Eur J Biochem, 1989 Sep 15, 184(2), 345 - 52 Interaction of elongation factor Tu from Escherichia coli with aminoacyl-tRNA carrying a fluorescent reporter group on the 3' terminus; Ott G et al.; Transfer ribonucleic acids containing 2-thiocytidine in position 75 ({s2C}tRNAs) were prepared by incorporation of the corresponding cytidine analogue into 3'-shortened tRNA using ATP(CTP):tRNA nucleotidyltransferase . {s2C}tRNA was selectively alkylated with fluorescent N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine (1,5-I-AEDANS) on the 2-thiocytidine residue . The product {AEDANS-s2C}aminoacyl-tRNA, forms a ternary complex with Escherichia coli elongation factor Tu and GTP, leading to up to 130% fluorescence enhancement of the AEDANS chromophore . From fluorescence titration experiments, equilibrium dissociation constants of 0.24 nM, 0.22 nM and 0.60 nM were determined for yeast {AEDANS-s2C}Tyr-tRNATyr, yeast Tyr-tRNATyr, and the homologous E . coli Phe-tRNAPhe, respectively, interacting with E . coli elongation factor Tu.GTP . The measurement of the association and dissociation rates of the interaction of {AEDANS-s2C}Tyr-tRNATyr with EF-Tu.GTP and the temperature dependence of the resulting dissociation constants gave values of 55 J mol-1 K-1 for delta S degrees' and -34.7 kJ mol-1 for delta H degrees' of this reaction. Biochem Biophys Res Commun, 1989 Sep 15, 163(2), 851 - 9 Tetramer formation of a variant type human transthyretin (prealbumin) produced by Escherichia coli expression system; Furuya H et al.; A variant of human transthyretin(TTR, prealbumin) with methionine for valine substitution at position 30 is a major component of amyloid fibrils found in patients of familial amyloidotic polyneuropathy(FAP) type I, an autosomal dominant genetic disease . But the molecular nature of the variant TTR has been obscure, because most of plasma TTR from FAP patients is a mixture of variant and wild type TTR and no pure preparation of the variant has been available . For this reason, we constructed a system in which the variant type TTR was efficiently synthesized . In this system, the recombinant variant TTR was first synthesized as a fusion protein with E . coli outer membrane protein A (ompA) signal peptide, processed to eliminate the signal peptide and finally secreted to the culture medium . The final concentration of the recombinant variant TTR in the medium was about 5 mg/l . SDS polyacrylamide gel electrophoresis and gel filtration analysis suggested that the recombinant variant TTR can form tetramer as seen for native one . Purification of the protein was accomplished by only two steps of chromatography. Biochim Biophys Acta, 1989 Sep 15, 992(3), 397 - 9 Branched-chain-amino-acid aminotransferase assay using radioisotopes; Airas RK; A method for the assay of the activity of branched-chain-amino-acid aminotransferase from Escherichia coli has been developed . Radioactive isoleucine is used, the radioactive oxoacid formed in the enzymic reaction is converted to its p-nitrophenylhydrazone, and the hydrazone is extracted into toluene based scintillation liquid . The small reaction tubes containing the toluene layer and the reaction mixture as a water layer are placed into liquid scintillation vials and counted for radioactivity . The radioactive amino acid remaining in the water layer causes only a rather low background. J Biol Chem, 1989 Sep 15, 264(26), 15674 - 80 Crystallographic quaternary structural analysis of AMP nucleosidases from Escherichia coli and Azotobacter vinelandii; Giranda VL et al.; Adenosine monophosphate nucleosidases from Azotobacter vinelandii and Escherichia coli have been studied crystallographically to determine their quarternary structures . Preliminary characterization of the A . vinelandii enzyme shows that the crystals are monoclinic, C2 with a = 347 A, b = 204 A, c = 114 A, and beta = 91.7 degrees . The asymmetric unit contains 12 or 9 subunits of Mr 54,000 . Self-rotation functions with data from the AMP nucleosidases from A . vinelandii and from E . coli (Giranda, V . L., Berman, H . M., and Schramm, V . L . (1986) J . Biol . Chem . 