J Biol Chem, 1986 Feb 25, 261(6), 2524 - 8 The rho-115 mutation in transcription termination factor rho affects its primary polynucleotide binding site; Sharp JA et al.; We have investigated the effect of the rho-115 mutation on the catalytic properties of the Escherichia coli termination protein, rho . Comparison of the primary and secondary polynucleotide binding sites activities reveals dramatic differences between the mutant and wild-type molecules . Wild-type rho must bind single-stranded polynucleotides to activate its nucleotide triphosphatase (NTPase) activity, and either poly(C), or poly(dC) plus oligo(C), will suffice . In contrast, attempted activation of the rho-115 NTPase with poly(C) in the presence of poly(dC) showed the latter to be a potent inhibitor . Inclusion of small oligonucleotides such as oligo(C) in the activation assay does not inhibit the poly(C)-induced NTPase reaction of either wild-type rho or rho-115 . This would indicate, in the two polynucleotide binding site model for rho proposed by Richardson (Richardson, J.P . (1982) J . Biol . Chem . 251, 5760-5766), that the mutation in rho-115 affects the primary polynucleotide binding site . Transcription termination in vitro at the rho-dependent site trp t' showed dramatically reduced termination with rho-115 protein compared to wild-type rho . In the presence of rho-115, the transcript is longer and termination occurs over a narrower range of nucleotides than with wild-type rho . This suggests that the primary polynucleotide binding site is important not only for efficient termination of transcription but may also be involved in determining the terminal end point of the transcript itself. J Biol Chem, 1986 Feb 25, 261(6), 2654 - 9 Structural and functional properties of colicin B; Pressler U et al.; Colicin B was isolated in pure form from cells of Escherichia coli that contained the colicin activity and immunity genes cloned on a multi-copy plasmid . Active colicin B consisted of a single polypeptide with Mr of about 60,000 . The sequence of 44 amino acids from the amino-terminal portion is presented . The isoelectric point of the protein was at 4.5 . Colicin B inhibited the membrane potential-dependent transport of proline and enhanced the uptake of alpha-methylglucoside via the phosphoenolpyruvate-dependent phosphotransferase system . Colicin B formed small, ion permeable channels with an average single-channel conductance of 13.7 pS (1 pS = 10(-12) siemens) in 1 M KCl . Channel formation was voltage-dependent in the pH range between 4.5 and 6 . At pH 7 the channels were voltage independent . Voltage-dependent channels were only formed when the trans compartment (the protein was added to the cis compartment) was negative by at least 70 mV . Evidence for an asymmetric single channel conductance was obtained . With KCl a hyperbolic conductance-concentration relationship was observed . The conductance for monovalent cations was minimal for Li+ and was maximal for NH+4 . The single channel conductance of colicin B was larger than that of colicin A as judged from lipid bilayer experiments under otherwise identical conditions. Biochim Biophys Acta, 1986 Feb 24, 866(1), 15 - 8 Regulation of acetohydroxy acid synthase activities in Escherichia coli K-12 by small metabolites; Williams AL; The effects of several metabolites (indole acetic acid, imidazole acetic acid and indole) on acetohydroxy acid synthase activities have been examined in both cya+ and cya- strains . Specifically, indole acetic acid caused an increase in the rate of acetohydroxy acid synthase synthesis under both in vivo and in vitro conditions . Taken together, these data suggest that small metabolites, other than cAMP, can alter acetohydroxy acid synthase gene expression. J Mol Biol, 1986 Feb 20, 187(4), 603 - 15 Salt-dependent changes in the DNA binding co-operativity of Escherichia coli single strand binding protein; Lohman TM et al.; The co-operative nature of the binding of the Escherichia coli single strand binding protein (SSB) to single-stranded nucleic acids has been examined over a range of salt concentrations (NaCl and MgCl2) to determine if different degrees of binding co-operativity are associated with the two SSB binding modes that have been identified recently . Quantitative estimates of the binding properties, including the co-operativity parameter, omega, of SSB to single-stranded DNA and RNA homopolynucleotides have been obtained from equilibrium binding isotherms, at high salt (greater than or equal to 0.2 M-NaCl), by monitoring the fluorescence quenching of the SSB upon binding . Under these high salt conditions, where only the high site size SSB binding mode exists (65 +/- 5 nucleotides per tetramer), we find only moderate co-operativity for SSB binding to both DNA and RNA, (omega = 50 +/- 10), independent of the concentration of salt . This value for omega is much lower than most previous estimates . At lower concentrations of NaCl, where the low site size SSB binding mode (33 +/- 3 nucleotides/tetramer) exists, but where SSB affinity for single-stranded DNA is too high to estimate co-operativity from classical binding isotherms, we have used an agarose gel electrophoresis technique to qualitatively examine SSB co-operativity with single-stranded (ss) M13 phage DNA . The apparent binding co-operativity increases dramatically below 0.20 M-NaCl, as judged by the extremely non-random distribution of SSB among the ssM13 DNA population at low SSB to DNA ratios . However, the highly co-operative complexes are not at equilibrium at low SSB/DNA binding densities, but are formed only transiently when SSB and ssDNA are directly mixed at low concentrations of NaCl . The conversions of these metastable, highly co-operative SSB-ssDNA complexes to their equilibrium, low co-operativity form is very slow at low concentrations of NaCl . At equilibrium, the SSB-ssDNA complexes seem to possess the same low degree of co-operativity (omega = 50 +/- 10) under all conditions tested . However, the highly co-operative mode of SSB binding, although metastable, may be important during non-equilibrium processes such as DNA replication . The possible relation between the two SSB binding modes, which differ in site size by a factor of two, and the high and low co-operativity complexes, which we report here, is discussed. Biochim Biophys Acta, 1986 Feb 19, 880(2-3), 242 - 4 A pathway for putrescine catabolism in Escherichia coli; Prieto-Santos MI et al.; Escherichia coli mutants able to grow in putrescine have been isolated from gamma-aminobutyrate mutants . These mutants show putrescine-alpha-ketoglutarate transaminase and gamma-aminobutyraldehyde dehydrogenase activities . Both enzymes have been characterized, the first of them showing an apparent Km for putrescine of 22.5 microM and the second an apparent Km of 37 microM for NAD and 18 microM for delta-1-pyrroline; the optimum pH values were 7.2 and 5.4, respectively, for the two enzymes. FEBS Lett, 1986 Feb 17, 196(2), 357 - 60 Production of a novel tryptophan analog, beta-1-indazole-L-alanine with tryptophan synthase of Escherichia coli; Tanaka H et al.; The tryptophan synthase alpha 2 beta 2 complex from Escherichia coli has been found to catalyze the beta-replacement reaction of L-serine with indazole, an indole analog which has a nitrogen atom at the 2-position (pyrazole ring) . The reaction product was isolated and identified as beta-indazolealanine by mass spectrometric, elemental and NMR analyses . Careful assignment of 1H- and 13C-signals with several NMR techniques revealed that the beta-carbon of the product alanine moiety was bound to the 1-N-position of the indazole ring . This is the first example of the beta-replacement reaction catalyzed by tryptophan synthase occurring at any other position than the 3-position of indole analogs. FEBS Lett, 1986 Feb 17, 196(2), 215 - 8 Fluorescence study of the RecA-dependent proteolysis of LexA, the repressor of the SOS system in Escherichia coli; Takahashi M et al.; The fluorescence of the LexA protein, the common repressor of the SOS system in Escherichia coli decreases by about 30% upon incubation with the RecA protein, and its cofactors ATP {or its non-hydrolysable analogue adenosine-5'-O-(3-thiotriphosphate), ATP gamma S} Mg2+ and single-stranded DNA . In the absence of any one of these elements required for the RecA-dependent proteolysis of LexA, this fluorescence change was not observed . The final fluorescence change depends only upon the concentration of LexA regardless of that of RecA . The time course of the fluorescence decrease corresponds well with the kinetics of the decrease of intact LexA protein and the increase of its 2 proteolytic fragments as determined by SDS-polyacrylamide gel electrophoresis . These results allow us to use the fluorescence change as a signal for a detailed kinetic analysis . The velocity of the proteolysis (d{LexA}/dt) is proportional to the concentration of LexA and RecA indicating that the formation of the LexA-RecA complex is the limiting step. Eur J Biochem, 1986 Feb 17, 155(1), 167 - 71 The primary structure of the alpha subunit of human elongation factor 1 . Structural aspects of guanine-nucleotide-binding sites; Brands JH et al.; The primary structure of the alpha subunit of elongation factor 1 (EF-1 alpha) from human MOLT 4 cells was determined by cDNA sequencing . The data show that the conservation of the amino acid sequence is more than 80% when compared with yeast and Artemia EF-1 alpha . An inventory of amino acid sequences around the guanine-nucleotide-binding site in elongation factor Tu from Escherichia coli and homologous amino acid sequences in G proteins, initiation and elongation factors and proteins from the RAS family shows two regions containing conserved sequence elements . Region I has the sequence apolar-Xaa-Xaa-Xaa-Gly-Xaa-Xaa-Yaa-Xaa-Gly-LYs-Thr(Ser)- -Xaa-Xaa-Xaa-Xaa-X-apolar . Except for RAS proteins, Yaa is always an acidic amino acid residue . Region II is characterized by the invariant sequence apolar-apolar-Xaa-Xaa-Asn-Lys-Xaa-Asp . In order to facilitate sequence comparison we have used a graphic display, which is based on the hydrophilicity values of individual amino acids in a sequence. Eur J Biochem, 1986 Feb 17, 155(1), 57 - 68 Imino proton NMR assignments and ion-binding studies on Escherichia coli tRNA3Gly; Hyde EI; The imino region of the proton NMR spectrum of Escherichia coli tRNA3Gly has been assigned mainly by sequential nuclear Overhauser effects between neighbouring base pairs and by comparison of assignments of other tRNAs . The effects of magnesium, spermine and temperature on the 1H and 31P NMR spectra of this tRNA were studied . Both ions affect resonances close to the G15 . C48 tertiary base pair and in the ribosylthymine loop . The magnesium studies indicate the presence of an altered tRNA conformer at low magnesium concentrations in equilibrium with the high magnesium form . The temperature studies show that the A7 . U66 imino proton (from a secondary base pair) melts before some of the tertiary hydrogen bonds and that the anticodon stem does not melt sequentially from the ends . Correlation of the ion effects in the 1H and 31P NMR spectra has led to the tentative assignment of two 31P resonances not assigned in the comparable 31P NMR spectrum of yeast tRNAPhe . 31P NMR spectra of E . coli tRNA3Gly lack resolved peaks corresponding to peaks C and F in the spectra of E . coli tRNAPhe and yeast tRNAPhe . In the latter tRNAs these peaks have been assigned to phosphate groups in the anticodon loop . Ion binding E . coli tRNA3Gly and E . coli tRNAPhe had different effects on their 1H NMR spectra which may reflect further differences in their charge distribution and conformation. Arch Biochem Biophys, 1986 Feb 15, 245(1), 114 - 24 beta-Sulfur substituted alpha-ketoglutarates as inhibitors and alternate substrates for isocitrate dehydrogenases and certain other enzymes; Plaut GW et al.; The RS-isomers of beta-mercapto-alpha-ketoglutarate, beta-methylmercapto-alpha-ketoglutarate and beta-methylmercapto-alpha-hydroxyglutarate have been synthesized . Beta-Mercapto-alpha-ketoglutarate was a potent inhibitor, competitive with isocitrate and noncompetitive with NADP+, of the mitochondrial NADP-specific isozyme from pig heart (Ki = 5 nM; Km (DL-isocitrate)/Ki(RS-beta-mercapto-alpha-ketoglutarate) = 650) and pig liver, the cytosolic isozyme from pig liver (I0.5 = 23 nM), and the NADP-linked enzymes from yeast (Ki = 58 nM) and Escherichia coli (Ki = 58 nM) at pH 7.4 and with Mg2+ as activator . beta-Mercapto-alpha-ketoglutarate was also an effective inhibitor of NADP-isocitrate-dehydrogenase activity in intact liver mitochondria . beta-Mercapto-alpha-ketoglutarate was a much less potent inhibitor for heart NAD-isocitrate dehydrogenase (Ki = 520 nM) than for the NADP-specific enzyme . beta-Methylmercapto-alpha-ketoglutarate (I0.5 = 10 microM) was a much less effective inhibitor than the beta-mercapto derivative for heart NADP-isocitrate dehydrogenase . The beta-sulfur substituted alpha-ketoglutarates were substrates for the oxidation of NADPH by heart NADP-isocitrate dehydrogenase without requiring CO2 . beta-Methylmercapto-alpha-hydroxyglutarate, the expected product of reduction of beta-methylmercapto-alpha-ketoglutarate, did not cause reduction of NADP+ but it was an inhibitor competitive with isocitrate for NADP-isocitrate dehydrogenase . The beta-sulfur substituted alpha-ketoglutarate derivatives were alternate substrates for alpha-ketoglutarate dehydrogenase and the cytosolic and mitochondrial isozymes of heart aspartate aminotransferase but had no effect on glutamate dehydrogenase or alanine aminotransferase. Biochem J, 1986 Feb 15, 234(1), 49 - 57 The serC-aro A operon of Escherichia coli . A mixed function operon encoding enzymes from two different amino acid biosynthetic pathways; Duncan K et al.; Sub-cloning experiments aimed at precisely locating the E . coli aroA gene, which encodes the shikimate pathway enzyme 5-enolpyruvylshikimate 3-phosphate synthase, showed that in certain constructions, which remain capable of complementing an auxotrophic aroA mutation, expression of aroA is reduced . DNA sequence analysis revealed that a sequence approx . 