in the estrogen binding domain but not with the steroid domains of the progesterone and glucocorticoid receptors or with thioredoxin, which is insensitive to estrogens . We propose the hypothesis that enzymes can acquire sensitivity to a hormone via exon shuffling to the enzyme gene from the DNA region coding for the hormone binding domain of the hormone's receptor.

Nucleic Acids Res, 1989 Aug 25, 17(16), 6515 - 22
Tet repressor binding induced curvature of tet operator DNA; Tovar K et al.; Tet repressor dimer binds to two tet operator sites spaced by 30 bp in the Tn10 encoded tet regulatory DNA . The effect of repressor binding on the gel mobility of circular permutated DNA fragments containing either one or both operator sequences is reported . The EcoRI induced bending of DNA is used to compare the results with other protein binding induced structural perturbations of DNA . Tet repressor bends a DNA fragment with a single tet operator to an angle of 42 degrees +/- 7 degrees . The apparent bend angle of DNA fragments containing the tandem tet operator arrangement occupied by two Tet repressor dimers turns out to be 52 degrees +/- 9 degrees . These results are interpreted with respect to the end to end distances of the bent DNA fragments . They indicate that either the intervening tet regulatory DNA between the operators or the bound operator sequences themselves contain additional perturbations from the canonical B-DNA structure . This finding is discussed in the light of previously obtained results from CD, neutron scattering, and electrooptical studies.

J Biol Chem, 1989 Aug 25, 264(24), 14531 - 42
The Escherichia coli primosome can translocate actively in either direction along a DNA strand; Lee MS et al.; The primosome is a mobile multiprotein DNA replication-priming apparatus that requires seven Escherichia coli proteins (replication factor Y (protein n'), proteins n and n", and the products of the dnaB, dnaC, dnaT, and dnaG genes) for assembly at a specific site (termed a primosome assembly site) on single-stranded DNA binding protein-coated single-stranded DNA . Two of the protein components of the primosome have intrinsic DNA helicase activity . The DNA B protein acts in the 5'----3' direction, whereas factor Y acts in the 3'----5' direction . The primosome complex has DNA helicase activity when present at a replication fork in conjunction with the DNA polymerase III holoenzyme . In this report, evidence is presented that the multiprotein primosome per se can act as a DNA helicase in the absence of the DNA polymerase III holoenzyme . The primosome DNA helicase activity can be manifested in either direction along the DNA strand . The directionality of the primosome DNA helicase activity is modulated by the concentration and type of nucleoside triphosphate present in the reaction mixture . This DNA helicase activity requires all the preprimosomal proteins (the primosomal proteins minus the dnaG-encoded primase) . Preprimosome complexes must assemble at a primosome assembly site in order to be loaded onto the single-stranded DNA and act subsequently as a DNA helicase . The 5'----3' primosome DNA helicase activity requires a 3' single-stranded tail on the fragment to be displaced, while the 3'----5' activity does not require a 5' single-stranded tail on the fragment to be displaced . Multienzyme preprimosomes moving in either direction are capable of associating with the primase to form complete primosomes that can synthesize RNA primers.

J Biol Chem, 1989 Aug 25, 264(24), 14389 - 95
Cloning and expression of the yeast plasma membrane ATPase in Escherichia coli; Holzer KP et al.; The yeast plasma membrane ATPase gene PMA1 was cloned into Escherichia coli using the high expression tac and T7 promoters . The gene product is toxic to the bacterial cell leading to very low expression levels and arrested growth of the host cell within minutes of induction . The expressed protein is immunologically cross-reactive with the yeast ATPase, comigrates with the original protein in sodium dodecyl sulfate-polyacrylamide gels, and is isolated in the E . coli membrane fraction . The partially purified protein exhibits ATPase activity.

Cell, 1989 Aug 25, 58(4), 741 - 53
The proline-rich transcriptional activator of CTF/NF-I is distinct from the replication and DNA binding domain; Mermod N et al.; Human CTF/NF-I consists of a family of CCAAT box binding proteins that activate both transcription and DNA replication . Analysis of cDNA mutants expressed in E . coli and Drosophila cells reveals that the N-terminal portion of CTF-1 is sufficient for site-specific DNA recognition, protein dimerization, and adenovirus replication . In contrast, transcriptional activation requires an additional C-terminal domain . Furthermore, this transcription domain efficiently activates a heterologous promoter, such as SV40, when fused to the DNA binding domain of Sp1 . The CTF C-terminal region consists of an unusual type of transcriptional activation domain containing approximately 25% proline residues . We propose that this proline-rich domain represents a novel class of activators which are distinct from those containing either acidic or glutamine-rich activation motifs . This indicates that transcriptional activation is likely to be mediated by several different mechanisms . In addition, these results suggest that the interactions, and consequently the mechanisms, governing transcriptional activation by CTF are distinct from those mediating DNA replication.

J Biol Chem, 1989 Aug 25, 264(24), 13975 - 8
Co-expression of the subunits of the heterodimer of HIV-1 reverse transcriptase in Escherichia coli; Muller B et al.; Expression of the 66-kDa form of human immunodeficiency virus, type 1 reverse transcriptase in Escherichia coli leads to isolation of small amounts of a 2 x 66-kDa homodimer and larger amounts of a heterodimer form of the enzyme in which the 66-kDa protein is complexed with its carboxyl-terminally truncated is complexed with its carboxyl-terminally truncated 51-kDa form . The latter arises via proteolysis by contaminating proteases . The heterodimer, which was characterized by gel filtration (apparent native molecular mass of 120-130 kDa), was the most active form of the enzyme (specific activity, 5000 units/mg, cf . less than 2000 for the 66-kDa fragment) . The 66-kDa fragment alone was shown to be only partially dimerized, with the activity residing mainly in the dimer fraction . Proteolysis of the 66-kDa form resulting partially in the 51-kDa form led to an increase in reverse transcriptase activity . Expression of a truncated version of the protein containing the first 428 amino acids of the reverse transcriptase coding region led to a protein which had low but measurable reverse transcriptase activity (400-500 units/mg) . Co-expression of the two proteins on a single plasmid led to expression in a 1:1 ratio . The 1:1 mixture behaved as a heterodimer, as shown by its chromatographic properties . It is likely that the mechanism for the production of heterodimers in vivo involves cleavage of 66-kDa monomers followed by rapid dimerization of the 51- and 66-kDa forms to give the heterodimeric form, which is stable toward further proteolysis.

Biochemistry, 1989 Aug 22, 28(17), 7088 - 97
A proton nuclear magnetic resonance assignment and secondary structure determination of recombinant human thioredoxin; Forman-Kay JD et al.; Two-dimensional 1H NMR spectroscopy has been applied to a structural analysis of the reduced form of a recombinant human thioredoxin, a ubiquitous dithiol oxidoreductase recently isolated from an immunocompetent lymphoblastoid cell line . The sequential assignment of the spectrum, including all proline residues, has been accomplished by using experiments to demonstrate through-bond and through-space connectivities . The secondary structure has been determined by a qualitative interpretation of nuclear Overhauser effects, NH exchange data, and 3JHN alpha coupling constants . The secondary structure was found to be similar to that of the X-ray structure of Escherichia coli thioredoxin, consisting of a mixed five-stranded beta-sheet surrounded by four alpha-helices . The assignment and structural characterization of human thioredoxin was facilitated by the increased resolution and sensitivity afforded by a magnetic field strength of 600 MHz and required the use of two temperatures and two pH conditions to resolve ambiguities caused by a duplication of resonances . This duplication, extending from Phe-41 to Val-59, and including Lys-3-Ile-5, Val-24, Val-25, Asn-39, and Ile-101-Glu-103, appears to be due to heterogeneity arising from the presence or absence of the N-terminal methionine.