261, 15307-15309) are consistent with the monomers arranged as hexamers with point symmetry 32 . The hexamers are arranged in the unit cells so that crystallographic 2-fold axes are coincident with the local 2-folds of the point group 32. J Biol Chem, 1989 Sep 15, 264(26), 15535 - 41 Purification and characterization of SopA and SopB proteins essential for F plasmid partitioning; Mori H et al.; Mini-F plasmid has the trans-acting genes sopA and sopB and the cis-acting site sopC which are essential for accurate partitioning of plasmid DNA molecules into both daughter cells . In this study, we purified independently SopA and SopB proteins, analyzed the in vitro DNA-binding activity of these proteins by the gel retardation assay, and determined the precise binding sites of DNA by the footprinting method . SopA binds to four repeated sequences (CTTTGC) located in the promoter-operator region of the sopAB operon . The SopA binding activity is enhanced by the addition of SopB protein . SopB protein itself does not bind to this DNA region . These results suggest that the complex of SopA and SopB proteins autoregulate the expression of the sopA-sopB operon . On the other hand, SopB protein binds to the sopC region, in which 12 direct repeats of 43-base pairs nucleotides exist . SopB protein recognizes the inverted repeats of 7 base pairs in each direct repeats . SopA protein does not affect the SopB binding activity to the sopC DNA segment. J Biol Chem, 1989 Sep 15, 264(26), 15169 - 72 Essential role of T4 phage deoxycytidylate hydroxymethylase in a multienzyme complex for deoxyribonucleotide synthesis; Thylen C et al.; Several enzymes of deoxyribonucleoside triphosphate (dNTP) biosynthesis interact in T4 phage-infected Escherichia coli to form a multienzyme aggregate, the T4 dNTP synthetase complex . To test the specificity of enzyme interactions seen in vitro with this complex, we analyzed bacteria infected with four T4 gene 42 amber mutants, which specify truncated forms of dCMP hydroxymethylase, one of the constituent enzymes in the complex . Mutants that specify a nearly full length gene 42 product can form an intact complex, as revealed by two criteria: kinetic coupling among constituent enzymes in crude extracts of infected bacteria, and co-elution of enzyme activities from a gel filtration column . By these criteria, mutations that specify truncated proteins less than half the size of the full length protein cause disruption of the complex . These findings suggest that an enzymatically inactive form of dCMP hydroxymethylase can contribute toward assembly of an intact complex, so long as the incomplete protein is of sufficient size to fold normally, allowing interaction with other proteins in the complex. Biochem Biophys Res Commun, 1989 Sep 15, 163(2), 780 - 7 A novel ubiquitous protein 'chaperonin' supports the endosymbiotic origin of mitochondrion and plant chloroplast; Gupta RS et al.; The deduced amino acid sequences for a major mitochondrial protein (designated P1, related to the 'chaperonin' family of proteins) from human and Chinese hamster cells show extensive similarity (greater than 60% identity observed over the entire length) with a related protein present in evolutionarily as divergent organisms as Escherichia coli, Coxiella burnetii, Mycobacterium species, cyanobacteria as well as in yeast mitochondria and higher plant chloroplasts . Of the different groups of bacteria for which sequence data is available, maximum similarity of the mammalian/yeast P1 protein is observed with the corresponding protein from purple bacteria (especially C . burnetii) while the protein from plant chloroplasts exhibited highest similarity with the corresponding protein from cyanobacteria . The sequence data for this protein thus support the contention that the endosymbiont that gave rise to mitochondrion was a member of purple bacteria, while plant chloroplast originated from a member of the cyanobacterial lineage. Eur J Biochem, 1989 Sep 15, 184(2), 361 - 5 Synthesis of a gene for the protein kinase domain of the epidermal growth factor receptor and its expression in Escherichia coli; Farrow SN et al.; A gene encoding the protein kinase domain of the epidermal growth factor receptor has been chemically synthesised, cloned and expressed in Escherichia coli . The 942-base-pair gene was constructed by enzymatic ligation of 56 oligonucleotides and cloned into an expression vector downstream of the E . coli trp promoter . Production of active gene product was confirmed by means of a protein kinase assay, demonstrating that the enzymatic activity of the protein kinase domain of the epidermal growth factor receptor is retained after expression in E . coli. J Biol Chem, 1989 Sep 15, 264(26), 15475 - 82 Synthesis in Escherichia coli of GTPase-deficient mutants of Gs alpha; Graziano MP et al.; We have reduced the GTPase activity of the alpha subunit of Gs, the guanine nucleotide-binding regulatory protein that stimulates adenylyl cyclase, by introduction of point mutations