1200 base pairs (bp) upstream of aroA is necessary for its expression . An open reading frame was identified in this region which encodes a protein of 362 amino acids with a calculated Mr of 39,834 and which ends 70 bp before the start of the aroA coding sequence . This gene has been identified as serC, the structural gene for 3-phosphoserine aminotransferase, an enzyme of the serine biosynthetic pathway . Both genes are expressed as a polycistronic message which is transcribed from a promotor located 58 bp upstream of serC . Evidence is presented which confirms that the aroA and serC genes constitute an operon which has the novel feature of encoding enzymes from two different amino acid biosynthetic pathways. Clin Chim Acta, 1986 Feb 15, 154(3), 203 - 11 Highly sensitive enzyme immunoassay for human brain aldolase C; Haimoto H et al.; A sensitive sandwich-type enzyme immunoassay for brain-type isozyme of human aldolase C4 was developed using purified antibodies specific to the C subunit . The antibodies were raised in rabbits by injecting the purified aldolase C4, and purified by means of immunoaffinity chromatography on a column of aldolase C4-coupled Sepharose . The assay system consisted of polystyrene balls with immobilized antibody F(ab')2 fragments and the same antibody Fab' fragments labelled with beta-D-galactosidase from Escherichia coli . The assay was highly sensitive and the minimum detection limit of aldolase C4 was 3 pg/tube . The assay was specific to the C subunit of aldolase (aldolase C) . It cross-reacted about 60% with aldolase AC3, 30% with aldolase A2C2, and 4% with aldolase A3C, but showed no cross-reactivity with aldolase A4, the muscle-type isozyme . Coefficients of variation in within-run and between-run precision studies for serum aldolase C were less than or equal to 11% . Serum aldolase C levels in healthy adults of various ages (16-59 yr old) and both sexes ranged from 8.74-18.9 ng/ml . Immunoreactive aldolase C in the extracts of various human tissues was determined . It was distributed at high concentrations in the central nervous tissue and heart and at significant levels in liver, adrenal glands and testis . The assay of aldolase C in cerebrospinal fluid or serum by employing this sensitive immunoassay might be useful in the diagnosis of neurological disorders or acute myocardial damage. Biochem Pharmacol, 1986 Feb 15, 35(4), 679 - 85 Effects of the antitumor drugs 3-nitrobenzothiazolo{3,2-alpha}quinolinium and fagaronine on nucleic acid and protein synthesis; Torres CA et al.; 3-Nitrobenzothiazolo{3,2-alpha}quinolinium perchlorate (NBQ) has been shown to be active against in vivo experimental tumors of P388 and Ehrlich ascites cells . Furthermore, it has been established that NBQ binds to DNA by intercalation . In this work we describe its effects on DNA, RNA and protein syntheses both in KB cells and in cell-free synthesizing systems . Fagaronine, an alkaloid structurally related to NBQ, was studied also in an attempt to establish the basis for future studies on structure-activity relationships . Both NBQ and fagaronine inhibited DNA, RNA and protein syntheses in KB cells, with essentially equal effectiveness . Exposure of KB cells to NBQ for 2 hr caused irreversible inhibition of DNA, RNA and protein syntheses . Studies in cell-free systems showed that NBQ strongly inhibited Escherichia coli DNA polymerase I, whereas RNA polymerase activities were moderately affected . Furthermore, both drugs inhibited protein synthesis in cell-free systems derived from rabbit reticulocytes and Saccharomyces cerevisiae . Our results indicate that NBQ and fagaronine exert their cytotoxic activity by at least two independent mechanisms: inhibition of DNA activity by binding to this molecule, and inhibition of protein synthesis probably by interacting with the ribosomal system. J Immunol, 1986 Feb 15, 136(4), 1415 - 7 Antibodies isolated by using cloned surface antigens recognize antigenically related components of Legionella pneumophila and other Legionella species; Engleberg NC et al.; We studied the antigenic cross-reactivity of surface proteins among various strains of Legionella pneumophila and other Legionella species by using a novel method of antibody purification . Anti-bacterial antibodies in hyperimmune sera were adsorbed to and eluted from the surface of recombinant E . coli cells that express individual L . pneumophila antigens on their surface . These affinity-purified antibodies were then used to probe protein immunoblots prepared from the test strains to detect cross-reactive domains . We found that antigenic proteins are generally conserved in all L . pneumophila serogroups . Although some of these antigenic domains are shared with members of other Legionella species, they are associated with proteins of different molecular mass . Our approach to the study of antigenic cross-reactivity has potential advantages over similar studies that use either monoclonal antibodies or monospecific antibodies prepared by immunization with purified antigens. J Biol Chem, 1986 Feb 15, 261(5), 2289 - 93 The ribosomal binding domain of the Escherichia coli release factors . Modification of tyrosine in the N-terminal domain of ribosomal protein L11 affects release factors 1 and 2 differentially; Tate WP et al.; Ribosomal protein L11 is one of only two ribosomal proteins significantly iodinated when Escherichia coli 50 S subunits are modified by immobilized lactoperoxidase, and the major target has been shown previously to be tyrosine at position 7 in the N-terminal domain . This modification reduces in vitro termination activity with release factor (RF)-1 by 70-90%, but RF-2 activity is less affected (30-50%) . The loss of activity parallels incorporation of iodine into the subunit . The 50 S subunits from L11-lacking strains of bacteria have highly elevated activity with RF-2 and low activity with RF-1 . The iodination does not affect RF-2 activity but reduces the RF-1 activity further . Ribosomal proteins, L2, L6, and L25, are significantly labeled in L11-lacking ribosomes in contrast to the control 50 S subunits . L11 has been modified in isolation and incorporated back efficiently into L11-lacking ribosomes . This L11, iodinated also predominantly at Tyr 7, is unable to restore RF-1 activity to L11-lacking ribosomes in contrast to mock-iodinated protein . These results suggest the involvement of the N terminus of L11 in the binding domain of the bacterial release factors and indicate that there are subtle differences in how the two factors interact with the ribosome. J Immunol, 1986 Feb 15, 136(4), 1283 - 7 Interleukin 1 activates phospholipase A2 in rabbit chondrocytes: a possible signal for IL 1 action; Chang J et al.; We examined the effects of Interleukin 1 (IL 1) on rabbit articular chondrocytes with particular emphasis on arachidonic acid metabolism in these cells . Articular chondrocytes were isolated from the knee joints of normal New Zealand white rabbits and were cultured in vitro until confluent . Addition of 5 U/ml of purified IL 1 to chondrocytes led to an early increase in cell-associated phospholipase A2 (PLA2; measured by hydrolysis of {14C}arachidonic acid-labeled E . coli) . Within 1 hr after IL 1 addition, cell-associated PLA2 activity was increased by more than threefold relative to basal PLA2 activity, and further increases in cellular enzyme activity were observed up to 48 hr of IL 1 treatment . IL 1 stimulation also led to a time- and dose-related release of extracellular PLA2 and PGE2, but IL 1-induced PLA2 and PGE2 secretion occurred after the initial burst of intracellular PLA2 activity . Similar PLA2 and PGE2 responses were also observed when purified human IL 1 or IL 1-containing conditioned medium from LPS-stimulated human monocytes were used, but recombinant IL 2 or IL 3 were inactive . IL 1-induced chondrocyte PLA2 did not release radiolabeled free fatty acid from phosphatidylethanolamine labeled at the C-1 position with {14C}stearic acid, confirming the identity of this enzyme as PLA2 . These data, therefore, provide the first direct evidence that IL 1 activates cellular PLA2, and we propose that PLA2 activation may be an early signal that initiates the inflammatory actions of IL 1. Biochem J, 1986 Feb 15, 234(1), 111 - 7 Insertion of transposon Tn7 into the Escherichia coli glmS transcriptional terminator; Gay NJ et al.; The transposon Tn7 is unusual as it transposes at high frequencies from episomal elements to a unique site in the Escherichia coli chromosome . This unique site is within a region of dyad symmetry that we have demonstrated to be the transcriptional terminator of the glmS gene which encodes the glutamine amidotransferase, glucosamine synthetase . Transposition of Tn7 abolishes termination of glmS transcription at this site; the transcripts now extend into the left end of Tn7 and terminate at a new site, tm, 127 base pairs from the left end of Tn7 . This region of the transposon contains a long open reading frame which encodes a protein sequence that is significantly related to the transposase proteins of the transposable elements IS1 and Tn3 . A weak transcript has been identified that emanates from a promoter on the 5' side of this reading frame . This promoter is over-run by glmS transcripts and so it appears that expression of the Tn7 transposase may be regulated by promoter occlusion. J Biol Chem, 1986 Feb 15, 261(5), 2299 - 303 Effects of secA mutations on the synthesis and secretion of proteins in Escherichia coli . Evidence for a major export system for cell envelope proteins; Liss LR et al.; We have followed the synthesis and secretion of a number of periplasmic and outer membrane proteins in three strains of Escherichia coli, a secA amber mutant, a secA temperature-sensitive mutant, and a strain that blocks protein secretion due to a high level of expression of an export-defective hybrid protein between maltose-binding protein and beta-galactosidase (MalE-LacZ) . Our results show that after several hours under nonpermissive conditions the specificity and extent of the export blocks in the secA temperature-sensitive mutant and the strain producing the MalE-LacZ hybrid protein are identical, affecting at least four major outer membrane proteins and most but not all periplasmic proteins . The secA gene product, therefore, appears to be an essential component of the major export pathway in E . coli which is used by many envelope proteins independent of whether they are cotranslationally or post-translationally secreted . In contrast, the synthesis of only a subset of these envelope proteins is reduced in the secA amber mutant after shift to the nonpermissive condition . These results indicate that the SecA protein serves roles both in the synthesis and the secretion of certain cell envelope proteins. J Biol Chem, 1986 Feb 15, 261(5), 2284 - 8 Lipoprotein-28, a cytoplasmic membrane lipoprotein from Escherichia coli . Cloning, DNA sequence, and expression of its gene; Yu F et al.; Escherichia coli contains several lipoproteins in addition to the major outer membrane lipoprotein (Ichihara, S., Hussain, M., and Mizushima, S . (1981) J . Biol . Chem . 256, 3125-3129) . We cloned the gene for one of these new lipoproteins by using a synthetic 15-mer oligonucleotide probe identical to the DNA sequence at the signal peptide cleavage site of the major lipoprotein . The DNA sequence of the cloned gene revealed an open reading frame encoding a 272-amino acid protein with a signal peptide of 23 amino acid residues . The amino acid sequence of the putative cleavage site region of the signal peptide, -Leu-Leu-Ala-Gly-Cys-, is identical to that of the major lipoprotein . When the cloned gene was expressed in E . coli, a gene product with an apparent molecular weight of approximately 29,000 was identified which agrees well with the calculated molecular weight (27,800) . The product was labeled with {3H}glycerol, and a precursor molecule of increased molecular weight was accumulated when cells were treated with globomycin, a specific inhibitor for prolipoprotein signal peptidase . We thus designed the gene product as lipoprotein-28 . Unlike the major lipoprotein, lipoprotein-28 was found to be localized in the cytoplasmic membrane . A possible orientation of lipoprotein-28 in the E . coli envelope is discussed. Arch Biochem Biophys, 1986 Feb 15, 245(1), 51 - 65 Characterization of intestinal brush border guanylate cyclase activation by Escherichia coli heat-stable enterotoxin; ElDeib MM et al.; Intestinal brush border guanylate cyclase was previously reported to be activated by the Escherichia coli enterotoxin (STa) . This system was reexamined in order to develop a hypothesis for the mechanism of activation . The extent of activation was previously underestimated, since by using sodium azide to inhibit competing reactions and ethylene glycol bis(beta-aminoethyl ether) N,N-tetraacetic acid to chelate Ca2+, which is inhibitory, maximal activations of 30- to 50-fold were obtained . Ca2+ inhibition was only partially relieved by the calmodulin inhibitor calmidazolium . Inhibitors of the O2-dependent activation of soluble guanylate cyclase had no effect on STa activation; hence, it was concluded that STa activation did not involve arachidonate release and oxidation . STa was able to further increase activity already elevated by the nonionic detergent Lubrol PX . The membrane-active agent filipin, which was previously reported to inhibit both basal and agonist-stimulated adenylate cyclase, did not inhibit STa activation of guanylate cyclase . Digitonin, another cholesterol binder, inhibited STa activation at low concentrations, which disappeared at higher concentrations . Both of these agents stimulated basal activity . Dimethyl sulfoxide produced a concentration-dependent inhibition of STa activation, while increasing basal activity 7-fold . Ethanol inhibited both basal and STa-stimulated activity, with the former