Biochemistry, 1989 Aug 22, 28(17), 7074 - 87
Assignment of the proton NMR spectrum of reduced and oxidized thioredoxin: sequence-specific assignments, secondary structure, and global fold; Dyson HJ et al.; Complete proton assignments are reported for the 1H nuclear magnetic resonance (NMR) spectrum of Escherichia coli thioredoxin in the oxidized (with active-site disulfide bridge) and reduced (with two sulfhydryl groups) states . The assignments were obtained by using an integrated assignment strategy in which spin systems were identified from a combination of relayed and multiple quantum NMR techniques prior to sequential assignment . Elements of secondary structure were identified in each protein from characteristic nuclear Overhauser effects (NOE), coupling constants, and slowly exchanging amide protons . In both oxidized and reduced thioredoxin, approximately 33% of the 108 amino acid residues participate in a beta-sheet containing four major strands (three antiparallel and one parallel) . A further short beta-strand is connected in a parallel fashion at the N-terminal end of the sheet . Two of the antiparallel beta-strands are connected by a 7-residue beta-bulge loop . Three helical segments, also containing approximately 33% of the amino acid residues, are well-defined in both oxidized and reduced thioredoxin . The remaining third of the molecule apparently consists of reverse turns and loops with little defined secondary structure . The global folds of oxidized and reduced thioredoxin are shown to be essentially identical . Both NOE connectivities and chemical shift values for the two proteins are very similar, except in the immediate vicinity of the active site where significant variations in the chemical shift indicate subtle conformational changes . While the overall fold of oxidized thioredoxin is the same in solution and in the crystalline state, some small differences in local conformation are apparent.

Biochemistry, 1989 Aug 22, 28(17), 6800 - 4
Evidence that a major determinant for the identity of a transfer RNA is conserved in evolution; Hou YM et al.; We observed recently that a single G3.U70 base pair in the amino acid acceptor stem of an Escherichia coli alanine tRNA is a major determinant for its identity . Inspection of tRNA sequences shows that G3.U70 is unique to alanine in E . coli and is present in eucaryotic cytoplasmic alanine tRNAs . We show here that single nucleotide changes of G3.U70 to A3.U70 or to G3.C70 eliminate in vitro aminoacylation of an insect and of a human alanine tRNA by the respective homologous synthetase . Compared to the influence of G3.U70, other sequence variations in tRNAAla have a relatively small effect on aminoacylation by the insect and human enzymes . In addition, while these eucaryotic tRNAs have nucleotide differences from E . coli alanine tRNA, they are heterologously charged only with alanine when expressed in E . coli . The results indicate a functional role for G3.U70 that is conserved in evolution . They also suggest that the sequence differences between E . coli and the eucaryotic alanine tRNAs at sites other than the conserved G3.U70 do not create major determinants for recognition by any other bacterial enzyme.

Biochemistry, 1989 Aug 22, 28(17), 6914 - 24
Escherichia coli cAMP receptor protein: evidence for three protein conformational states with different promoter binding affinities; Heyduk T et al.; Cyclic AMP receptor protein (CRP) from Escherichia coli is assumed to exist in two states, namely, those represented by the free protein and that of the ligand-protein complex . To establish a quantitative structure-function relation between cAMP binding and the cAMP-induced conformational changes in the receptor, protein conformational change was quantitated as a function of cAMP concentration up to 10 mM . The protein conformation was monitored by four different methods at pH 7.8 and 23 degrees C, namely, rate of proteolytic digestion by subtilisin, rate of chemical modification of Cys-178, tryptophan fluorescence, and fluorescence of the extrinsic fluorescence probe 8-anilino-1-naphthalenesulfonic acid (ANS) . Each of these techniques reveals a biphasic dependence of protein conformation on cAMP concentration . At low cAMP concentrations ranging from 0 to 200 microM, the rates of proteolytic digestion and that of Cys-178 modification increase, whereas the fluorescence intensity of the ANS-protein complex is quenched, and there is no change in the fluorescence intensity of the tryptophan residues in the protein . At higher cAMP concentrations, the rates of proteolytic and chemical modification of the protein decrease, while the fluorescence intensity of the ANS-protein complex is further quenched but there is an increase in the intensity of tryptophan fluorescence . These results show unequivocally that there are at least three conformational states of the protein . The association constants for the formation of CRP-cAMP and CRP-(cAMP)2 complexes derived from conformational studies are in good agreement with those determined by equilibrium dialysis, nonequilibrium dialysis, and ultrafiltration . Therefore, the simplest explanation would be that the protein exhibits three conformational states, free CRP and two cAMP-dependent states, which correspond to the CRP-cAMP and CRP-(cAMP)2 complexes . The binding properties of CRP-cAMP and CRP-(cAMP)2 to the lac promoter were studied by using the gel retardation technique . At a high concentration of cAMP which favors the formation of the CRP-(cAMP)2 complex, binding of the protein to DNA is decreased . This, together with conformational data, strongly suggests that only the CRP-cAMP complex is active in specific DNA binding whereas CRP and CRP-(cAMP)2 are not.

Biochemistry, 1989 Aug 22, 28(17), 6841 - 7
Dissection of the effector-binding site and complementation studies of Escherichia coli phosphofructokinase using site-directed mutagenesis; Lau FT et al.; A systematic study by site-directed mutagenesis has been conducted on the effector site of phosphofructokinase from Escherichia coli to delineate the role of side chains in binding the allosteric activator, GDP, and inhibitor, PEP, and to search for key residues in the allosteric transtion . Target residues were identified from the crystal structure of the enzyme-nucleoside diphosphate complex . It is found that both activator and inhibitor bind to the same set of amino acid side chains . Deletion of positively charged groups (Arg21, Arg25, Arg54, Arg154, and Lys213 mutated to alanine) weakens binding of both effectors by 2-3 kcal/mol, consistent with the disruption of charged hydrogen bonds . Residue Glu187, which is known from the crystal structure to bind the coordinated Mg2+ ion of GDP, is found to have a unique behavior on mutation and appears to be crucial in triggering the allosteric transition . All other residues mutated simply weaken binding of both PEP and GDP in a parallel manner . However, mutation of Glu----Ala187 reverses the roles of GDP and PEP, causing GDP to become an allosteric inhibitor and PEP an activator . Mutation of Glu----Gln187 has only a small effect on the binding of PEP, and both PEP and GDP are inhibitors . Studies are described in which mutations in different subunits of a tetrameric complex complement each other . The effector site is composed of residues from two subunits . In particular, Arg21 and Lys213 in each site are from different subunits . Mutations of either one of these residues abolishes activation by GDP of the homotetramer.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1989 Aug 22, 28(17), 6836 - 41
Urea-induced inactivation, dissociation, and unfolding of the allosteric phosphofructokinase from Escherichia coli; Bras GL et al.; The influence of urea on the allosteric phosphofructokinase from Escherichia coli has been studied by measuring the changes in enzymatic activity, protein fluorescence, circular dichroism, and retention in size-exclusion chromatography . Tetrameric, dimeric, and monomeric forms of the protein can be discriminated by their elution from a high-performance liquid chromatography gel filtration column . Three successive steps can be detected during the urea-induced denaturation of phosphofructokinase: (i) the dissociation of the native tetramer into dimers which abolishes the activity; (ii) the dissociation of dimers into monomers which exposes the unique tryptophan, Trp-311, to the aqueous solvent; (iii) the unfolding of the monomers which disrupts most of the secondary structure . This pathway involves the ordered dissociation of the interfaces between subunits and supports a previous hypothesis (Deville-Bonne et al., 1989) . Phosphofructokinase can be quantitatively renatured from urea solutions, provided that precautions are taken to avoid the aggregation of one insoluble monomeric state . The renaturation of phosphofructokinase from urea implies three steps: an initial folding reaction within the monomeric state is followed by two successive association steps . The faster association step restores the native fluorescence, and the slower regenerates the active enzyme . The renaturation and denaturation of phosphofructokinase correspond to the complex pathway: tetramer in equilibrium dimer in equilibrium folded monomer in equilibrium unfolded monomer . It is found that the subunit interface which forms the regulatory site is more stable and associates 40 times more rapidly than the subunit interface which forms the active site.