analogous to those described in p21ras . Mutants G49V and Q227L differ from the wild type protein in the substitution of Val for Gly49 and Leu for Gln227, respectively (analogous to positions 12 and 61 in p21ras) . Wild type and mutant proteins were synthesized in Escherichia coli, purified, and characterized . The rate constants for dissociation of GDP from G49V recombinant Gs alpha (rGs alpha) (0.47/min) and Q227L rGs alpha (0.23/min) differ by no more than 2-fold from that observed for the wild type protein (0.5/min) . In marked contrast, the rate constants for hydrolysis of GTP by G49V rGs alpha (0.78/min) and Q227L rGs alpha (0.03-0.06/min) are 4-fold and roughly 100-fold slower than that for wild type rGs alpha (3.5/min) . These reductions in the rate of hydrolysis of GTP result in significant fractional occupancy of these proteins by GTP in the presence of the nucleotide, 0.37 for G49V rGs alpha and 0.78 for Q227L rGs alpha, compared to 0.05 for wild type rGs alpha . When reconstituted with cyc- (Gs alpha-deficient) S49 cell membranes or purified adenylyl cyclase, both mutant proteins stimulate adenylyl cyclase activity in the presence of GTP to a much greater extent than does wild type Gs alpha; their maximal ability to activate the enzyme is largely unaltered . The fractional ability of a given Gs alpha polypeptide to active adenylyl cyclase in the presence of GTP correlates well with the fractinal occupancy of the protein by the nucleotide . The mutant subunits appear to interact normally with G protein beta gamma subunits, and their ability to activate adenylyl cyclase is enhanced by interaction with beta-adrenergic receptors . These results indicate that the structural analogy that has been inferred between the guanine nucleotide-binding domains of G proteins and the p21ras family is at least generally correct . They also provide confirmation of the kinetic model of G protein function and document mutations that permit the expression in vivo of constitutively activated G protein alpha subunits. J Biol Chem, 1989 Sep 15, 264(26), 15398 - 403 Thermodynamic and spectroscopic characteristics of the cytochrome bc1 complex . Role of quinone in the behavior of cytochrome b562; Salerno JC et al.; Cytochrome b562 does not behave as a single independent thermodynamic component in preparations of purified quinol cytochrome c reductase . This effect is much more pronounced in quinone sufficient preparations; in such preparations, the epr spectrum of the cytochrome is Eh sensitive, with a peak shift from g = 3.42 to 3.48 occurring as the potential is lowered from 100 mV to 0 mV . The peak shift is dependent on the presence of quinone and can be restored to quinone-depleted preparations by supplementation with ubiquinol 2 if phospholipid depletion is not too severe . The results suggest that cytochrome b562 is strongly interacting with the Qc quinone binding site. J Biol Chem, 1989 Sep 15, 264(26), 15376 - 83 Kinetic characterization of the unisite catalytic pathway of seven beta-subunit mutant F1-ATPases from Escherichia coli; al-Shawi MK et al.; We have studied the kinetics of "unisite" ATP hydrolysis and synthesis in seven mutant Escherichia coli F1-ATPase enzymes . The seven mutations are distributed over a 105-residue segment of the catalytic nucleotide-binding domain in beta-subunit and are: G142S, K155Q, K155E, E181Q, E192Q, M209I, and R246C . We report forward and reverse rate constants and equilibrium constants in all seven mutant enzymes for the four steps of unisite kinetics, namely (i) ATP binding/release, (ii) ATP hydrolysis/synthesis, (iii) Pi release/binding, and (iv) ADP release/binding . The seven mutant enzymes displayed a wide range of deviations from normal in both rate and equilibrium constants, with no discernible common pattern . Notably, steep reductions in Kd ATP were seen in some cases, the value of Kd Pi was high, and K2 (ATP hydrolysis/synthesis) was relatively unaffected . Significantly, when the data from the seven mutations were combined with previous data from two other E . coli F1-beta-subunit mutations (D242N, D242V), normal E . coli F1, soluble and membranous mitochondrial F1, it was found that linear free energy relationships obtained for both ATP binding/release (log k+1 versus log K1) and ADP binding/release (log k-4 versus log K-4) . Two conclusions follow . 1) The seven mutations studied here cause subtle changes in interactions between the catalytic nucleotide-binding domain and substrate ATP or product ADP . 