being more affected . Benzyl alcohol, like ethanol, a "fluidizer" of cell membranes, also inhibited both basal and activated enzymes . We concluded that STa directly activates this guanylate cyclase and, because of the differential effects of inhibitors on basal and STa-stimulated activity, propose a receptor-mediated mechanism. Biochem Biophys Res Commun, 1986 Feb 13, 134(3), 1404 - 11 Functionally important conserved amino-acids in interferon-alpha 2 identified with analogues produced from synthetic genes; Camble R et al.; A gene was chemically synthesised and expressed in Escherichia coli to produce {Ala30,32,33}IFN-alpha 2, an analogue of human alpha 2-interferon (IFN-alpha 2) which is devoid of activity on human cells . Eight additional analogues provided single changes in IFN-alpha 2 at each of these three conserved positions . No one residue is essential for activity, but both antiviral and anti-proliferative activity are particularly sensitive to changes in the side-chain of Arg33. Biochem Biophys Res Commun, 1986 Feb 13, 134(3), 1086 - 92 5S RNA gene specific transcription factor (TFIIIA) changes the linking number of the DNA; Shastry BS; The purified 5S gene specific transcription factor A (TFIIIA), when incubated with the relaxed 5S gene in the presence of topoisomerase I alters the conformation of the DNA, resulting in a change in the linking number . This interaction introduces a change of one bp per TFIIIA binding site at a low concentration of DNA to protein (1:2) which increases to an extent of 0.9 turns (9 bp) per TFIIIA binding site at a higher protein concentration (1:12) . These analyses support the notion that the binding of TFIIIA to the 5S DNA introduces a minimal change in the topology of circular DNA molecules. Biochem Biophys Res Commun, 1986 Feb 13, 134(3), 1182 - 9 Induction of alpha 2u globulin mRNA by phenobarbital in rat liver: characterization of a cDNA clone; Osorio-Almeida ML et al.; A clone has been selected from a cDNA library previously constructed from phenobarbital pre-treated rat liver polysomal poly(A)+ RNA, which was reverse transcribed . The double-stranded cDNA was inserted by GC homopolymeric tailing in the Pst I site of pAT 153, and further cloned in E . coli HB101 . This clone, called 2A9, corresponds to a mRNA whose concentration is increased five fold 16 h after phenobarbital treatment . Its length is 1200 nucleotides as revealed by RNA dot and Northern blot analysis respectively . The two strands of a 450 bp fragment from the 2A9 580 bp double-stranded cDNA insert have been sequenced and proven to correspond to alpha 2u globulin mRNA . It shows one single bp difference from the sequence previously published by Unterman et al . (1981, PNAS, 78, 3478) . Thus, alpha 2u globulin, a hormone regulated gene product, is inducible by phenobarbital. J Immunol Methods, 1986 Feb 12, 86(2), 217 - 23 Identification of a particular antigen from a parasite cDNA library using antibodies affinity purified from selected portions of Western blots; Beall JA et al.; Portions of nitrocellulose filters containing blotted electrophoresed antigens of Schistosoma japonicum adult worms were reacted with polyclonal rabbit antisera raised to this human parasite . Eluted antibodies were used as probes for detection of antigen-positive clones in an Escherichia coli lambda gt11 amp3 expression library of adult worm cDNA . Several cloned antigens corresponding to a S . japonicum antigen of Mr 26 000, being sought as a candidate vaccine molecule in a mouse model of schistosomiasis japonica, were identified using this approach . The method provides an antibody reagent that is an attractive alternative to other more tedious means of producing oligospecific antibodies, including monoclonal antibodies, for screening of expression libraries. Biochemistry, 1986 Feb 11, 25(3), 733 - 9 Unpaired cysteine-54 interferes with the ability of an engineered disulfide to stabilize T4 lysozyme; Perry LJ et al.; We have introduced an intramolecular disulfide bond into T4 lysozyme and have shown this molecule to be significantly more stable than the wild-type molecule to irreversible thermal inactivation {Perry, L.J., & Wetzel, R . (1984) Science (Washington, D.C.) 226, 555-557} . Wild-type T4 lysozyme contains two free cysteines, at positions 54 and 97, and no disulfide bonds . By directed mutagenesis of the cloned T4 lysozyme gene, we replaced Ile-3 with Cys . Oxidation in vitro generated an intramolecular disulfide bond; proteolytic mapping showed this bond to connect Cys-3 to Cys-97 . While this molecule exhibited substantially more stability against thermal inactivation than wild type, its stability was further enhanced by additional modification with thiol-specific reagents . This and other evidence suggest that at basic pH and elevated temperatures Cys-54 is involved in intermolecular thiol/disulfide interchange with the engineered disulfide, leading to inactive oligomers . Mutagenic replacement of Cys-54 with Thr or Val in the disulfide-cross-linked variant generated lysozymes exhibiting greatly enhanced stability toward irreversible thermal inactivation. Biochemistry, 1986 Feb 11, 25(3), 728 - 32 Effects of guanidine hydrochloride on the refolding kinetics of denatured thioredoxin; Kelley RF et al.; The effect of guanidine hydrochloride concentration on the kinetics of the conformational change of Escherichia coli thioredoxin was examined by using fluorescence, absorbance, circular dichroic, and viscosity measurements . Native thioredoxin unfolds in a single kinetic phase whose time constant decreases markedly with increasing denaturant concentration in the denaturation base-line zone . This dependency merges with the time constant of the slowest refolding kinetic phase at the midpoint of the equilibrium transition in 2.5 M denaturant . The time constant of the slowest refolding phase becomes denaturant independent below 1 M denaturant in the native base-line region . The denaturant-independent slowest refolding phase has an activation energy of 16 kcal/mol and is generated in the denatured base-line zone in a denaturant-independent reaction having a time constant of 19 s at 25 degrees C . The fractional amplitude of the slowest refolding phase diminishes in the native base-line zone to a minimum value of 0.25 . This decrease is accompanied by an increase in the fractional amplitudes of two faster refolding kinetic phases, an increase describing a sigmoidal transition centered at about 1.6 M denaturant . Manual multimixing measurements indicate that only the slowest refolding kinetic phase generates a product having the stability of the native protein . We suggest that the two faster refolding phases reflect the transient accumulation of folding intermediates which can contain a nonnative isomer of proline peptide 76. Biochemistry, 1986 Feb 11, 25(3), 681 - 7 Substrate range of the 40,000-dalton DNA-photoreactivating enzyme from Escherichia coli; Sutherland BM et al.; We determined the ability of the 40 000-dalton Escherichia coli photoreactivating enzyme to act on a variety of pyrimidine-pyrimidine photoproduct substrates in nucleic acids . The enzyme is at least as active on cis-syn-cyclobutylpyrimidine dimers in supercoiled DNA as in linear DNA, but inactive on dimers in RNA . Both the phosphodiester bond internal to the deoxyriboses of the pyrimidines of the dimer and the N-glycosyl bond joining the pyrimidine to deoxyribose must be intact for enzyme action . The enzyme has no activity toward (6-4) pyrimidine-cytosine products in DNA. Biochemistry, 1986 Feb 11, 25(3), 590 - 8 Backbone dynamics of a model membrane protein: 13C NMR spectroscopy of alanine methyl groups in detergent-solubilized M13 coat protein; Henry GD et al.; The filamentous coliphage M13 possesses multiple copies of a 50-residue coat protein which is inserted into the inner membrane of Escherichia coli during infection . 13C nuclear magnetic resonance (NMR) spectroscopy has been used to probe the structure and dynamics of M13 coat protein solubilized in detergent micelles . A comparison of backbone dynamics within the hydrophobic core region and the hydrophilic terminal domains was obtained by biosynthetic incorporation of {3-13C}alanine . Alanine is distributed throughout the protein and accounts for 10 residues (i.e., 20% of the total) . Similar 13C NMR spectra of the protein have been obtained in two anionic detergents, sodium deoxycholate and sodium dodecyl sulfate, although the structures and physical properties of these solubilizing agents are quite different . The N-terminal alanine residues, assigned by pH titration, and the penultimate residue, assigned by carboxypeptidase A digestion, give rise to analogous peaks in both detergent systems . The pKa of Ala-1 (approximately 8.8) and the relaxation parameters of individual carbon atoms (T1, T2, and the nuclear Overhauser enhancement) are also generally similar, suggesting a similarity in the overall protein structure . Relaxation data have been analyzed according to the model-free approach of Lipari and Szabo {Lipari, G., & Szabo, A . (1982) J . Am . Chem . Soc . 104, 4546-4559} . The overall correlation times were obtained by fitting the three experimental relaxation values for a given well-resolved single carbon atom to obtain a unique value for the generalized order parameter, S2, and the effective correlation time, tau e . The former parameter reflects the spatial restriction of motion, and the latter, the rate.(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1986 Feb 11, 25(3), 529 - 39 Methionyl-tRNA synthetase induced 3'-terminal and delocalized conformational transition in tRNAfMet: steady-state fluorescence of tRNA with a single fluorophore; Ferguson BQ et al.; Five species of tRNAfMet labeled with a single fluorophore are prepared to analyze the conformational changes at the 3'-end, at dihydrouridine, and at thiouridine in tRNAfMet upon binding of methionyl-tRNA synthetase . The emission and excitation spectra, anisotropy, and solvent accessibility of the fluorophore in each of the modified tRNAfMet's are determined in the absence and presence of methionyl-tRNA synthetase . The results are consistent with the following . The probes at the 3'-end are in a nonpolar environment, mobile relative to the tRNA molecule, and fully exposed to the solvent . The probes at dihydrouridine are partially stacked over the neighboring bases, nearly immobile, and relatively inaccessible . The S8-C13 cross-linked product is rigid . Upon binding of methionyl-tRNA synthetase, the probes at the 3'-terminus become localized in a less polar environment, highly immobilized, and effectively shielded against solvent access, while the probes at dihydrouridine appear to be partially unstacked from the neighboring base and become slightly more accessible for solvent . Singlet-singlet energy transfer between the intrinsic protein fluorescence and the fluorophores in modified tRNA's was observed by sensitized emission for tRNAfMet modified at the 3'-end and for S8-C13 but not for tRNAfMet's modified at dihydrouridine . These results suggest that dihydrouridine in tRNAfMet is oriented away from methionyl-tRNA synthetase in the tRNA-enzyme complex. Nucleic Acids Res, 1986 Feb 11, 14(3), 1149 - 57 The binding of RecA protein to duplex DNA molecules is directional and is promoted by a single stranded region; Cassuto E et al.; RecA protein from E . coli binds more strongly to single stranded DNA than to duplex molecules . Using duplex DNA that contains single stranded gaps, we have studied the protection by RecA protein at various concentrations, of restriction sites as a function of their distance from the single stranded region . We show that the binding of RecA protein, initiated in the single stranded region, extends progressively along the adjoining duplex in the 5' to 3' direction with respect to the single stranded region . The strand exchange reaction is known to proceed in the same direction. Nucleic Acids Res, 1986 Feb 11, 14(3), 1131 - 48 A single tRNA (guanine)-methyltransferase from Tetrahymena with both mono- and di-methylating activity; Reinhart MP et al.; A tRNA (guanine-2) methyltransferase has been purified to homogeneity from the protozoan Tetrahymena pyriformis . The enzyme methylates purified E . coli tRNAs which have a guanine residue at position 26 from the 5' end; it also methylates tRNA prepared from the m22G- yeast mutant trm 1 . This methyltransferase is therefore equivalent to the guanine methyltransferase 2mGII found in mammalian extracts . The purified 2mGII from Tetrahymena is capable of forming both N2-methylguanine and N22-dimethylguanine on a single tRNA isoaccepting species; under conditions of limiting tRNA or long reaction times the predominant product is dimethylguanine . Analysis of the products formed under varying reaction conditions suggests that dimethylguanine formation is a two step process requiring dissociation of the enzyme-monomethylated tRNA intermediate. Nucleic Acids Res, 1986 Feb 11, 14(3), 1171 - 86 RNA-protein cross-linking in Escherichia coli ribosomal subunits: localization of sites on 16S RNA which are cross-linked to proteins S17 and S21 by treatment with 2-iminothiolane; Kyriatsoulis A et al.; Treatment of E . coli ribosomal subunits with 2-iminothiolane coupled with mild ultraviolet irradiation leads to the formation of a large number of RNA-protein cross-links . In the case of the 30S subunit, a number of sites on 16S RNA that are cross-linked to proteins S7 and S8 by this procedure have already been identified (see ref . 