Biochemistry, 1989 Aug 22, 28(17), 6827 - 35
Subcloning, expression, and purification of the enterobactin biosynthetic enzyme 2,3-dihydroxybenzoate-AMP ligase: demonstration of enzyme-bound (2,3-dihydroxybenzoyl)adenylate product; Rusnak F et al.; The gene coding for the enzyme 2,3-dihydroxybenzoate-AMP ligase (2,3DHB-AMP ligase), responsible for activating 2,3-dihydroxybenzoic acid in the biosynthesis of the siderophore enterobactin, has been subcloned into the multicopy plasmid pKK223-3 and overproduced in a strain of Escherichia coli . The protein is an alpha 2 dimer with subunit molecular mass of 59 kDa . The enzyme catalyzes the exchange of {32P}pyrophosphate with ATP, dependent upon aromatic substrate with a turnover number of 340 min-1 . The enzyme also releases pyrophosphate upon incubation with 2,3-dihydroxybenzoic acid and ATP; an initial burst corresponding to 0.7 nmol of pyrophosphate released per nanomole of enzyme is followed by a slower, continuous release with a turnover number of 0.41 min-1 . The 1000-fold difference in rates observed between ATP-pyrophosphate exchange and continuous pyrophosphate release, as well as the close to stoichiometric amount of pyrophosphate released, suggests that intermediates are accumulating on the enzyme surface . Such intermediates have been observed and correspond to enzyme-bond (2,3-dihydroxybenzoyl)adenylate product.

J Mol Biol, 1989 Aug 20, 208(4), 701 - 7
Biological consequences of a reduction in the non-target DNA scanning capacity of a DNA repair enzyme; Dowd DR et al.; Numerous DNA-interactive proteins have been shown to locate specific sequences within large domains of non-target DNA in vitro and in vivo by a one-dimensional diffusion mechanism; however, the biological significance of this process has not been evaluated . We have examined the biological consequences of sliding for the pyrimidine dimer-specific DNA repair enzyme T4 endonuclease V, an enzyme which scans non-target DNA both in vitro and in vivo . An endonuclease V mutant was constructed whose only altered biochemical characteristic, measured in vitro, was a loss in its ability to slide on non-target DNA . In contrast to the native enzyme, when the mutated endonuclease V was expressed in DNA repair-deficient Escherichia coli, no enhanced ultraviolet survival was conferred . These results suggest that the mechanisms which DNA-interactive proteins employ to enhance the probability of locating their target sequences are of significant biological importance.

J Mol Biol, 1989 Aug 20, 208(4), 567 - 74
Regulation of IS1 transposition by the insA gene product; Machida C et al.; The IS1 element contains two adjacent genes called insA and insB, both required for IS1 transposition and IS1-mediated plasmid cointegration . These two genes are transcribed polycistronically from the promoter in the left terminal inverted repeat of IS1 (insL) . We constructed overexpression systems of these genes with the tac promoter, which are regulated by an exogenous inducer, isopropyl-beta-D-thiogalactopyranoside (IPTG) . Then we have examined, under various conditions of induction with IPTG, how overexpression of these genes affects IS1 transposition, using an assay based on plasmid cointegration . When the insA and insB genes were organized identically to the wild-type IS1 genes and simultaneously expressed using low concentrations of IPTG, activity of a mutant IS1 in cis was restored, but not in trans . Higher IPTG concentrations resulted in lower transposition activity . Expression in trans of insA and insB results in a 50 to 100-fold reduction of the frequency of cointegration mediated by wild-type IS1 . Such a reduction is also observed when only the insA gene is overexpressed in trans . Overexpression of either mutant insA or insB does not affect the cointegration event . Tests with the insA-lacZ fusion gene showed that the InsA product inhibits the expression of IS1 genes directed by its own promoter in insL . These results suggest that the InsA product regulates IS1 transposition by inhibiting expression of IS1 transposition genes in addition to acting as part of a transposase complex.

J Mol Biol, 1989 Aug 20, 208(4), 623 - 33
Reductive acetylation of tandemly repeated lipoyl domains in the pyruvate dehydrogenase multienzyme complex of Escherichia coli is random order; Allen AG et al.; In vitro deletion and site-directed mutagenesis of the aceF gene of Escherichia coli was used to generate dihydrolipoamide acetyltransferase (E2p) polypeptide chains containing various permutations and combinations of functional and non-functional lipoyl domains . A lipoyl domain was rendered non-functional by converting the lipoylatable lysine residue to glutamine . Two- and three-lipoyl domain E2p chains, with lipoyl-lysine (Lys244) substituted by glutamine in the innermost lipoyl domains (designated +/- and +/+/-, respectively), and similar chains with lipoyl-lysine (Lys143) substituted by glutamine in the outer lipoyl domains (designated -/+ and -/-/+), were constructed . In all instances, pyruvate dehydrogenase complexes were assembled in vivo around E2p cores composed of the modified peptide chains . All the complexes were essentially fully active in catalysis, although the complex containing the -/-/+ version of the E2p polypeptide chain showed a 50% reduction in specific catalytic activity . Similarly, active-site coupling in the complexes containing the +/-, +/+/- and -/+ constructions of the E2p chains was not significantly different from that achieved by the wild-type complex . However, active-site coupling in the complex containing the -/-/+ version of the E2p chain was slightly impaired, consistent with the reduced overall complex activity . These results indicate that during oxidative decarboxylation there is no mandatory order of reductive acetylation of repeated lipoyl domains within E2p polypeptide chains, and strongly suggest that the three tandemly repeated lipoyl domains in the wild-type E2p chain function independently in the pyruvate dehydrogenase complex.

J Mol Biol, 1989 Aug 20, 208(4), 575 - 86
Differentially expressed trmD ribosomal protein operon of Escherichia coli is transcribed as a single polycistronic mRNA species; Bystrom AS et al.; The trmD operon is a four-cistron operon in which the first and fourth genes encode ribosomal proteins S16 (rpsP) and L19 (rplS), respectively . The second gene encodes a 21,000 Mr polypeptide of unknown function and the third gene (trmD) encodes the enzyme tRNA(m1G37)methyltransferase, which catalyzes the formation of 1-methylguanosine (m1G) next to the 3' end of the anticodon (position 37) of some tRNAs in Escherichia coli . Here we show under all regulatory conditions studied, transcription initiates at one unique site, and the entire operon is transcribed into one polycistronic mRNA . Between the promoter and the first gene, rpsP, an attenuator-like structure is found (delta G = -18 kcal; 1 cal = 4.184 J), followed by four uridine residues . This structure is functional in vitro, and terminates more than two-thirds of the transcripts . The different parts of the trmD operon mRNA decay at a uniform rate . The stability of the trmD mRNA is not reduced with decreasing growth rate, which is in contrast to what has been found for other ribosomal protein mRNAs . Furthermore, earlier experiments have shown the existence of differential expression as well as non-co-ordinate regulation within the operon . Our results are consistent with the regulation of the trmD operon being due to some mechanism(s) operating at the post-transcriptional level, and do not involve differential degradation of different mRNA segments, internal promoters or internal terminators.