2) The mitochondrial, normal E . coli, and nine total beta-subunit mutant enzymes represent a continuum in which subtle structural differences in the catalytic site resulted in changes in binding energy; therefore insights into the nature of energy coupling during ATP hydrolysis and synthesis by F1-ATPase may be ascertained by detailed studies of this group of enzymes. Biochem Biophys Res Commun, 1989 Sep 15, 163(2), 739 - 45 Expression of human poly(ADP-ribose) polymerase with DNA-dependent enzymatic activity in Escherichia coli; Ikejima M et al.; A cDNA for human poly(ADP-ribose) polymerase was inserted into a plasmid, transfected and expressed in E . coli . A lysate of the E . coli cells containing the expression plasmid reacted with antibody against human poly(ADP-ribose) polymerase and synthesized poly(ADP-ribose) . The partially purified poly(ADP-ribose) polymerase expressed in E . coli had the same molecular weight and enzymological properties as human placental poly(ADP-ribose) polymerase, including affinity for NAD, turnover number and DNA-dependency for activity . This expression system should be useful for structure-function analysis of poly(ADP-ribose) polymerase. Eur J Biochem, 1989 Sep 15, 184(2), 367 - 74 Human immunodeficiency virus reverse transcriptase expressed in transformed yeast cells . Biochemical properties and interactions with bovine tRNALys; Sallafranque-Andreola ML et al.; Human immunodeficiency virus (HIV) reverse transcriptase has been purified from yeast transformed by an autoreplicating plasmid containing the retroviral DNA polymerase gene . The previously described purification procedure for the yeast-expressed reverse transcriptase {Barr, P.J., Power, M.D., Chun Ting Lee-Ng, Gibson, H . & Luciw, P . (1987) Bio/Technology 5, 486-489} has been substantially modified, leading to an increased yield and a higher degree of purity . Several biochemical properties of the enzyme are described (template specificity, effect of DNA synthesis inhibitors); interestingly, HIV reverse transcriptase is highly resistant to N-ethylmaleimide . A complex between the human retroviral enzyme and the bovine tRNALys was shown, using a direct approach, by glycerol gradient centrifugation, as well as by the protective and specific effect of the tRNALys against enzyme inactivation by thermal denaturation and trypsin digestion . A competitive type of inhibition of HIV reverse transcriptase by tRNALys, but not by tRNAVal, is observed when viral RNA or activated DNA are used as templates. Eur J Biochem, 1989 Sep 15, 184(2), 353 - 9 Isolation and characterisation of a cDNA clone for a chlorophyll synthesis enzyme from Euglena gracilis . The chloroplast enzyme hydroxymethylbilane synthase (porphobilinogen deaminase) is synthesised with a very long transit peptide in Euglena; Sharif AL et al.; A cDNA expression library was constructed from light-grown Euglena gracilis poly(A)-rich RNA in lambda gt11 . Antibodies to Euglena hydroxymethylbilane synthase, the third enzyme in the porphyrin biosynthetic pathway, were used to screen the library and a clone encoding part of the sequence of hydroxymethylbilane synthase was identified . This was used to rescreen the library and a full-length clone was isolated, which encoded not only the entire mature protein (Mr 36,927), but also an N-terminal extension of 139 amino acids . The deduced Mr of the whole polypeptide is 51,744, which corresponds to the size of the protein immunoprecipitated from the translation products of Euglena poly(A)-rich RNA . The mature protein is 60-70% similar to hydroxymethylbilane synthase from human erythrocytes and Escherichia coli . The sequence of the N-terminal extension has similarities to both the transit peptides of chloroplast proteins and those for the endoplasmic reticulum . This is the first report both of a cDNA clone for an enzyme of the chlorophyll biosynthetic pathway and of a putative transit peptide for a nuclear-encoded Euglena protein. J Immunol, 1989 Sep 15, 143(6), 2006 - 12 The mapping of epitopes of the 18-kDa protein of Mycobacterium leprae recognized by murine T cells in a proliferation assay; Harris DP et al.; The 18-kDa protein of Mycobacterium leprae was purified from recombinant plasmids pUL108 and pML-3 grown in Saccharomyces cerevisiae and Escherichia coli, respectively . Significant lymphoproliferative responses were observed when T cells from immunized mice were challenged in culture with purified 18-kDa protein . Synthetic peptides have been prepared that span most of the 148 amino acid residues that constitute the sequence of the 18-kDa protein and used to map epitopes recognized