6) . Here, by using new or modified techniques for the partial digestion of the RNA and the subsequent isolation of the cross-linked RNA-protein complexes, three new iminothiolane cross-links have been localized: Protein S17 is cross-linked to the 16S RNA within an oligonucleotide encompassing positions 629-633, and protein S21 is cross-linked to two sites within oligonucleotides encompassing positions 723-724 and positions 1531-1542 (the 3'-end of the 16S RNA). J Theor Biol, 1986 Feb 7, 118(3), 351 - 65 A stochastic process determines the time at which cell division begins in Escherichia coli; Bremer H; The theoretical distributions of cell masses in exponential cultures of bacteria were derived for both total cells and cells having formed a constriction in preparation for division . The parameters used for this derivation include the mass doubling time, tau, the T-period, and 3 statistical parameters (h, sigma 1, sigma 2) which describe the variability of the cell cycle . The theoretical distributions were compared with observed distributions from E . coli B/rA growing in glucose minimal medium (45 min doubling time) to determine whether a stochastic process in the division pathway affects the time of initiation of constriction or the duration of the constriction process . The results indicate that the stochastic process determines the onset rather than the completion of constriction and that the timing of this process is coupled (6% variability, = sigma 1) to a given cell mass . The values obtained for the duration of the T-period, T = 9.3 min, and for a half-life parameter associated with the stochastic process, h = 4.3 min, agree with previously reported data. Nature, 1986 Feb 6-12, 319(6053), 516 - 8 Stimulation of bone resorption and inhibition of bone formation in vitro by human tumour necrosis factors; Bertolini DR et al.; When leukocytes are exposed to mitogens or antigens in vitro, they release bone-resorbing activity into the culture supernatants which can be detected by bioassay . Like many lymphocyte-monocyte products, this activity has been difficult to purify because of its low abundance in activated leukocyte cultures and the unwieldy bioassay required to detect biological activity . Partially purified preparations of this activity inhibit bone collagen synthesis in organ cultures of fetal rat calvariae . Recent data suggest that both activated lymphocytes and monocytes release factors which could contribute to this activity . Recently, monocyte-derived tumour necrosis factor alpha (TNF-alpha) and lymphocyte-derived tumour necrosis factor beta (TNF-beta) (previously called lymphotoxin), two multifunctional cytokines which have similar cytotoxic effects on neoplastic cell lines, have been purified to homogeneity and their complementary DNAs cloned and expressed in Escherichia coli . As both of these cytokines are likely to be present in activated leukocyte supernatants, we tested purified recombinant preparations for their effects on bone resorption and bone collagen synthesis in vitro, and report here that both cytokines at 10(-7) to 10(-9) M caused osteoclastic bone resorption and inhibited bone collagen synthesis . These data suggest that at least part of the bone-resorbing activity present in activated leukocyte culture supernatants may be due to these cytokines. J Mol Biol, 1986 Feb 5, 187(3), 341 - 8 Kinetic and equilibrium characterization of the Tet repressor-tetracycline complex by fluorescence measurements . Evidence for divalent metal ion requirement and energy transfer; Takahashi M et al.; The interaction of Tet repressor protein with the inducer tetracycline was studied by fluorescence measurements, equilibrium dialysis and nitrocellulose filter binding . The repressor-tetracycline complex was formed from two molecules of tetracycline and one Tet repressor dimer . Formation of the complex requires divalent cations, and results in drastic effects upon the fluorescence spectra of both compounds . The fluorescence of Tet repressor was quenched about 70%, while that of tetracycline was increased between three- and eightfold, depending upon pH . In addition, the emission maximum of the protein was shifted from 330 to 340 nm, and the excitation maximum of tetracycline dropped from 380 to 370 nm . The latter shift is accompanied by a similar change in the absorption spectra . An analogous effect was observed upon changing the environment of the drug by the addition of sodium dodecyl sulphate . These results suggest that tetracycline binds to a hydrophobic region of the protein . A new excitation band in the fluorescence spectrum of the complex is observed . This presumably arises from energy transfer from a tryptophan to the drug . The association rate constant for formation of the complex is 3.3(+/- 0.3) X 10(5) M-1 s-1, and the equilibrium association constant is 2.8(+/- 0.5) X 10(9) M-1 . These results are discussed with respect to the biological function of the Tet repressor. J Biol Chem, 1986 Feb 5, 261(4), 1878 - 82 A photoaffinity-labeled allosteric site in Escherichia coli ribonucleotide reductase; Eriksson S et al.; The B1 subunit of Escherichia coli ribonucleotide reductase is coded for by the nrdA gene, of determined structure . Protein B1 contains two types of allosteric binding sites . One type (h-sites) determines the substrate specificity while the other type (l sites) governs the overall activity . The effectors dGTP and dTTP bind only to the h-sites while dATP and ATP bind to both the h- and the l-sites . Protein B1 has been photoaffinity-labeled with radioactive dTTP and dATP using direct UV irradiation . Following tryptic digestion of labeled protein B1 only one peptide labeled with dTTP was found, while several peptides were labeled with dATP . One of the dATP-labeled peptides had chromatographic properties very similar to that labeled with dTTP and this peptide most likely forms part of the h-site of protein B1 . Labeling of the l-site could not be conclusively shown since substantial non-specific labeling occurred with dATP . CNBr fragments of dTTP-labeled protein B1 were used to localize the region of nucleotide binding in the deduced primary structure of the nrdA gene . The dTTP label was further localized to a tryptic octapeptide with the sequence Ser-X-Ser-Gln-Gly-Gly-Val-Arg . The labeled amino acid was found at position 2, but the residue itself could not be directly identified . Unexpectedly, this sequence was not found in the earlier reported primary structure of the nrdA gene . However, a recent revised structure of the gene identifies the labeled residue as Cys-289 and fully confirms the rest of the peptide sequence . Thus the present result clearly defines one of the allosteric binding sites in ribonucleotide reductase. J Biol Chem, 1986 Feb 5, 261(4), 1835 - 7 Effect of amino acid substitutions at the signal peptide cleavage site of the Escherichia coli major outer membrane lipoprotein; Pollitt S et al.; The requirement for the glycine residue at the COOH terminus of the signal peptide of the precursor of the major Escherichia coli outer membrane lipoprotein was examined . Using oligonucleotide-directed site-specific mutagenesis, this residue was replaced by residues of increasing side chain size . Substitution by serine had no effect on the modification or processing of the prolipoprotein . Substitution by valine or leucine resulted in the accumulation of the unmodified precursor, whereas threonine substitution resulted in slow lipid modification and no detectable processing of the lipid modified precursor . The results indicate that serine is the upper limit on size for the residue at the cleavage site . Larger residues at this position prevent the action of both the glyceride transferase and signal peptidase II enzymes, indicating that the cleavage site residue plays a role in events prior to proteolytic cleavage . The upper limit on size of the cleavage site residue is similar to that found for exported proteins cleaved by signal peptidase I, as well as eucaryotic exported proteins . The possibility that the cleavage site residue may have a role other than active site recognition by the signal peptidase is discussed. J Biol Chem, 1986 Feb 5, 261(4), 1808 - 14 Reconstitution of quinone reduction and characterization of Escherichia coli fumarate reductase activity; Cecchini G et al.; Resolution of the fumarate reductase complex (ABCD) of Escherichia coli into reconstitutively active enzyme (AB) and a detergent preparation containing peptides C and D resulted in loss of quinone reductase activity, but the phenazine methosulfate or fumarate reductase activity of the enzyme was unaffected . An essential role for peptides C and D in quinone reduction was confirmed by restoration of this activity on recombination of the respective preparations . Neither peptide C nor peptide D by itself proved capable of permitting quinone reduction and membrane binding by the enzyme when E . coli cells were transformed with plasmids coding for the enzyme and the particular peptides . Transformation of a plasmid coding for all subunits resulted in a 30-fold increase in membrane-bound complex, which exhibited, however, turnover numbers for succinate oxidation and fumarate reduction that were intermediate between the high values characteristic of chromosomally produced complex and the relatively low values found for the isolated complex . It is also shown that preparations of the isolated complex and membrane-bound form of the enzyme, as obtained from anaerobically grown cells, are in the deactivated state owing to the presence of tightly bound oxalacetate and thus must be activated prior to assay. J Biol Chem, 1986 Feb 5, 261(4), 1507 - 9 Characterization of the monovalent cation activator binding site of S-adenosylmethionine synthetase by 205Tl NMR of enzyme-bound Tl+; Markham GD; The structure of the binding site for the monovalent cation activator of S-adenosylmethionine (AdoMet) synthetase from Escherichia coli has been characterized by 205Tl NMR of enzyme-bound Tl+ . The chemical shift of the enzyme-Tl+ complex is 176 ppm downfield from aquo Tl+, a shift which is typical only of Tl+ complexes with solely oxygen ligands . The 205Tl resonance shifts upfield to 85 ppm in the enzyme-Mg(II)-Tl+ complex, to 38 ppm in the enzyme-Tl+-AdoMet complex and to 34 ppm in the enzyme-Tl+-AdoMet-Mg(II) complex . The 205Tl chemical shift of enzyme-bound Tl+ was not altered by binding of either methionine, or the Mg(II)-ATP analog Mg(II)-adenyl-5'-yl imidodiphosphate, or Mg(II)-pyrophosphate to the enzyme-Tl+-Mg(II) complex . The NMR data suggest that the substrates or products of the enzyme do not coordinate to the monovalent cation activator and imply that monovalent cation activation results from alterations in protein conformation. J Biol Chem, 1986 Feb 5, 261(4), 1778 - 81 Nucleotide sequence of the Escherichia coli dnaJ gene and purification of the gene product; Ohki M et al.; The dnaJ and dnaK genes are essential for replication of Escherichia coli DNA, and they constitute an operon, dnaJ being downstream from dnaK . The amount of the dnaJ protein in E . coli is substantially less than that of the dnaK protein, which is produced abundantly . In order to construct a system that over-produces the dnaJ protein, we started our study by determining the DNA sequence of the entire dnaJ gene, and an operon fusion was constructed by inserting the gene downstream of the lambda PL promoter of an expression vector plasmid, pPL-lambda . Cells containing the recombinant plasmid produced dnaJ protein amounting to 2% of the total cellular protein when cells were induced . The overproduced protein was purified, and Edman degradation of the protein indicated that the NH2-terminal methionine was found to be processed . From the DNA sequence of the dnaJ gene, the processed gene product is composed of 375 amino acid residues, and its molecular weight is calculated to be 40,975. J Mol Biol, 1986 Feb 5, 187(3), 399 - 416 Rapid chemical probing of conformation in 16 S ribosomal RNA and 30 S ribosomal subunits using primer extension; Moazed D et al.; We have investigated in detail the higher-order structure of 16 S ribosomal RNA, both in its naked form and in 30 S ribosomal subunits . Each base in the 16 S rRNA chain has been probed using kethoxal (which reacts with guanine at N1 and N2), dimethylsulfate (which reacts with adenine at N1 and cytosine at N3) and 1-cyclohexyl-3-(2-morpholinoethyl)-carbodiimide metho-p-toluenesulfonate (which reacts with uracil at N3 and guanine at N1) . The sites of reaction were identified by primer extension with reverse transcriptase using synthetic oligodeoxynucleotide primers . These results provide a detailed and rigorous experimental test of a model for 16 S rRNA secondary structure, which was derived mainly from comparative sequence analysis . Our data also provide information relevant to tertiary and quaternary structure of 16 S rRNA . Data obtained with naked 16 S rRNA show reasonably close agreement with the proposed model, and data obtained with 30 S subunits show nearly complete agreement . Apart from an apparent overall "tightening" of the structure (in which many weakly reactive bases become unreactive), assembly of the proteins with 16 S rRNA to form 30 S subunits brings about numerous local structural rearrangements, resulting in specific enhancements as well as protections . In many instances, the ribosomal proteins appear to "tune" the 16 S rRNA structure to bring it into accordance with the phylogenetically predicted model, even though the RNA on its own often seems to prefer a different structure in certain regions of the molecule . Extensive protection of conserved, unpaired adenines upon formation of 30 S subunits suggests that they play a special role in the assembly process, possibly providing signals for protein recognition. Eur J Biochem, 1986 Feb 3, 154(3), 523 - 7 Nucleotide sequence of cloned cDNA specific for rat ribosomal protein L35a; Tanaka T et al.; A cDNA clone specific for rat ribosomal protein L35a, which is known to be a tRNA-binding protein, was isolated by hybrid-selected translation from a cDNA library made for 8-9-S poly(A)-rich RNA from regenerating rat liver . The nucleotide sequence of the cDNA was determined . It consists of one base pair from the 5' leading sequence, the entire coding sequence of 333 base pairs and 14 base pairs from the 3' trailing sequence . The primary structure of protein L35a was deduced from the nucleotide sequence . It consists of 109 amino acids with a molecular mass of 12422 . The calculated amino acid composition is consistent