Vet Rec, 1989 Aug 19, 125(8), 181 - 4
Cystogastrostomy in the treatment of pancreatic pseudocyst/abscess in two dogs; Bellenger CR et al.; Two dogs with pancreatic pseudocyst or pancreatic abscess formation were treated by transgastric cystogastrostomy . In each case drainage of the cyst/abscess cavity into the stomach was followed by resolution of the primary lesion . One dog succumbed to an E coli bronchopneumonia after infection of the deep venous line used for total parenteral nutrition . The second dog recovered despite requiring additional surgery for biliary obstruction . Both dogs required intensive care during and after the operation.

Science, 1989 Aug 18, 245(4919), 738 - 40
Identification by ENDOR of Trp191 as the free-radical site in cytochrome c peroxidase compound ES; Sivaraja M et al.; The chemical identity of the amino acid free-radical site that represents one of the two oxidizing equivalents stored in the H2O2-oxidized intermediate (compound ES) of the mitochondrial heme enzyme, cytochrome c peroxidase (CcP) has been sought for almost a quarter of a century . Site-directed mutagenesis alone cannot yield this answer . Low-temperature 35-gigahertz (Q-band) electron nuclear double resonance (ENDOR) spectroscopy was used to examine compound ES prepared from proteins containing specifically deuterated methionine or tryptophan, as well as the amino acid replacement Trp51----Phe . The results definitely identify the site of the radical in compound ES as tryptophan, most likely Trp191.

Mol Cell Biochem, 1989 Aug 15, 89(1), 29 - 35
Phosphorylation of human interleukin-2 (IL-2); Kung HF et al.; Human interleukin-2 (IL-2) is a lymphokine which is capable of activating lymphocytes and supporting the long-term in vitro growth of activated T cell clones . Recombinant human IL-2, expressed in either E . coli or cos cells, was shown to be phosphorylated by protein kinase C . Phosphorylated IL-2 synthesized in E . coli was analyzed by SDS-PAGE, reverse phase HPLC, and tryptic peptide mapping . The phosphorylated tryptic peptide was identified as the N-terminal fragment containing a single phosphorylation site at the serine residue at position 7 . There was no difference in biological activity between non-phosphorylated and phosphorylated IL-2, as determined by a T cell growth assay . Although the physiological role of phosphorylation of IL-2 is unclear, IL-2 can be labeled with {gamma-32P} ATP and protein kinase C to a high specific radioactivity, and the synthesis of biologically active 32p-labeled IL-2 may be useful for receptor-binding studies of the cells containing low level of phosphoprotein phosphotases.

J Biol Chem, 1989 Aug 15, 264(23), 13751 - 9
Purification, characterization, and transcriptional analyses of RNA polymerases from Rhodobacter sphaeroides cells grown chemoheterotrophically and photoheterotrophically; Kansy JW et al.; RNA polymerase was purified from Rhodobacter sphaeroides cells grown both chemoheterotrophically and photoheterotrophically . Both preparations of polymerase were comprised of five major subunits designated: beta' (160,000 Da), beta (150,000 Da), sigma (93,000 Da), alpha (41,000 Da), and a 35,000-Da protein, designated epsilon . All five subunits of the polymerase isolated from photoheterotrophically grown cells were found to be serologically related to the major subunits (beta', beta, sigma, and alpha) of the RNA polymerase from Escherichia coli; however, only four of the subunits of the RNA polymerase isolated from chemoheterotrophically grown cells were serologically related to the four major E . coli RNA polymerase subunits . The enzyme isolated from photoheterotrophically grown cells had a lower specific activity and was considerably less stable than the enzyme isolated from chemoheterotrophically grown cells . However, RNA synthesis by both enzyme preparations was dependent upon the presence of DNA template and MgCl2, and the RNA synthetic activity was inhibited by rifampicin . The transcriptional activities of both samples of polymerase were studied using different DNA templates, and the sizes of the run-off products compared to expected values obtained from analysis of mRNA's produced in vivo . These results are discussed in light of DNA sequences expected to contain promoter-like regions as determined from in vivo studies.

Biochem Biophys Res Commun, 1989 Aug 15, 162(3), 1425 - 30
Design and chemical synthesis of a 32 residues chimeric microprotein inhibiting both trypsin and carboxypeptidase A; Le-Nguyen D et al.; A chimeric peptide, 32 amino acids long, bearing two active sites, one inhibiting trypsin (Kd = 1.8 10(-9) M), one carboxypeptidase A (Kd = 3 10(-9) M), was designed and synthesized . It is a "squash inhibitor" (EETI II, 28 amino acids) elongated with the 4 amino acids from the C-terminus of the potato carboxypeptidase inhibitor . It has 3 disulfide bridges assembled in the particular knotted topology shared by the two inhibitors, by conotoxin omega, and possibly by E . coli enterotoxin ST1b.

Gene, 1989 Aug 15, 80(2), 305 - 14
Cloning and expression of a collagen-analog-encoding synthetic gene in Escherichia coli; Goldberg I et al.; A family of totally synthetic genes coding for multiple tandem repeats of the amino acid sequence (Gly-Pro-Pro) has been prepared and inserted into the ClaI cloning site of the expression vector pJL6 . A representative recombinant plasmid, pAC1, with an insert of about 340 bp was established in an Escherichia coli strain bearing a defective lambda prophage, to study expression of the CII-collagen analog fusion protein produced from pAC1 upon heat induction . Authentic fusion protein production was demonstrated by nucleotide sequencing, Northern-blot analysis, and in vivo synthesis . Conversion of a wild-type rpoH allele to the rpoH165 mutation was shown to suppress proteolysis of the unstable fusion protein.

Eur J Biochem, 1989 Aug 15, 183(3), 545 - 53
NMR studies of the Escherichia coli trp aporepressor . Sequence-specific assignment of the aromatic proton resonances; Hyde EI et al.; The resonances in the aromatic region of the 1H-NMR spectrum of the Escherichia coli trp aporepressor have been assigned to amino acid type by two-dimensional correlated spectroscopy (COSY), homonuclear Hartmann-Hahn (HOHAHA) spectroscopy and nuclear Overhauser enhancement spectroscopy (NOESY) techniques and studies of the pH dependence of the chemical shifts, in combination with selective deuteration of the protein . Complete sequence-specific assignments of the aromatic resonances have been made by comparing the observed inter-residue NOEs with those expected on the basis of the crystal structure of the protein {Zhang, R.-G., Joachimiak, A., Lawson, C.L., Shevitz, R.W., Otwinowski, Z . & Sigler, P.B . (1987) Nature 327, 591-597} . The latter experiments have also permitted the sequence-specific assignment of some of the high-field methyl resonances . The complete assignment of the aromatic region of the spectrum, in particular of resonances from residues at the dimer interface, opens the way to detailed studies of the conformational effects of corepressor and operator binding.