with that reported for the hydrolysate of L35a . The amino acid sequence showed marked homology with the reported partial sequence of Xenopus leavis ribosomal protein L32, but not significant homology with Escherichia coli ribosomal proteins that bind to tRNA. FEBS Lett, 1986 Feb 3, 196(1), 103 - 7 The excess GTP hydrolyzed during mistranslation is expended at the stage of EF-Tu-promoted binding of non-cognate aminoacyl-tRNA; Kakhniashvili DG et al.; The system of translation of Sepharose-bound poly(U) in which all ribosomes are active in peptide elongation was used to determine the stoichiometry of GTP hydrolysis at the stage of EF-Tu-promoted aminoacyl-tRNA binding . The ratio of GTP hydrolyzed at this stage per peptide bond was assayed during codon-specific elongation (polyphenylalanine synthesis) and misreading (polyleucine synthesis) . It was demonstrated directly that the excess GTP hydrolyzed during misreading {(1984) FEBS Letters 178, 283-287} is expended at the stage of Ef-Tu-promoted binding of non-cognate aminoacyl-tRNA. Eur J Biochem, 1986 Feb 3, 154(3), 665 - 72 Analysis of human p53 proteins and mRNA levels in normal and transformed cells; Matlashewski G et al.; p53 mRNA and proteins were examined in a variety of human transformed cells and in normal human foreskin fibroblast cells . Both the steady-state and translatable levels of p53 mRNA were the same in normal and transformed human cells . In vitro synthesized p53, programmed by mRNA from normal and transformed human cells, revealed that there was heterogeneity in the primary structure of p53 from these cells . Pulse labeling of cells and immunoprecipitation analysis with a panel of human reactive anti-p53 antibodies demonstrated that the types of p53 synthesized in vitro corresponded to the types made in vivo from SV80 and COLO 320 cells . No p53 was detectable by similar pulse-labeling analysis of HeLa and normal foreskin fibroblast cells . Since it was necessary to use anti-p53 sera from cancer patients to carry out much of the immunoprecipitation analysis in this study we therefore further characterised these sera to determine if they reacted with one or more than one epitope . p53-beta-galactosidase fusion proteins were synthesized in Escherichia coli and used to analyse the anti-p53 antibodies produced by cancer patients . We demonstrate that the antisera contain antibodies directed against epitopes in both the N-terminal and C-terminal regions of the p53 molecule. J Lipid Res, 1986 Feb, 27(2), 158 - 65 Stereospecificity of monoacylglycerol acyltransferase activity from rat intestine and suckling rat liver; Coleman RA et al.; The stereospecificity of monoacylglycerol acyltransferase from rat intestinal mucosa and suckling rat liver microsomes was examined using sn-1,2-diacylglycerol kinase from Escherichia coli . With 2-monooleoyl glycerol and palmitoyl-CoA, 88 and 87.9% of the diacylglycerol synthesized by the intestinal mucosa and suckling liver, respectively, was demonstrated to be the sn-1,2-isomer . Analysis of similar preparations of these diacylglycerol products by gas-liquid chromatography-mass spectrometry indicated that most of the remaining diacylglycerol was the 1,3-isomer that probably arose via acyl-migration . These results indicate that monoacylglycerol acyltransferase is stereospecific . Measurement of acyltransferase activities in microsomes using 1- and 2-monoacyl- and monoalkylglycerols as substrates indicated that the monoacylglycerol acyltransferases from suckling liver and intestinal mucosa have different substrate specificities. J Appl Physiol, 1986 Feb, 60(2), 486 - 93 Respiratory muscle energetics during endotoxic shock in dogs; Hussain SN et al.; Respiratory muscle O2 consumption, lactate production, and endogenous substrate utilization during endotoxic shock were assessed in two groups of anesthetized spontaneously breathing dogs . In the endotoxin group (Escherichia coli endotoxin 10 mg/kg iv) and the sham group (saline iv), we sampled diaphragm, external intercostal, and gastrocnemius muscle tissue for glycogen and lactate concentrations before and after 3 h of the experimental period . Only in the endotoxin group did blood pressure and cardiac output decline significantly . Arterial O2 content did not change significantly during shock, whereas mixed venous, phrenic venous, and femoral venous O2 contents dropped to 8.0 +/- 1.1, 5.8 +/- 0.8, and 3.6 +/- 0.6 ml/dl at 60 min of shock, respectively, with little change thereafter . At 30 min of shock, femoral venous lactate rose higher than arterial values, whereas at 90 min of shock, onward, phrenic venous lactate was significantly higher than arterial concentrations . All muscle tissues showed significant lactate production and glycogen depletion after shock . In a second set of experiments we measured respiratory muscle blood flow during shock with radioactive microspheres . At 60 min of shock, diaphragmatic and intercostal blood flow rose by six- and twofold, respectively, whereas gastrocnemius blood flow declined significantly . We conclude that during endotoxin shock 1) the increased demands of the respiratory muscles are met by increasing blood flow and O2 extraction; 2) anaerobic metabolism and respiratory muscle substrate depletion, or both, may contribute to the observed fatigue. Arch Biochem Biophys, 1986 Feb 1, 244(2), 630 - 40 Synthesis of large subunit of ribulosebisphosphate carboxylase by thylakoid-bound polyribosomes from spinach chloroplasts; Hattori T et al.; Intact chloroplasts were isolated from developing first leaves of spinach . The chloroplasts were broken and separated into an extensively washed membrane (thylakoid) fraction and a soluble (stroma) fraction . The membrane fraction contained polyribosomes with properties similar to those of thylakoid-bound polyribosomes of other organisms . The distribution of mRNA for large-subunit ribulosebisphosphate carboxylase (LS) was determined by translating RNA from chloroplasts, thylakoids, and stroma in a wheat germ cell-free translation system . LS translation product was identified by immunoprecipitation with antibody to LS from spinach, electrophoresis of the immunoprecipitated product, and fluorography . At least 44% of translatable chloroplast LS-mRNA was in the washed thylakoid fraction . Thylakoid-bound LS-mRNA was in polyribosomes since LS was produced by thylakoids in an Escherichia coli cell-free translation system under conditions where initiation did not take place . Our results demonstrate that membrane-bound polyribosomes can synthesize the stroma-localized polypeptide LS, and suggest that the thylakoids may be an important site of its synthesis. Am J Physiol, 1986 Feb, 250(2 Pt 2), H240 - 6 In vitro myocardial performance after lethal and nonlethal doses of endotoxin; McDonough KH et al.; The present study was initiated to determine whether the myocardial effects of an in vivo injection of endotoxin into rats were correlated with the dose and thus the lethality of the endotoxin administered . All animals in this study were used 4 h after a bolus injection of 1,000, 100, 10, or 1 microgram/100 g body wt of Escherichia coli endotoxin . At this time, mean arterial blood pressure had returned to control levels but cardiac output was still depressed at the three higher doses as previously reported {Am . J . Physiol . 248 (Regulatory Integrative Comp . Physiol . 17): R471-R478, 1985} . Intrinsic function of the myocardium was assessed using the isolated perfused working heart preparation . Cardiac output and pressure development were measured at varying preloads and at two levels of aortic outflow resistance . In vitro approaches were chosen for this study to eliminate peripheral vascular changes and humoral or neural alterations that might influence myocardial performance in vivo . Results indicate that coronary vascular resistance was increased in all hearts from endotoxin-treated animals compared with controls . In addition, myocardial performance was impaired at several doses of endotoxin, and the degree of dysfunction was dependent on the dose of endotoxin administered . Dysfunction, i.e., a depression in cardiac output times peak systolic pressure, was evident at the two higher doses in which there was 50 and 10% lethality by 24 h and also in the lower dose of 10 micrograms/100 g, which was nonlethal . Cardiac output appeared to be very sensitive to the consequences of endotoxin administration . Defects in myocardial performance could be revealed by increasing preload or afterload stress on the hearts.(ABSTRACT TRUNCATED AT 250 WORDS) Infect Immun, 1986 Feb, 51(2), 699 - 703 Molecular characterization of Chlamydia trachomatis and Chlamydia psittaci plasmids; Joseph T et al.; Plasmids from Chlamydia trachomatis LGV-434 (serotype L2) and Chlamydia psittaci meningopneumonitis strain Cal-10 were cloned into the BamHI and EcoRI sites of pBR322, respectively . The recombinant plasmids pCTL2 and pCPMn, each containing an entire respective chlamydial plasmid, were transformed into Escherichia coli . The sizes of the plasmids of C . trachomatis and C . psittaci were 7.3 and 6.2 kilobases, respectively . The two plasmids were found to be distinct by restriction endonuclease analysis, DNA-DNA hybridization, and electron microscopic heteroduplex analysis . However, partial homology was observed between restriction fragments of pCTL2 and pCPMn by Southern blot analysis . Polypeptide products encoded by these plasmids were synthesized in vitro by an E . coli-directed transcription-translation system and in vivo in E . coli maxicells and minicells . None of these polypeptides was immunoreactive with anti-chlamydial sera by immunoblotting or immunoprecipitation . Based on the comparative analysis data, the C . trachomatis and C . psittaci plasmids were found to share little genetic relatedness. Radiat Res, 1986 Feb, 105(2), 227 - 39 Radiosensitization, pharmacokinetics, and toxicity of a 2-nitroimidazole nucleoside (RA-263); Agrawal KC et al.; A 2-nitroimidazole nucleoside, 1-(2',3'-dideoxy-alpha-D-erythro-hex-2'-enopyranosyl)-2-nitroimida zole (RA-263), has been investigated for its radiosensitization, pharmacokinetics, and toxicity properties . The in vitro radiosensitization tests against hypoxic Chinese hamster (V-79) cells demonstrated that RA-263 was a more potent radiosensitizer than misonidazole and at 2 mM concentration approached the oxic curve . Significant in vitro radiosensitization activity was also observed in EMT6 mammary tumor cells . The in vitro cytotoxicity data suggested that RA-263 is considerably more toxic to hypoxic cells than misonidazole . The increased cytotoxicity may be related to its higher depletion of nonprotein thiols (NPSH) than misonidazole . The combined effects of radiosensitization and hypoxic cell toxicity were measured by preincubation of the V-79 cells for 4 h under hypoxic conditions before irradiation . The results demonstrated a synergistic response by causing a significant decrease in the extrapolation number with loss of shoulder of the radiation survival curves . The in vivo radiosensitization experiments conducted by the in vivo-in vitro cloning assay with the EMT6 mammary tumor indicate that RA-263 is an effective sensitizer . Pharmacokinetic data suggested that RA-263 was eliminated from plasma by a rapid alpha phase and a slower beta phase with T 1/2 of 36 and 72 min, respectively . The concentration in the brain was approximately one-sixth of tumor concentration, suggesting that RA-263 is excluded from the CNS . Moreover, RA-263 was two times less toxic than misonidazole on equimolar basis by acute LD50 tests . This agent was also significantly less mutagenic than misonidazole in a strain of Escherichia coli. Biochem Cell Biol, 1986 Feb, 64(2), 133 - 8 Hybrid gene synthesis: its application to the assembly of DNA sequences encoding the human parathyroid hormones and analogues; Sung WL et al.; Bypassing any intermediate steps of purification and gene assembly, several synthetic oligonucleotides constituting a DNA duplex with a small base-mismatching region were phosphorylated, annealed, and ligated directly into a linearized plasmid vector . After transformation in bacteria, the two plasmid strands individually yielded two different plasmids bearing altered versions of the same gene . Via this approach, DNA coding sequences of the human parathyroid hormone and analogues were synthesized and cloned in Escherichia coli. J Biochem (Tokyo), 1986 Feb, 99(2), 503 - 11 Solubilization and reconstitution of iodide counterflow activity from the thyroid plasma membranes into soybean phospholipid vesicles; Saito K et al.; The activity of I- counterflow was solubilized with n-octyl beta-D-glucopyranoside from the thyroid plasma membranes and reconstituted into phospholipid vesicles made from soybean phospholipids by a detergent dilution procedure . The vesicles exhibited apparent "entrance" I- counterflow but no apparent Na+-dependent I- transport activity . The activity of I- counterflow was saturated by external I- at the concentration of 50-200 microM, indicating the substrate specificity of the counterflow-mediating activity for I- . The optimal concentration of the detergent for solubilization was near 12.5 mg/ml, as reported in solubilization of some sugar transport carriers of E . coli . The counterflow of I- was inhibited by externally added SCN- but not by ClO4- . When solubilized protein was treated with 0.2-1 mM dithiothreitol and with 2-10 mM 2-mercaptoethanol prior to reconstitution, dose-dependent inactivation of I- counterflow was observed . However, the activity of I- counterflow was fully preserved when solubilized protein was treated with these agents in the presence of high concentrations of I- (40 and 80 mM) . In contrast, treatment with N-ethylmaleimide at a concentration of as high as 1 mM did not decrease the activity . In conclusion, it appears that the I- counterflow activity can be solubilized and reconstituted into phospholipid vesicles from the thyroid plasma membranes . The counterflow activity may be susceptible to disulfide-reducing agents. Mol Cell Biol, 1986 Feb, 6(2), 730 - 4 Biochemical characterization of polypeptides encoded by mutated human Ha-ras1 genes; Colby WW et al.; We expressed six forms of p21-ras polypeptides in Escherichia coli with differing transformation potentials resulting from amino acid substitutions at position 12 . The ability of the encoded p21's to autophosphorylate, bind guanine nucleotides, and hydrolyze GTP was assessed . All versions of p21 bound GTP equivalently; the kinase activity, while dependent upon residue 12, did not correlate with the transforming potential of the polypeptide . All transforming versions exhibited an impaired GTPase activity, while a novel nontransforming derivative {p21(pro-12)} possessed an enhanced GTPase activity . These results provide strong support for the proposal that an impairment of the cellular p21 GTPase activity can unmask its transforming potential. Mol Cell Biol, 1986 Feb, 6(2), 653 - 62 New, small circular DNA in transfected mammalian cells; Wiberg FC et al.; Circular DNA isolated by the Hirt procedure from transfected mammalian cells was examined by electron microscopy . Typically, the number of small (1- to 5-kilobase) DNA circles increased about fivefold even though DNA of larger size classes (5 to 15 kilobases) has been transferred . In one case, where extensive rearrangement of the transferred DNA was observed, the rearrangement products were cloned and analyzed . In most cases, however, no rearrangement could be detected, but the amount of small circular DNA was still increased . This effect was seen with two transfection procedures (erythrocyte ghost fusion and calcium phosphate precipitation) and with various combinations of transfecting DNA and recipient cell type . The origin of the new small circular DNA is discussed. J Trop Med Hyg, 1986 Feb, 89(1), 29 - 32 Asymptomatic bacteriuria in Nigerian diabetics; Abu-Bakare A et al.; One hundred and ninety diabetic patients and an equal number of non-diabetic patients, age- and sex-matched, were screened for occurrence of asymptomatic bacteriuria . Of the diabetics 6.3% were found to have covert bacteriuria as were 5.3% of the control group . Asymptomatic bacteriuria was found to occur more commonly in females than males and in the patients who were over 40 years old . The incidence of bacteriuria was not found to be related to the degree of control in the diabetic patients . The most common organism cultured was E . coli. J Gen Microbiol, 1986 Feb, 132 ( Pt 2), 403 - 10 Effects of microcin B17 on microcin B17-immune cells; Herrero M et al.; When microcin B17-immune cells are treated with microcin B17 they show many of the physiological effects displayed by microcin B17-sensitive cells treated in the same way . DNA replication stops immediately and several SOS functions are subsequently induced . In sensitive cells these effects are irreversible and lead to cell death, whereas in immune cells they are reversible and there is no loss of viability . This is an unusual mechanism of immunity because it does not prevent the primary action of the microcin . The implications of this mechanism concerning the mode of action of microcin B17 and the induction of the SOS system are discussed. Appl Biochem Biotechnol, 1986 Feb, 12(1), 1 - 15 Immobilized respiratory chain activities from Escherichia coli utilized to measure D- and L-lactate, succinate, L-malate, 3-glycerophosphate, pyruvate, or NAD(P)H; Burstein C et al.; The respiratory chain (membranous, multienzymatic system) from Escherichia coli, was coimmobilized with gelatin and insolubilized in film form by tanning with glutaraldehyde . The film was fixed onto an oxygen sensor . The enzyme electrode can be used for measuring NAD(P)H, D- and L-lactate, succinate, L-malate, 3-glycerophosphate, or pyruvate . The range of metabolites concentrations was from 1 to 50 mM . It was possible to discriminate between the different metabolites (if mixed): By inducing during bacterial growth the specific flavoproteins necessary for L-lactate, succinate, L-malate, and 3-glycerophosphate respirations . The constitutive activities are unaltered on glucose or glycerol, namely D-lactate, NAD(P)H, and pyruvate respiration . When intact bacteria were immobilized (with or without induction), D- and L-lactate, succinate, 3-glycerophosphate, and L-malate respiration were measured, no activities of pyruvate and NAD(P)H respiration were obtained . For these last activities, French press breakage (see section on Membrane Preparations) of bacteria prior to immobilization was necessary . Products of reactions can be used as enzyme inhibitors: Pyruvate inhibits D- and L-lactate; fumarate inhibits succinate, and oxaloacetate inhibits L-malate respirations . Heat denaturation of the bacteria at 55 degrees C for 1 h maintains full activity of succinate and pyruvate respiration . On the other hand, no activity of D- and L-lactate, L-malate, or NAD(P)H respiration was measurable . These enzyme electrodes have many applications in basic and applied research. Vet Microbiol, 1986 Feb, 11(1-2), 153 - 61 Screening of pig intestines for K88 non-adhesive phenotype by enzyme immunoassay; Chandler DS et al.; An adhesion test for binding of porcine brush border membranes to Escherichia coli cells that possess the K88 antigen (K88+) has been developed using enzyme immunoassay procedures . K88 pilus protein or K88+ E . coli cells were immobilized in the wells of polystyrene microtitre plates . These plates were incubated in the presence of material obtained by scraping the villous surface of pig small intestines . Adhesion of membrane material to immobilized K88 was detected by adding rabbit anti-brush border IgG followed by urease-labelled sheep anti-rabbit IgG conjugate . Action of bound enzyme on urea/bromo-cresol purple substrate solution (pH 4.8) produced an intense colour change from yellow to purple, enabling the test to be read visually . This test enables simple, rapid testing of large numbers of intestial samples and gives results that agree well with the more cumbersome microscopic adhesion test for adhesion of K88+ E . coli to purified brush border membranes. Vet Microbiol, 1986 Feb, 11(1-2), 103 - 15 Bacteriostasis of Escherichia coli by bovine lactoferrin, transferrin and immunoglobulins (IgG1, IgG2, IgM) acting alone or in combination; Rainard P; The effect on Escherichia coli strain B117 of bovine lactoferrin (LF), transferrin (Tr) and immunoglobulins (Ig), acting alone or in combination, was investigated in vitro . IgG1 and IgM, which, in contrast to IgG2 possessed a marked antibody activity, as assessed by enzyme-linked immunosorbent assay, induced the appearance of microcolonies in suspension, a phenomenon which could lead to misleading interpretations in the absence of sonic treatment, but none of these three isotypes influenced bacterial growth . Both LF and Tr promoted a strong inhibition of bacteria without requirement for antibodies . After a short period of growth, the multiplication of bacteria was almost completely prevented by the iron-binding proteins acting alone . A significant (p less than 0.02) but moderate (0.5 less than log10) further growth reduction was obtained when Ig of either class was added to Tr, while addition of Ig to LF revealed no significant cooperative effect . All of 11 strains of E . coli isolated from bovine mastitis were sensitive to LF action in the absence of Ig . It therefore appeared that antibodies were not required for iron-binding proteins to exert a potent bacteriostatic effect on mastitis isolates of E . coli. Thromb Res, 1986 Feb 1, 41(3), 309 - 17 Chromogenic assay of endotoxin in platelet poor or rich plasma; Tachiyama G et al.; In order to determine which sample preparation, platelet rich plasma (PRP) or platelet poor plasma (PPP), is more suitable for clinical endotoxin assay, we investigated the binding of endotoxins to platelets by comparing the amount of endotoxin in PRP with that in PPP, using a newly developed colorimetric assay with chromogenic substrate (Boc-Leu-Gly-Arg-pNA) . When purified endotoxins were added to human whole blood, the amount of endotoxin recovered in PPP was significantly lower than that in PRP for all endotoxins tested except that from E . coli 0111:B4 and their ability to bind to platelets was varied depending on the species of bacteria from which they were purified . However, the amount of endotoxin in PRP obtained from surgical patients (n = 50) was almost same as that in PPP with a correlation coefficient r = 0.95, indicating that natural endotoxins circulating in human blood may not bind to platelets and that PPP can be used for endotoxin assay as well as PRP. Mol Gen Genet, 1986 Feb, 202(2), 257 - 64 Pleiotropic mutations in appR reduce pH 2.5 acid phosphatase expression and restore succinate utilisation in CRP-deficient strains of Escherichia coli; Touati E et al.; Several strains of Escherichia coli K12 were compared for activity of the periplasmic "pH 2.5 acid phosphatase", an enzyme whose expression is regulated negatively by cyclic AMP . Two distinct enzyme levels differing by about four-fold were observed . This strain-dependent difference does not involve modifications in the structure of the enzyme, but results from a difference in its expression . We show that strains with a high- or a low level of enzyme differ in the gene locus appR located in the 59 min region of the chromosome, a site remote from the structural gene appA; the appR+ versus appR enzyme ratio is 3-4 in wild-type strains, adenylate cyclase-deficient strains (cya) or cyclic AMP receptor protein-deficient strains (crp) grown in rich medium or in glucose minimal medium, but is close to 1 in cya strains in the presence of 0.1 mM cyclic AMP and in wild-type strains grown with succinate as carbon source; in a crp genetic background, appR strains, contrary to appR+ strains, are able to grow on minimal medium with succinate as the sole carbon source . The selection, from an appR+ crp strain, of clones growing on succinate-minimal medium, yielded mutations in the same region of the chromosome and showing the same phenotype as "naturally-occurring" appR strains . All appR strains analysed so far showed other similar deficiencies . The possibility that mutated appR gene products might function as weak substitutes for a functional cAMP-CRP complex is discussed. Mol Gen Genet, 1986 Feb, 202(2), 186 - 93 Isolation of nuclear encoded plastid ribosomal protein cDNAs; Gantt JS et al.; A pea leaf cDNA library was constructed in the expression vector lambda gt11 and screened with antisera raised against proteins extracted from 30S and 50S ribosomal subunits and 70S ribosomes prepared from isolated pea chloroplasts . Six recombinant phage were identified that encoded fusion proteins containing plastid ribosomal protein antigenic determinants . Phage-induced cell lysate proteins, containing the fusion proteins, were bound to nitrocellulose membranes and used as affinity matrices to prepare monospecific antibodies . These antibodies were then used to identify by Western blotting which plastid ribosomal protein shared antigenic determinants with the fusion proteins . cDNA inserts from the antigen-producing phage were used to hybrid-select complementary mRNAs . The cell-free translation products of these mRNAs were added to a pea chloroplast in vitro transport system and imported proteins analyzed by two-dimensional gel electrophoresis . The imported proteins comigrated with the plastid ribosomal proteins that were identified as being antigenically related to the fusion proteins produced by the corresponding recombinant phage . The imported proteins were 3,500-5,500 daltons smaller than their precursors. J Biochem (Tokyo), 1986 Feb, 99(2), 591 - 6 Primary structures of the ColE2-P9 and ColE3-CA38 lysis genes; Toba M et al.; The lysis genes of plasmids ColE2-P9 and ColE3-CA38 were identified by DNA sequencing and electrophoretic analysis of the products of both wild type and artificially introduced ochre mutant genes . The E2 and E3 lysis genes had identical primary structures and were shown to encode 47 amino acids with a calculated molecular weight of 4,861, which is much smaller than that proposed previously for the ColE3-CA38 lysis protein . They are homologous with ColDF13 gene H, except in their 3'-portions . The nine C-terminal amino acids of the E2 and E3 lysis proteins proved to be non-essential for the lysis phenotype. J Biochem (Tokyo), 1986 Feb, 99(2), 365 - 74 Acetyl-CoA-dependent chain elongation of fatty acids in Escherichia coli K-12; Nishimaki T et al.; Crude extract of Escherichia coli was found to elongate medium chain acyl-CoA primers . The reaction products were fatty acids one or two C2 units longer than the primer . Acetyl-CoA acted as the condensing unit in this reaction, while malonyl-CoA did not . The optimal pH for the reaction was 5.0 in 0.1 M citrate-phosphate buffer . NADH was the predominant electron donor for the incorporation of acetyl-CoA into fatty acids, and NADPH was one-third as effective as NADH at pH 5.0 . Acyl carrier protein and cerulenin had no effect on the acetyl-CoA incorporation into the chain elongation products . Acyl-CoA compounds with medium carbon chain lengths proved to be the best as primers, and the maximum incorporation was observed with octanoyl-CoA . N-Ethylmaleimide and p-hydroxymercuribenzoate blocked the chain elongation reaction by inhibiting either condensation or 3-ketoacyl reduction. Gastroenterol Clin Biol, 1986 Feb, 10(2), 117 - 21 C3-mediated phagocytosis induced in murine Kupffer cells by "in vitro" activation with endotoxin; Steffan AM et al.; Binding and phagocytosis of red blood cells by Kupffer cells, mediated by C3 receptors, have been studied by incubating sheep red blood cells opsonized with C3, and Kupffer cells isolated from rat and mouse livers . Sheep red blood cell-binding was followed by internalization and phagocytosis only after preincubation of Kupffer cells with bacterial endotoxin . In both species of cells there was a direct relationship between the value of the phagocytic index