Eur J Biochem, 1989 Aug 15, 183(3), 519 - 28
Carbamoyl phosphate biosynthesis and partition in pyrimidine and arginine pathways of Escherichia coli . In situ properties of carbamoyl-phosphate synthase, ornithine transcarbamylase and aspartate transcarbamylase in permeabilized cells; Robin JP et al.; A procedure for the permeabilization of Escherichia coli cells was adapted to the in situ determination of the catalytic and regulatory properties of the enzymes responsible for the biosynthesis of carbamoyl phosphate and its utilization in the pyrimidine and arginine pathways . Differences in enzyme sensitivity to effectors and changes in pH dependence were observed . Partition of carbamoyl phosphate in the two metabolic pathways could be measured under conditions of substrate saturation . The results obtained will allow to test experimentally the theoretical predictions made by A . Goldbeter (1973) PhD thesis, Universite Libre de Bruxelles, on the distribution of carbamoyl phosphate and the oscillation of its intracellular concentration.

Biochem Biophys Res Commun, 1989 Aug 15, 162(3), 1376 - 81
A specific inhibition of induction of adaptive response by o-vanillin, a potent comutagen; Takahashi K et al.; The present study revealed that potent comutagenic activity of o-vanillin in mutagenesis by N-methyl-N-nitrosourea (MNU) is a consequence of inhibition of the transcriptional promoting capacity of methylated O6-methylguanine-DNA methyltransferase (MGTase) . As evidence of this, in the presence of o-vanillin, there were (i) dose-dependent decreases in MGTase induced in E . coli exposed to MNU, (ii) inhibition of ada gene promotion determined in the ada'-lacZ' assay system, and (iii) minimal inhibition of the methyl transfer capacity of MGTase in a subcellular system.

Biochem Biophys Res Commun, 1989 Aug 15, 162(3), 1025 - 9
Can ribozymes be used to regulate procaryote gene expression?
Chuat JC, Galibert F.
The in vivo activity of ribozymes designed against mRNA coding for E . coli beta-galactosidase was tested both in intramolecular and in intermolecular conditions . When recombinant M13 phage DNA carrying on the same molecule the information for both the ribozyme and the target was transfected into bacterial cells, ribozyme activity was observed . Conversely, a ribozyme coded by a recombinant M13 vector, but targeted against an mRNA transcribed from the F episome including the remaining part of the beta-galactosidase gene, was inefficient.

J Immunol Methods, 1989 Aug 15, 122(1), 25 - 32
A new tool for the serodiagnosis of acute Plasmodium falciparum malaria in individuals with primary infection; Fruh K et al.; We have developed an ELISA which detects, with high specificity, antibodies against a major surface protein of P . falciparum merozoites which is a processing product of the precursor glycoprotein gp190 . This assay can be used in the diagnosis of acute malaria in individuals with primary infection . Two partial sequences of gp190 were expressed in E . coli as beta-galactosidase (beta-Gal) fusion proteins . The same sequences fused to chloramphenicol acetyltransferase (CAT) or mouse dihydrofolate reductase (DHFR) react with high frequency when sera of acute malaria patients are analyzed in immunoblots . Antibodies from such sera crosslink, via their antigen binding sites, the beta-Gal fusions to the corresponding CAT or DHFR fusions adsorbed to a solid phase as demonstrated by the captured beta-Gal activity . The assay is highly specific, shows extremely low cut off values and should therefore be widely applicable.

J Biol Chem, 1989 Aug 15, 264(23), 13888 - 95
Cloning and sequence analysis of the Escherichia coli metH gene encoding cobalamin-dependent methionine synthase and isolation of a tryptic fragment containing the cobalamin-binding domain; Banerjee RV et al.; A gene encoding cobalamin-dependent methionine synthase (EC 2.1.1.13) has been isolated from a plasmid library of Escherichia coli K-12 DNA by complementation to methionine prototrophy in an E . coli strain lacking both cobalamin-dependent and -independent methionine synthase activities (RK4536:metE, metHH) . Maxicell expression of a series of plasmids containing deletions in the metH structural gene was employed to map the position and orientation of the gene on the cloned DNA fragment . A 6.3-kilobase EcoRI-SalI fragment containing the gene was cloned into the sequencing vector pGEM3B for double-stranded DNA sequencing; the MetH coding region consists of 3372 nucleotides . The enzyme was purified from an overproducing strain of E . coli harboring the recombinant plasmid, in which the level of methionine synthase was elevated 30- to 40-fold over wild-type E . coli . Recombinant enzyme is a protein of 123,640 molecular weight and has a turnover number of 1,450 min-1 in the standard assay . These values are to be compared with previously reported values of 133,000 for the molecular weight and 1,240-1,560 min-1 for the turnover number of the homogenous enzyme purified from a wild-type strain of E . coli B (Frasca, V., Banerjee, R . V., Dunham, W . R., Sands, R . H., and Matthews, R . G . (1988) Biochemistry 27, 8458-8465) . Limited proteolysis of the native enzyme with trypsin resulted in loss of enzyme activity but retention of bound cobalamin on a peptide fragment of 28,000 molecular weight . This fragment has been shown to extend from residue 643 to residue 900 of the 1124-residue deduced amino acid sequence.

J Biol Chem, 1989 Aug 15, 264(23), 13880 - 7
Purification and properties of Methanobacterium thermoautotrophicum DNA photolyase; Kiener A et al.; We have purified DNA photolyase from the autotrophic anaerobic archaebacterium Methanobacterium thermoautotrophicum to near homogeneity by a two-column affinity chromatography . The purified enzyme has an Mr = 60,000 and shows near UV absorption peak at 440 nm and a fluorescence emission maximum at 462 nm indicating that it contains 8-hydroxy-5-deazaflavin (coenzyme F420) as an intrinsic chromophore . The photolyase binds with high specificity to thymine dimer in DNA with an equilibrium binding constant, KA = 1.4 x 10(9) M-1, and a dissociation rate constant, koff = 1.4 x 10(-4) s-1 (t1/2 = 43 min) . Despite 6-fold higher affinity compared to the folate-containing Escherichia coli photolyase the two enzymes apparently contact the same phosphates around the thymine dimer: the phosphate immediately 5' and the three phosphates immediately 3' to the dimer on the damaged strand and the phosphate across from the dimer in the minor groove on the complementary strand . The absolute action spectrum of the Methanobacterium photolyase in the 400-500-nm region closely matches the absorption of the enzyme-bound F420 . The quantum yield (phi) over this region is constant and is approximately 0.2 . The value is measurably smaller than the quantum yields reported for other DNA photolyases.