and the amount of endotoxin needed to stimulate the cells . Increased phagocytosis was related to an increase in the number of phagocytosing cells; it did not depend on an increase in the number of red blood cells internalized per Kupffer cell . C3-mediated phagocytosis, together with the intrinsic and extrinsic antiviral activities, and the synthesis of interferon which take place in activated cells may play an important role in non-specific immunity. Anal Biochem, 1986 Feb 1, 152(2), 232 - 8 Dideoxy sequencing method using denatured plasmid templates; Hattori M et al.; The dideoxy sequencing method in which denatured plasmid DNA is used as a template was improved . The method is simple and rapid: the recombinant plasmid DNA is extracted and purified by rapid alkaline lysis followed by ribonuclease treatment . The plasmid DNA is then immediately denatured with alkali and subjected to a sequencing reaction utilizing synthetic oligonucleotide primers . It takes only several hours from the start of the plasmid extraction to the end of the sequencing reaction . We examined each step of the procedure, and several points were found to be crucial for making the method reproducible and powerful: (i) the plasmid DNA should be free from RNA and open circular (or linear) DNA; (ii) a heptadecamer rather than a pentadecamer is recommended as a primer; and (iii) the sequencing reaction should be done at 37 degrees C or higher rather than at room temperature . The method enabled us to determine the sequence of more than a thousand nucleotides from a single template DNA. Anal Biochem, 1986 Feb 1, 152(2), 215 - 20 A new rapid procedure for the preparation of plasmid DNA; Edwardson PA et al.; This report describes a simple and efficient procedure for the isolation of plasmid DNA free from chromosomal DNA, cellular RNA, and protein . The technique comprises a modified cleared lysate procedure of D.B . Clewell and D.R . Helinski (1969, Proc . Natl . Acad . Sci . USA, 62, 1159-1166) followed by high-performance liquid chromatography on a Dupont Bioseries GF250 surface stable diol-coated silica gel permeation column (Zorbax) for the final purification of the plasmid DNA . The use of HPLC facilitates rapid and high-resolution separations within 3-4 h . Plasmid DNA produced in this manner retains its biological activity and exhibits yields equal to those obtained by the conventional cesium chloride-ethidium bromide density centrifugation method. Zh Mikrobiol Epidemiol Immunobiol, 1986 Feb, (2), 30 - 4 {Mechanism of the metabolic death of Escherichia coli B cells following freezing and thawing}; Trofimenko AF et al.; The state of E . coli B genome after low-temperature freezing and subsequent thawing (F--T) has been studied . The study has shown that in the process of F--T the DNA of the cells remains intact . But after thawing the incubation of the cells at 37 degrees C leads to the accumulation of ruptures in their DNA and its low-molecular decomposition products . At the same time the processes of the synthesis of DNA and proteins are suppressed to a considerable extent . Repeated F--T cycles enhance, on one hand, the inhibition of the processes of synthesis and, on the other hand, the activation of DNA metabolic decomposition . Such unbalance of the competitive enzymatic systems governing the synthesis and decomposition of DNA, which appears in the process of the revival of the cell from anabiosis, leads supposedly to the accumulation of ruptures and "gaps" in its DNA and, as a result, to the fragmentation of the genome and the death of the cell. J Anim Sci, 1986 Feb, 62(2), 315 - 26 Cold resistance and environmental temperature preference in diarrheic piglets; Balsbaugh RK et al.; Piglets aged 12 to 72 h in which diarrhea had been induced by enteric Escherichia coli infection or sucrose gavage were studied with respect to cold resistance and thermal-circulation index in a 90-min test in a 6 C environment (Exp . 1) and free-choice environmental-temperature preference during a 60-min test in a 24 to 44 C thermocline (Exp . 2) . In Exp . 1, diarrhea lowered the piglet's ability to maintain body temperature during the cold test . Also, diarrheic piglets tended to have lower thermal circulation index values at the end of the cold test, indicative of a greater vasoconstrictive response to the cold environment . In Exp . 2, mean preferred environmental temperatures were 35.7, 34.9 and 34.5 C, respectively, for piglets in sham-control, E . coli-infected and sucrose-gavaged groups . For reason(s) still unknown, diarrheic piglets did not choose to locate themselves in a warmer niche than did normal piglets; in fact, they did the opposite . Results of the two experiments indicate that diarrheic neonatal piglets need even more attention and care in terms of the thermal environment than do healthy ones. J Anim Sci, 1986 Feb, 62(2), 307 - 14 Body weight, total body water and hematocrit in diarrheic piglets; Balsbaugh RK et al.; Piglets aged 12 to 72 h in which diarrhea had been induced by enteric Escherichia coli infection or sucrose gavage were studied with respect to body weight, total body water concentration (determined by tritiated-water dilution) and hematocrit . Sucrose-induced diarrhea reduced body weight by 13 to 17%, and E . coli diarrhea, by 8 to 9% . Neither age nor diarrheal treatment affected total body water concentration, although diarrheic piglets tended to have higher hematocrit values at all ages . There was a significant daily cycle in the piglets' hematocrit values, so hematocrit might be a less valid reflector of neonates' whole body hydration status than of adults' . It was concluded that diarrheic neonatal piglets lose body water and dry matter in a ratio similar to that of normal body water and dry matter concentrations, thus their bodies have normal total body water concentrations and normal average specific heat values. DNA, 1986 Feb, 5(1), 37 - 51 Construction of a systematic set of tRNA mutants by ligation of synthetic oligonucleotides into defined single-stranded gaps; Cline SW et al.; A series of mutant tRNA genes has been constructed by site-directed mutagenesis in pOP203, a colE1 derivative carrying a transcription unit under control of the lacUV5 promoter . These mutant genes include all possible amber suppressing variants of tRNATrp with single nucleotide substitutions at anticodon loop positions 32, 37, and 38 (numbered from the 5' end), and all possible paired base substitutions in the three base pairs nearest the anticodon loop . G at position 38 was not recovered as a single mutation, but rather in conjunction with an undirected mutation to T at position 32 . The singly mutated G38 tRNA may not be active, though all the other tRNA derivatives are functional in the translation of amber codons . To construct the mutants, we ligated a synthetic deoxyoligonucleotide into a precisely formed single-stranded gap covering the anticodon arm region DNA, in an otherwise double-stranded fragment containing the tRNATrp gene . The resulting heteroduplex was then ligated into the plasmid and introduced into Escherichia coli . This method of mutagenesis is simple, reproducible, and highly tolerant of varying degrees of heteroduplex in the gap, variations in temperature of ligation, and changes in the oligonucleotide concentration . Mutagenesis does not require a 5'-phosphorylated oligonucleotide . These qualities suit the gap method for intensive study of a region by site-directed mutagenesis. DNA, 1986 Feb, 5(1), 21 - 8 Synthesis of bovine prolactin in Escherichia coli; Luck DN et al.; Transformation of Escherichia coli cells with a recombinant plasmid (pESP4) containing a modified bovine prolactin cDNA clone in a pEMBL vector resulted in efficient expression of prolactin . The cDNA was modified by removal of a 5' nontranslated sequence as well as the sequence that specified the signal peptide of preprolactin . To achieve a high level of synthesis, a sequence of 30 nucleotides in the cDNA, which included the ATG initiation codon and the first 7 codons of mature bovine prolactin, was replaced by a chemically synthesized oligonucleotide duplex . The sequence of this duplex was chosen from the consensus sequence around the initiation codon of E . coli genes and by the amino acid sequence of the protein . Prolactin, a single-chain polypeptide of molecular weight 24,000, was identified by Coomassie Blue staining of NaDodSO4-polyacrylamide gels of total protein from transformed E . coli cells, and by reaction with specific antibody . Increased levels of expression of the hormone, corresponding to the form secreted from the pituitary, were observed in the presence of isopropyl-beta-D-thiogalactopyranoside (IPTG). Biophys J, 1986 Feb, 49(2), 425 - 35 Theoretical evaluation of transcriptional pausing effect on the attenuation in trp leader sequence; Suzuki H et al.; The effect of transcriptional pausing on attenuation is investigated theoretically on the basis of the attenuation control mechanism presented by Oxender et al . (Oxender, D . L., G . Zurawski, and C . Yanofsky, 1979, Proc . Natl . Acad . Sci . USA . 76:5524-5528) . An extended stochastic model including the RNA polymerase pausing in the leader region is developed to calculate the probability of relative position between the RNA polymerase transcribing the trp leader sequence and the ribosome translating the transcript . The present study results in a new rationale that the transcriptional pausing site in the leader sequence makes the attenuation control both more sensitive as an on/off switch and less sensitive to variations in the concentration of cellular metabolites not connected with the need for expressing, or not expressing, the particular operon . It is also proposed that the transcriptional pausing diminishes the dependence of attenuation control characteristics on the number of nucleotides in the leader sequence . This result may be useful for understanding the attenuation control efficiencies of other amino acid leader sequences with different lengths of nucleotides. Am J Vet Res, 1986 Feb, 47(2), 385 - 8 Adhesion of K99-positive Escherichia coli to intestinal brush borders of pigs; Grimes SD et al.; Ileal samples from 242 pigs, collected at 3 Michigan slaughterhouses, were studied to determine the prevalence of intestinal receptors for K99-positive Escherichia coli . A brush border adhesion test was used to identify the receptors . Of the 242 samples examined, receptors were demonstrated in 230 (95%) . After storage of brush border preparations at 4 C, bacterial aggregates lacking identifiable brush border fragments were present in samples tested for adhesion, indicating that K99 receptors may be released from brush border membranes . Seemingly, most, if not all, pigs have intestinal receptors for K99 pili, and an inheritance pattern similar to that observed for K88 receptors probably does not exist for K99 receptors. Am J Vet Res, 1986 Feb, 47(2), 210 - 2 Comparative prevalence of four enterotoxin genes among Escherichia coli isolated from swine; Moon HW et al.; Presence of Escherichia coli enterotoxin genes LT (heat-labile enterotoxin), STaP (heat-stable enterotoxin a, porcine genotype), STaH (heat-stable enterotoxin a, human genotype), and STb (heat-stable enterotoxin b) among 874 swine isolates of E coli was determined, using DNA probes and the DNA colony hybridization technique . Of the 874 isolates evaluated, 45% hybridized with at least one of the enterotoxin gene probes and were designated as enterotoxigenic E coli (ETEC) . Eighty-five percent of the ETEC were from pigs with enteric colibacillosis . The remaining 15% were from pigs with edema disease or various other diseases, and from healthy swine . Seventy-four percent of the ETEC hybridized with the STb probe, 52% with STaP, and 31% with LT; ETEC did not hybridize with the STaH probe . Most of the ETEC hybridized with more than one enterotoxin gene probe . Isolates that hybridized with the LT probe also hybridized with STb . The most prevalent gene combination was LT-STb . However, 35% of the ETEC from neonatal (less than or equal to 1 week old) swine with enteric colibacillosis were of the STaP-only genotype, and 33% of the ETEC from older swine with enteric colibacillosis were of the STb-only genotype. Proc Natl Acad Sci U S A, 1986 Feb, 83(4), 952 - 6 Mutant ras-encoded proteins with altered nucleotide binding exert dominant biological effects; Sigal IS et al.; We report that residues Lys-16 and Asp-119 play critical roles in the guanine nucleotide binding and, consequently, the biological function of the Ha-ras-encoded protein (Ha) . Substitution of an asparagine residue for Lys-16 reduces the affinity of Ha for GDP and GTP by a factor of 100 but does not alter the specificity of nucleotide binding . The replacement of Asp-119 with an alanine residue reduces the affinity of Ha for GDP and GTP by a factor of 20 and reduces the relative affinity of Ha for GDP over IDP from 200-500 to 10 . Based on these observations, a structural model for the GDP/GTP-binding site of Ha is proposed . By microinjecting purified proteins into NIH 3T3 cells, we observed that the ability of {Ala119}Ha to induce changes characteristic of cellular transformation was much greater than that of normal Ha and similar to that of the oncogenic {Val12, Thr59}Ha . In this assay, {Asn16}Ha and {Val12, Asn16, Thr59}Ha were similar in potency to normal Ha . In yeast cells, Ha proteins with reduced nucleotide affinity exert a dominant temperature-dependent lethality that is avoided by the coexpression of the activated yeast ras gene {Ala18, Val19}RAS2 . We interpret the biological consequences of reducing the nucleotide affinity of ras proteins in terms of two opposing factors: a growth-promoting effect, resulting from an increase in the GDP-GTP exchange rate, and a growth-limiting effect, resulting from an increase in the nucleotide-free ras protein species. Proc Natl Acad Sci U S A, 1986 Feb, 83(4), 877 - 81 Isolation of the gene encoding yeast single-stranded nucleic acid binding protein 1; Jong AY et al.; A yeast gene encoding SSB-1, a single-stranded nucleic acid binding protein, has been isolated by screening a lambda gt11 genomic DNA library . The gene is located on a 1.84-kilobase chromosomal Bgl II-BamHI fragment . Yeast strains carrying the high-copy-number vector YEp24 with an SSB1 gene insert overproduce SSB-1 3-fold and SSB-1 mRNA 10-fold . A typical haploid cell contains about 20,000 molecules of SSB-1; thus, the cells can tolerate up to 60,000 copies . Yeast SSB-1 was expressed in Escherichia coli cells by using a phage T7 expression system . Spores containing the gene disrupted at a point within the coding sequence germinate and grow normally; thus, the gene is not essential . Protein blots show that no SSB-1 or novel immunologically related species that might retain SSB-1 activity are present in cells containing the disrupted SSB1 genes . Southern analysis and protein blots suggest the presence in yeast of a second, related, but nonidentical gene and two immunologically related proteins of 55 kDa and 75 kDa. Proc Natl Acad Sci U S A, 1986 Feb, 83(4), 867 - 71 Evolution in biosynthetic pathways: two enzymes catalyzing consecutive steps in methionine biosynthesis originate from a common ancestor and possess a similar regulatory region; Belfaiza J et al.; The metC gene of Escherichia coli K-12 was cloned and the nucleotide sequence of the metC gene and its flanking regions was determined . The translation initiation codon was identified by sequencing the NH2-terminal part of beta-cystathionase, the MetC gene product . The metC gene (1185 nucleotides) encodes a protein having 395 amino acid residues . The 5' noncoding region was found to contain a "Met box" homologous to sequences suggestive of operator structures upstream from other methionine genes that are controlled by the product of the pleiotropic regulatory metJ gene . The deduced amino acid sequence of beta-cystathionase showed extensive homology with that of the MetB protein (cystathionine gamma-synthase) that catalyzes the preceding step in methionine biosynthesis . The homology strongly suggests that the structural genes for the MetB and MetC proteins evolved from a common ancestral gene. Biull Eksp Biol Med, 1986 Feb, 101(2), 147 - 9 {Efficacy of using litonit in the combined therapy of infectious-inflammatory kidney lesions in alcoholism}; Kostev FI; The results of 250 experiments suggest the advisability of using a new antialcoholic metabolite drug, litonit, in combined therapy of infectious-inflammatory kidney disorders occurring in alcoholism . The course of litonit injections does not only increase considerably the content of oxidized and reduced nicotinamide coenzymes, but also normalizes the number of IgA, IgG and IgM . Certain drug-induced functional and immunological shifts are now believed to indicate the increase in the functional renal capacity and enhanced kidney resistance to inflammation. Am J Pathol, 1986 Feb, 122(2), 268 - 76 The reduction of inflammatory responses in lipopolysaccharide-tolerant eyes; Bhattacherjee P et al.; The development of tolerance induced by subcutaneous or intraocular injections of lipopolysaccharide (LPS, Escherichia coli) in rat eyes has been studied . In addition, the ocular inflammatory responses to the reversed passive Arthus (RPA) reaction in the tolerant eyes were investigated . The tolerance in the eye after a single injection of LPS persisted for at least 42 days . Up to 42 days, vasodilatation, disruption of the blood-aqueous barrier, and leukocyte accumulation in the anterior chamber after a second injection of LPS were inhibited . Unilateral intraocular injection of LPS produced local tolerance, which was not observed in the contralateral eyes . The inflammatory reactions in response to RPA in the LPS-tolerant eyes were also significantly attenuated . It was also found that inflammatory reactions induced by RPA or 16,16-dimethyl prostaglandin E2 had no inhibitory effect on the responses to subsequent RPA or LPS administration, which indicated that initial inflammatory reactions do not render the tissues refractory to the response to a second stimuli . The results of this study suggest that some, as yet unknown, local changes in the ocular tissues caused by LPS may be involved in the development of tolerance. Virology, 1986 Feb, 149(1), 83 - 90 Functional expression in Escherichia coli of cloned reovirus S1 gene encoding the viral cell attachment protein sigma 1; Masri SA et al.; A cDNA clone encompassing the complete reovirus (serotype 3) S1 gene was constructed using two partial clones containing overlapping sequences . The gene (with the first 15 bases at the 5' end up to and including the first ATG removed) was then inserted in frame into the lac cloning site of the pUC13 plasmid and expressed in Escherichia coli as a fusion product under control of the lac promoter . The expressed product can be immunoprecipitated as a 47,000-mol wt (47K) protein using several monoclonal anti-sigma 1 antibodies . Like authentic soluble sigma 1 from reovirus-infected cells, the expressed protein is capable of attaching to mammalian cells (mouse L fibroblasts) in a specific manner and of competing with reovirus particles for cell surface receptors . Lysates prepared from the recombinant plasmid-transformed, but not those from pUC13-transformed E . coli cells, were also found to exhibit hemagglutinating (HA) activity . Such hemagglutination was inhibited by a monoclonal anti-sigma 1 antibody previously shown to inhibit reovirus HA activity . It is concluded that both the host cell attachment domain and the hemagglutination domain on the expressed protein are functionally intact. Proc Natl Acad Sci U S A, 1986 Feb, 83(3), 561 - 5 Short synthetic oligodeoxyribonucleotide leader sequences enhance accumulation of human proinsulin synthesized in Escherichia coli; Sung WL et al.; Enhanced accumulation of human proinsulin synthesized in Escherichia coli has been achieved by inserting a short leader of homooligopeptide at the amino end of proinsulin . Out of 20 amino acid oligomers studied, (Ala)6, (Asn)6, (Cys)7, (Gln)7, (His)6, (Ser)6, and (Thr)6 leaders were the most effective, with the yield of proinsulin ranging between 6% and 26% of the total bacterial protein . These constructions were made by inserting a synthetic oligodeoxyribonucleotide duplex, coding for a small homooligopeptide, between a synthetic proinsulin gene and an eight-codon beta-galactosidase gene residue in vector pUC8 . Cyanogen bromide cleavage of the 102 amino acid fused polypeptide yielded a species identical to authentic proinsulin, as judged by NaDodSO4/PAGE and radioimmunoassay. Mutat Res, 1986 Feb, 173(2), 89 - 91 The relationship between survival and mutagenesis in Escherichia coli after fractionated ultraviolet irradiation; Dzidic S et al.; The relationship between survival and mutagenesis in Escherichia coli after fractionated ultraviolet (UV) irradiation was studied . The cells were incubated either in buffer or nutrient media . Regardless of incubation conditions, greater survival is observed after fractionated irradiation than after acute irradiation . When the cells are incubated in buffer, UV mutagenesis decreases with an increase in the number of dose fractions . However, when the cells are cultivated in nutrient media, the increased survival (i.e., the enhanced capacity for repair) is coupled with the enhanced capacity for UV mutagenesis . We, therefore, assume that during incubation in nutrient media, fractionated irradiation leads to full and prolonged expression of all UV-inducible (SOS) genes, including those required for mutagenesis. J Surg Res, 1986 Feb, 40(2), 95 - 104 Extravascular lung water measurement in septic sheep; Andreasson S et al.; Sheep were prepared with a chronic lung lymph fistula and studied unanesthetized following septicemia by infusion of live Escherichia coli 10(9) ml/kg bw or injection of oleic acid 0.05 ml/kg bw . Extravascular lung water (EVTV) was measured with thermal-dye technique and compared to gravimetrically measured lung water (EVWV) . Septic sheep had increased pulmonary artery pressure, reduced mean arterial blood pressure and reduced cardiac output . In control animals there was a correlation between EVTV-EVWV of r = 0.70 . In animals given oleic acid lungs were macroscopically edematous and the correlation was r = 0.93 . In septic sheep, however, no correlation could be found between EVTV and EVWV (r = -0.25) . The thermal-dye technique was found to give erroneously high values . This finding could probably be due to erythrostasis and leukocyte plugging with uneven perfusion and prolonged transit times due to reduced cardiac output. J Trauma, 1986 Feb, 26(2), 157 - 62 Topical ibuprofen decreases thromboxane release from the endotoxin-stimulated burn wound; Katz A et al.; A full-thickness burn wound in adult sheep releases prostanoids when they are injected locally with E . coli endotoxin, 2 micrograms/kg, resulting in an increase in pulmonary artery pressure (Ppa) from 20 +/- 3 to 34 +/- 5 mm Hg, and a decrease in mean arterial oxygen tension (PaO2) from 88 +/- 6 to 70 +/- 5 torr; this corresponds to an increase in venous plasma TxB2 content from a baseline of 220 +/- 79 pg/ml to 440 +/- 90 pg/ml . Burn prostanoid production, measured in lymph, increased ten- to fifteen-fold for both thromboxane A2, measured as TxB2, and prostacyclin, 6-keto-PGF1 alpha . The intravenous administration of ibuprofen, 12.5 mg/kg, eliminated both the increase in Ppa and decrease in PaO2 as well as the increase in burn lymph prostanoids . However, plasma prostanoids were also decreased below baseline, a potentially deleterious effect . A topical ibuprofen cream, 5% ibuprofen in a water-soluble ester, was applied to the burn hide every 6 hrs x 4 after which endotoxin was again injected below the hide . The pulmonary dysfunction was prevented as was the increase in plasma TxB2 with the value remaining at baseline . Burn lymph levels were only increased three- to five fold . Ibuprofen levels in burn lymph were maintained at 1-2 mcg/ml . The addition of the cream to the burn, however, did increase the wound bacterial content to 10(5)-10(7) bacteria/gram of tissue compared to 10(2)-10(3) for the dry, untreated burn, probably due to softening of the burn . Topically applied ibuprofen, therefore, can decrease burn wound prostanoid production from local endotoxin, preventing lung dysfunction.(ABSTRACT TRUNCATED AT 250 WORDS) J Bacteriol, 1986 Feb, 165(2), 631 - 7 Discontinuity in DNA replication during expression of accumulated initiation potential in dnaA mutants of Escherichia coli; Helmstetter CE et al.; Potential for initiation of chromosome replication present in temperature-sensitive, initiation-defective dnaA5 mutants of Escherichia coli B/r incubated at nonpermissive temperature was expressed by shifting to a more permissive temperature (25 degrees C) . Upon expression of initiation potential, the rate of {3H}thymidine incorporation varied in a bimodal fashion, i.e., there was an initial burst of incorporation, which lasted 10 to 20 min, then a sudden decrease in incorporation, and finally a second rapid increase in incorporation . Analyses of this incorporation pattern indicated that a round of replication initiated upon expression of initiation potential, but DNA polymerization stopped after replication of 5 to 10% of the chromosome . This round of replication appeared to resume about 30 min later coincident with initiation of a second round of replication . The second initiation was unusually sensitive to low concentrations of novobiocin (ca . 1 microgram/ml) when this inhibitor was added in the presence of chloramphenicol . In the absence of chloramphenicol, novobiocin at this concentration had no detectable effect on DNA replication . It is suggested that cis-acting inhibition, attributable to an attempted second initiation immediately after the first, caused the first round to stall until both it and the second round could resume simultaneously . This DNA replication inhibition, probably caused by overinitiation, could be a consequence of restraints on replication in the vicinity of oriC, possibly topological in nature, which limit the minimum interinitiation interval in E . coli. J Bacteriol, 1986 Feb, 165(2), 608 - 11 Effect of a 2-methylthio-N6-isopentenyladenosine deficiency on peptidyl-tRNA release in Escherichia coli; Petrullo LA et al.; We examined the effect of miaA, a mutation conferring a deficiency in 2-methylthio-N6-isopentenyladenosine in tRNA, on patterns of peptidyl-tRNA accumulation in Escherichia coli strains deficient in peptidyl-tRNA hydrolase activity . A specific reduction in peptidyl-tRNA accumulation was seen for tRNAs which normally contain the 2-methylthio-N6-isopentenyladenosine modification . These results provide new evidence in support of the ribosome editor model, which links peptidyl-tRNA release to mistranslation events. J Bacteriol, 1986 Feb, 165(2), 474 - 82 Precursor for elongation factor Tu from Escherichia coli; Lifson ER et al.; The tufA gene, one of two genes in Escherichia coli encoding elongation factor Tu (EF-Tu), was cloned into a ColE1-derived plasmid downstream of the lac promoter-operator . In cells carrying this plasmid, the synthesis of EF-Tu was increased four- to fivefold upon the addition of isopropyl-beta-D-thiogalactopyranoside (an inducer of the lac promoter) . This condition led to the synthesis of a novel protein, called pTu, which comigrated with EF-Tu on a sodium dodecyl sulfate-polyacrylamide gel but could be separated on an isoelectric focusing gel, since pTu is slightly more basic than EF-Tu . The synthesis of pTu could also be induced by the synthesis of a hybrid protein containing just the amino-terminal half of the EF-Tu protein . Genetic data suggest that pTu is the product of the tufA and tufB genes . The pTu protein was shown to be related to EF-Tu by gel electrophoresis of tryptic peptides . Pulse-chase experiments suggest that pTu is a precursor of EF-Tu . Interestingly, in a classic membrane fractionation procedure, EF-Tu was found in the cytosolic frac