J Biol Chem, 1989 Aug 15, 264(23), 13599 - 604
Fumarate reductase mutants of Escherichia coli that lack covalently bound flavin; Blaut M et al.; Menaquinol-fumarate oxidoreductase of Escherichia coli is a four-subunit membrane-bound complex that catalyzes the final step in anaerobic respiration when fumarate is the terminal electron acceptor . The catalytic domain of fumarate reductase consists of the FrdA subunit, which contains the active site, and a FAD prosthetic group covalently attached to His44, plus the FrdB subunit which contains at least two of the three nonidentical iron-sulfur clusters of the enzyme . To examine the role of covalently bound FAD in enzyme activity and electron transfer during anaerobic cell growth, site-directed mutagenesis was used to alter His44 of the FrdA subunit to a Ser, Cys, or Tyr residue . The resulting mutant enzyme complexes that were synthesized associated normally with the cytoplasmic membrane, but had decreased ability (greater than 70%) to reduce fumarate with reduced benzyl viologen, an artificial electron donor of low redox potential (Em = -359 mV; Clark, W . M . (1972) Oxidation-Reduction Potentials of Organic Systems, Robert E . Kreiger Publishing Co., Melbourne, FL) . Even lower activities were measured when the higher potential, natural electron donor menaquinol was used, which, however, correlated with the slower growth rates of the different mutant complexes . In contrast to the normal enzyme, the mutant enzyme complexes were unable to oxidize succinate . Substitution of Arg for His44 produced a totally inactive enzyme complex that permitted no cell growth on nonfermentable substrates with fumarate as electron acceptor . All four mutant complexes contained noncovalently bound FAD in stoichiometric amounts . These data indicate a unique role of the 8 alpha-{N(3)-histidyl} FAD linkage in enzyme activity, by raising the redox potential of free FAD to permit reduction by both menaquinol and succinate.

J Biol Chem, 1989 Aug 15, 264(23), 13440 - 7
The structure of a molybdopterin precursor . Characterization of a stable, oxidized derivative; Johnson JL et al.; An oxidized pterin species, termed compound Z, has been isolated from molybdenum cofactor-deficient mutants of Escherichia coli and shown to be the direct product of oxidation of a molybdopterin precursor which accumulates in these mutants . The complete structural characterization of compound Z has been accomplished . A carbonyl function at C-1' of the 6-alkyl side chain can be reacted with 2,4-dinitrophenylhydrazine to yield a phenylhydrazone and can be reduced with borohydride, producing a mixture of two enantiomers, each with a hydroxyl group on C-1' . Compound Z contains one phosphate/pterin and no sulfur . The phosphate group is insensitive to alkaline phosphatase and to a number of phosphodiesterases but is quantitatively released as inorganic phosphate by mild acid hydrolysis . From 31P and 1H NMR of compound Z it was inferred that the phosphate is bound to C-2' and C-4' of a 4-carbon side chain, forming a 6-membered cyclic structure . Mass spectral analysis showed an MH+ ion with an exact mass of 344.0401 corresponding to the molecular formula C10H11N5O7P, confirming the proposed structure.

J Biol Chem, 1989 Aug 15, 264(23), 13381 - 2
New crystal form of recombinant murine interferon-beta; Matsuda S et al.; Although we have reported (Matsuda, S., Kawano, G., Itoh, S., Mitsui, Y., and Iitaka, Y . (1986) J . Biol . Chem . 261, 16207-16209) that recombinant murine interferon-beta produced in Escherichia coli was crystallized in an orthorhombic space group C222(1) using polyethyleneglycol 8000 as precipitant, the crystals had an insufficient resolution and a marked tendency for orientational disorder around the c axis . We now report that another form of murine interferon-beta crystals with little disorder was obtained in the presence of dioxane using ammonium sulfate as precipitant . The new crystals belong to a hexagonal space group P6(1) or P6(5) with a = b = 71.4 A and c = 79.6 A having only one murine interferon-beta molecule in an asymmetric unit . The crystals are reasonably stable to x-rays and significantly diffract up to 2.2 A resolution when a synchrotron beam is used.

Experientia, 1989 Aug 15, 45(8), 722 - 6
Thermoadaptive influence on reactivity pattern of vasopressinergic neurons in the guinea pig; Merker G et al.; In cold-adapted guinea pigs, increased amounts of arginine-vasopressin (AVP) immunoreactive material could be visualized in neurons of the supraoptic and paraventricular nucleus, in fibers projecting to the neurohypophysis and in fiber terminals in the ventral lateral septum and in the amygdala . In warm-adapted animals the reactivity to AVP antiserum was poor in all neuronal structures examined . High AVP-immunoreactivity was accompanied by a reduced febrile response to bacterial pyrogen in cold-adapted guinea pigs.

J Immunol, 1989 Aug 15, 143(4), 1175 - 82
Analysis of human IL-6 mutants expressed in Escherichia coli . Biologic activities are not affected by deletion of amino acids 1-28; Brakenhoff JP et al.; We have constructed and analyzed amino terminally deleted analogs of IL-6 . Progressively shortened variants of mature IL-6 were constructed at the cDNA level and expressed in Escherichia coli . Mutant proteins were recovered from refractile bodies by solubilizing in 6 M guanidine-HCl . The mutant protein concentration in these preparations was estimated by Western blotting by using an IL-6-specific mAb and the biologic activity was measured in the B9 (hybridoma growth factor) assay . The first 28 amino acids of mature IL-6 could be removed without significantly affecting biologic activity . A further removal of amino acids 29 and 30 resulted in an approximately 50-fold decrease, whereas removal of amino acids 31 to 34 virtually abolished the activity . The mutants showed the same reaction pattern in three other IL-6 assays: induction of murine thymocyte proliferation, induction of fibrinogen synthesis by a human hepatoma cell line (HepG2), and the induction of IgM synthesis by an EBV-transformed B cell line . This suggests that a single functional domain might be responsible for all four activities of IL-6.

Gene, 1989 Aug 15, 80(2), 269 - 78
Nucleotide sequence of the Rickettsia prowazekii ATP/ADP translocase-encoding gene; Williamson LR et al.; The Rickettsia prowazekii ATP/ADP translocase (Tlc) gene (tlc), previously cloned in Escherichia coli was localized to a 1.6-kb chromosomal fragment . Nucleotide sequence analysis of this fragment revealed an open reading frame of 1494 bp that could encode a hydrophobic protein of 497 amino acids (aa) with an Mr of 56,668 . Analysis of the deduced aa sequence revealed that it contained twelve potential membrane-spanning regions . Comparisons between the deduced aa sequence of the R . prowazekii ATP/ADP Tlc and the sequences of mitochondrial (mt) Tlc revealed no detectable homologies between the rickettsial and mt sequences . The major protein synthesized in E . coli minicells containing the rickettsial gene exhibited and Mr of approx . 34,000.

Biochem J, 1989 Aug 15, 262(1), 241 - 4
Studies on some specific Ap4A-degrading enzymes with the use of various methylene analogues of P1P4-bis-(5',5'''-adenosyl) tetraphosphate; Guranowski A et al.; Six new methylenephosphonate analogues of P1P4-bis-(5',5'''-adenosyl) tetraphosphate, Ap4A, having P2-P3 carbon bridges CF2, CCl2 and CH2CH2 or P1-P2 and P3-P4 carbon bridges CF2, CCl2 and CH2CH2 in the tetraphosphate chain, were examined as substrates or inhibitors for two specific Ap4A-degrading enzymes: (asymmetrical) Ap4A hydrolase (EC 3.6.1.17) from yellow-lupin seeds and (symmetrical) Ap4A hydrolase (EC 3.6.1.41) from Escherichia coli . All analogues in which the central oxygen atom was replaced by a stable carbon bridge were hydrolysed by the asymmetrical hydrolase (CF2 greater than CCl2 greater than O greater than CHBr greater than CH2 greater than CH2CH2) . As expected, these analogues were not hydrolysed by the symmetrical hydrolase, which was also unable to act on analogues having P1-P2 and P3-P4 carbon bridges.

Anal Biochem, 1989 Aug 15, 181(1), 66 - 74
A solid-phase extraction procedure for DNA purification; McCormick RM; The preparation and use of particulate materials for the removal of proteins from nucleic acid samples by solid-phase extraction procedures are described . The solid-phase extraction procedure is analogous to the classical phenol extraction for DNA purification, with the exception that the phenol is replaced with insoluble particulate materials that are chemically similar to phenol and thus function in an analogous manner . These particulate materials have a very high affinity for proteins and a very low affinity for nucleic acids . With these materials, it is possible to remove large quantities of proteins (i.e., tens of milligrams) from minute quantities (submicrogram) of nucleic acid and quantitatively recover the latter in a biologically active state . Compared to other procedures that are currently used to purify nucleic acids, the protocols using these materials offer the advantages of speed, quantitative DNA recovery, safety, and convenience.

Eur J Pharmacol, 1989 Aug 15, 172(3), 263 - 71
Activation of type I cyclic AMP-dependent protein kinases is impaired by a point mutation in cyclic AMP binding sites; Kuno T et al.; The type I regulatory (R-I) subunit of cyclic AMP-dependent protein kinase (A-kinase) was expressed in E . coli, and a single amino acid substitution in cyclic AMP binding sites A or B was introduced by site-directed mutagenesis . The cyclic AMP binding activity and cyclic AMP-stimulated phosphotransferase activity of the holoenzymes formed by wild-type or mutant R-Is and the purified bovine catalytic subunit of A-kinase were then examined . The wild-type holoenzyme was activated by low concentrations of cyclic AMP, a finding in accord with its high-affinity binding to cyclic AMP . In contrast, although the two mutant holoenzymes showed high-affinity cyclic AMP binding at their non-mutated sites, both holoenzymes were resistant to activation by cyclic AMP . Thus, binding of cyclic AMP to the non-mutated cyclic AMP binding site is not sufficient to dissociate the catalytic subunit from the mutant R-Is upon cyclic AMP binding . These results suggest that both A and B cyclic AMP binding sites are required for efficient coupling between cyclic AMP binding and activation of the enzyme.

J Biol Chem, 1989 Aug 15, 264(23), 13775 - 9
Mutation of the predicted ATP binding site inactivates both activities of isocitrate dehydrogenase kinase/phosphatase; Stueland CS et al.; In Escherichia coli, the reversible phosphorylation of isocitrate dehydrogenase (IDH) is catalyzed by a bifunctional protein: IDH kinase/phosphatase . Although both IDH kinase and IDH phosphatase require ATP, the amino acid sequence of IDH kinase/phosphatase contains a single sequence that matches the consensus for ATP binding sites . A mutation that converted the "invariant" lysine (residue 336) of this consensus sequence to a methionine reduced the activities of both IDH kinase and IDH phosphatase by factors of greater than 500, to levels below the detection limits of the assays . The apparent elimination of both IDH kinase and IDH phosphatase by this mutation is consistent with the proposal that these activities share a common ATP binding site and that these reactions may occur at the same active site . Although conversion of Lys336 to a methionine eliminated detectable IDH kinase activity as measured in vitro, the mutant allele retained the ability to complement an aceK deletion mutation, restoring the ability of these cells to grow on minimal acetate medium . Complementation apparently resulted because the mutant protein retained sufficient activity to phosphorylate IDH in vivo . To determine whether the enzymatic assays performed in vitro had correctly reflected the activity of the mutant protein in vivo, we measured the rates at which mutant and wild-type cultures could incorporate {32P}inorganic phosphate into IDH . The wild-type culture achieved maximal incorporation in less than 3 min . In contrast, 32P incorporation was only barely detectable after 30 min in the mutant culture, indicating that the activity of the mutant protein is, indeed, greatly reduced in vivo . The ability of the mutant allele to complement an aceK null mutation thus suggests that IDH kinase/phosphatase levels in wild-type cells are in great excess over what is required for steady-state growth on acetate medium.

Gene, 1989 Aug 15, 80(2), 345 - 51
Expression of the rat interferon-alpha 1 gene in Escherichia coli controlled by the secondary structure of the translation-initiation region; Spanjaard RA et al.; A synthetic ribosome-binding site (RBS) containing a 7-nucleotide-long Shine-Dalgarno (SD) sequence was placed ahead of the rat interferon (IFN)-alpha 1 coding region . The translational efficiency of this construct was extremely low . Structural probing of transcripts with RNases T1 and U2 combined with computer predictions revealed the presence of a stable hairpin in which the SD region was base-paired to codons 3, 4 and 5 of the IFN mRNA . Each mutation in this stem changing an A-U to an A.C or a G-C a G.U pair increased translational efficiency about fourfold and this effect could be reversed by a compensating stabilizing substitution in the other strand of the stem . We conclude that the strength of an RBS is to a major degree determined by its involvement in secondary structure . We also show that the negative effect of secondary structure on the efficiency of an RBS can be overcome by allowing upstream translation to terminate within the base-paired region . In our clones, termination-dependent restarts occur at a frequency comparable to that taking place in constructs containing destabilized hairpins.

Biochem J, 1989 Aug 15, 262(1), 119 - 24
Evidence that pyridoxal phosphate modification of lysine residues (Lys-55 and Lys-59) causes inactivation of hydroxymethylbilane synthase (porphobilinogen deaminase); Miller AD et al.; A recombinant strain of Escherichia coli has been constructed that produces approx . 200 times the amount of hydroxymethylbilane synthase found in wild-type E . coli {Hart, Abell & Battersby (1986) Biochem . J . 240, 273-276} . Enzyme purified from this strain is shown to be permanently inactivated by pyridoxal 5'-phosphate/NaB1H3(3)H1 . The inactivation is not complete despite the fact that approx . 1 mol of lysine residues is modified per mol of enzyme . Evidence is gained showing that (a) modification of one of two conserved lysine residues (Lys-55 or Lys-59) results in inactivation of hydroxymethylbilane synthase and (b) these lysine residues are present in or close to the active site.

J Immunol, 1989 Aug 15, 143(4), 1223 - 7
Endotoxin-macrophage interaction: post-translational regulation of tumor necrosis factor expression; Zuckerman SH et al.; Thioglycollate-elicited murine peritoneal macrophages produce significant quantities of TNF 2 to 4 h after induction with bacterial endotoxin, LPS . However, macrophages exposed to a second LPS stimulus are refractory and the amount of TNF detected in these supernatants is reduced 10- to 50-fold . The acquisition of the refractory state in vitro or in vivo requires the continued presence of LPS for a minimum of 6 to 8 h, is optimal by 20 h, and is reversible . Refractory macrophages incubated for an additional 48 h in the absence of LPS produce significant quantities of TNF after reexposure to endotoxin . Although LPS refractory macrophages do not release TNF in response to a secondary endotoxin challenge, riboprobe ribonuclease protection assays demonstrated amplification of TNF message, suggesting that post-transcriptional events are involved in the regulation of TNF production in endotoxin refractory macrophages . Immunoprecipitation studies revealed the accumulation of the 26-kDa TNF precursor in lysates of refractory macrophages, thus demonstrating a post-translational regulatory process . Although LPS refractory macrophages do not release TNF in response to a second LPS stimulus, ingestion of zymosan by these cells results in the release of significant quantities of TNF . Furthermore, LPS-refractory macrophages do not demonstrate a reduction in other effector functions including Fc-mediated erythrophagocytosis . Therefore, the LPS refractory state is a metabolically dependent post-translational regulatory process, which requires continuous LPS exposure, is specific in which macrophage effector functions are inhibited, and is reversible with further incubation or by non-LPS-related stimuli.

Anal Biochem, 1989 Aug 15, 181(1), 153 - 62
Nonisotopic detection of RNA in an enzyme immunoassay using a monoclonal antibody against DNA-RNA hybrids; Coutlee F et al.; A sensitive nonisotopic solution hybridization assay for detection of RNA is described and characterized using a pSP65 plasmid model system . The assay procedure is based on a hybridization reaction in solution between a biotinylated DNA probe and a target RNA . The biotin-labeled hybrids are captured on a microtiter plate coated with an antibody to biotin . Bound DNA-RNA hybrids are detected by an immunoreaction with an enzyme-labeled monoclonal antibody specifically directed against DNA-RNA heteropolymers and the hybrids are quantitatively measured with the addition of a fluorogenic substrate . Optimal conditions under which to perform the assay were hybridization time, 1000 min; temperature, 75 degrees C; probe concentration, 0.2 microgram/ml; extent of probe biotinylation, 6.7%; buffer stringency, 2x SSC . A bisulfite-modified DNA probe was compared to nick-translated probes synthesized with reporter groups of different lengths (bio-11-dUTP or bio-19-dUTP) . All probes could detect 10 pg/ml of target RNA . The presence of nonhomologous DNA or RNA sequences reduced the sensitivity of RNA detection by one half-log to 32 pg/ml (1.6 pg/assay).

Biochem Biophys Res Commun, 1989 Aug 15, 162(3), 1311 - 6
EF-hands calcium binding regulates the thioredoxin reductase/thioredoxin electron transfer in human keratinocytes; Schallreuter KU et al.; Thioredoxin reductases purified from Escherichia coli from human metastatic melanoma tissue and from human keratinocytes are subject to allosteric inhibition by calcium . 45Calcium has been used to show that this enzyme contains a single binding site . Bound calcium does not exchange from thioredoxin reductase upon dialysis for 48 hours or upon exposure to 10(-3) M EGTA . An intelligenetics computer analysis yielded a single EF-hands calcium binding site on E . coli thioredoxin reductase with homology to the first EF-hands site on calmodulin . Calcium exchange from the enzyme requires the addition of the natural electron acceptor oxidized thioredoxin which causes a concentration dependent slow exchange . Due to the large conformational change caused by calcium binding to thioredoxin reductase it has been possible to separate Calcium-free and Calcium-bound enzyme by FPLC chromatography . Human keratinocytes contain 5% thioredoxin reductase in their acidic protein cytosol fraction . The influence of extracellular calcium concentration on the intracellular equilibrium between calcium bound versus calcium free thioredoxin reductase has been assessed . This equilibrium was shown to determine the redox status of keratinocytes via the reduction of thioredoxin . Our results provide the first evidence for calcium dependent regulation of redox conditions in the human epidermis.

Biochim Biophys Acta, 1989 Aug 14, 1008(3), 355 - 6
Nucleotide sequence of a wheat mitochondrial lysine tRNA gene; Joyce PB et al.; We present the sequence of a wheat mitochondrial (mt) lysine tRNA gene (trnK-UUU) . This gene more closely resembles its E . coli counterpart than it does the corresponding gene in fungal or mammalian mtDNA . Hybridization experiments with a trnK-specific probe suggest that at least two copies of this tRNALys gene are present in the wheat mitochondrial genome.

FEBS Lett, 1989 Aug 14, 253(1-2), 281 - 6
Conformational changes occurring in N-ras p21 in response to binding of guanine nucleotide and metal ions probed by proteolysis performed under controlled conditions; Grand RJ et al.; Variations in susceptibility to proteolysis by trypsin and chymotrypsin have been used as indicators of conformational changes taking place in N-ras p21 in response to ligand binding . It has been observed that changes occur in undenatured protein, rendering it more resistant to degradation, in the presence of divalent cations such as Mg2+ and Ca2+ (suggesting direct binding of metals to the polypeptide) and even more markedly in the presence of GDP and/or Mg2+ GDP . Monovalent cations (Na+ or K+) cannot substitute for Mg2+ or Ca2+ . Some capacity to bind guanine nucleotide is also retained by p21 treated with 7 M urea, as evidenced by increased resistance to proteolytic degradation, but the ability to bind divalent cations is irreversibly lost following denaturation . Protein prepared under denaturing conditions from a eukaryotic source, however, never regains the resistance to proteolysis shown by the bacterial p21 indicating irreversible changes in secondary and tertiary structure produced under these conditions.

FEBS Lett, 1989 Aug 14, 253(1-2), 221 - 5
Membrane-binding sites for acyl carrier protein in Escherichia coli; Bayan N et al.; We report that membrane vesicles of Escherichia coli contain protein-binding sites for acyl carrier protein . Scatchard analysis of the binding indicates a dissociation constant around 0.35 micrometers and a maximum number of protein-binding sites around 50 pmol per mg of membrane protein . Binding is on the inner membrane while the outer membrane is devoid of binding sites . These results are consistent with the fact that some acyl carrier protein-dependent enzymes implicated in phospholipid- and membrane-derived oligosaccharide biosynthesis are localized in the cytoplasmic membrane.

FEBS Lett, 1989 Aug 14, 253(1-2), 67 - 70
Regulation of DNA supercoiling in Escherichia coli: genetic basis of a compensatory mutation in DNA gyrase; McEachern F et al.; Bacterial DNA supercoiling is controlled by balancing the supercoiling activity of DNA gyrase and the relaxing activity of DNA topoisomerase I . We have characterized the gyrB gene from a top A deletion mutant of Escherichia coli (DM800) that has a compensatory mutation in gyrB, lowering the activity of gyrase 10-fold, and thereby redressing the intracellular level of supercoiling . The mutant gene differs from the wild type in carrying three rather than two direct tandem repeats of a 6 bp sequence encoding Ala-Arg . We suggest this novel mutation affects domain spacing and was generated by an unequal crossing over event, possibly involving gyrase.

Cell, 1989 Aug 11, 58(3), 545 - 51
Single and double loop formation when deoR repressor binds to its natural operator sites; Amouyal M et al.; Distal effects on the in vivo repression of the deo operon are thought to be mediated by the deoR repressor with DNA loop formation . Such loops are easily observed by electron microscopy when the oligomeric deoR repressor is added to a DNA fragment carrying the three genetically defined operators at their chromosomal distances . Upon binding of deoR to any two operators, single loops are formed, 280, 600, and 880 bp in size . With the deo operon, double loops are also formed, which are the combination of the 280 bp and 600 bp loops and the result of simultaneous binding of the protein to its three sites . The formation of both single and double loops is consistent with the long-range effects observed in vivo and with the cooperative involvement of all three operator sites in the repression.

Nucleic Acids Res, 1989 Aug 11, 17(15), 6217 - 27
A chloroplast gene encoding a protein with one zinc finger; Sasaki Y et al.; We have sequenced a pea chloroplast gene encoding a protein with a zinc finger (zfp A) . The putative protein is conserved in chloroplast DNA and shows sequence homology to the E . coli protein controlling fol C expression . Exposure of etiolated pea leaves to light leads to the accumulation of zfp A transcripts . The accumulation of these transcripts does not require de novo protein synthesis in the chloroplast, although the expression of highly inducible genes such as rbc L requires it . These properties suggest that zfp A encodes a protein involved in the regulation of chloroplast gene expression.

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