by T cells . When mice were immunized with 18-kDa protein and lymph node cells subsequently prepared and challenged in microculture proliferative assays by using synthetic peptides, only one region of the intact protein appeared stimulatory . This T cell epitope was located between residues 116 and 121, adjacent to an epitope between residues 110 and 115 which we have previously shown to bind the L5 mAb . Immunization of mice with peptides, and subsequent challenge of lymph node cells in assays by using the 18-kDa protein as Ag revealed that residues 111-125 were the most effective in priming responses . Furthermore, the ability of 18-kDa primed lymph node cells to recognize determinants on both M . leprae and Mycobacterium tuberculosis indicates that in addition to possessing an M . leprae-specific B cell determinant, the 18-kDa protein contains a cross-reactive T cell epitope(s). Biochim Biophys Acta, 1989 Sep 14, 998(1), 32 - 42 Isolation and characterization of biologically active murine interleukin-1 alpha derived from expression of a synthetic gene in Escherichia coli; Daumy GO et al.; A murine interleukin-1 alpha (mIL-1 alpha) gene coding for amino acids 115 to 270 of the precursor protein (Lomedico, P.T., Gubler, U., Hellmann, C.P., Dukovich, M., Giri, J.G., Pan, Y.E., Collier, K., Semionow, R., Chua, A.O . and Mizel, S.B . (1984) Nature 312, 458-462) was chemically synthesized and expressed in Escherichia coli . mIL-1 alpha, in the form of insoluble inclusion bodies, accounted for approx . 30% of total cellular protein produced by the recombinant strain . A simple isolation protocol was developed in which inclusion body material was first solubilized in 3 M guanidine hydrochloride, and the mIL-1 alpha was then simultaneously purified and allowed to fold to its active conformation by dialysis against distilled water . This procedure yielded pure, biologically active mIL-1 alpha with 41% recovery of the mIL-1 alpha present in the guanidine hydrochloride extract . The purified preparation had the expected amino acid composition, a molar absorptivity of 28,200 M-1.cm-1 and a pI of 5.2 . No methionyl-mIL-1 alpha was detected by N-terminal sequence analysis, and the endotoxin level was less than 10 pg per micrograms of mIL-1 alpha . The specific biological activity was 3.10(7) units/mg in a co-mitogenic thymocyte proliferation assay . In addition to full-length mIL-1 alpha, the preparation contained N-terminally truncated mIL-1 alpha species (mainly des-4 and des-6 amino acid forms) . The truncated species were isolated and found to have the same biological activity as the complete polypeptide . Thus, the active fragment of mIL-1 alpha appears to consist of a proteinase-sensitive N-terminal region which is not essential for activity, and a proteinase-resistant core which harbors the essential determinants of its cytokine function. Nucleic Acids Res, 1989 Sep 12, 17(17), 6781 - 94 The recR locus of Escherichia coli K-12: molecular cloning, DNA sequencing and identification of the gene product; Mahdi AA et al.; The recR gene of Escherichia coli, which is associated with recBC-independent mechanisms of recombination and DNA repair, has been located between dnaZX and htpG on a 6.4 kb EcoRI fragment of DNA that has been cloned and analysed in lambda and plasmid vectors . Nucleotide sequencing of this interval revealed two open reading frames that constitute an operon lying immediately downstream of dnaZX . The second of these two reading frames was identified as recR . It encodes a polypeptide with a predicted molecular weight of 21,965 Daltons that migrates on SDS gels as a 26 kDa protein . The first gene of the operon encodes a polypeptide of 12,015 daltons . Its function is not known. Nucleic Acids Res, 1989 Sep 12, 17(17), 6865 - 81 Characterization of yeast mitochondrial RNase P: an intact RNA subunit is not essential for activity in vitro; Morales MJ et al.; We have previously described a mitochondrial activity that removes 5' leaders from yeast mitochondrial precursor tRNAs and suggested that it is a mitochondrial RNase P . Here we demonstrate that the cleavage reaction results in a 5' phosphate on the tRNA product and thus the activity is analogous to that of other RNase Ps . A mitochondrial gene called the tRNA synthesis locus encodes an A + U-rich RNA required for this activity in vivo . Two regions of this RNA display sequence similarity to conserved sequences in bacterial RNase P RNAs . This sequence similarity coupled with the analogous activities of the enzymes has led us to conclude that the RNAs are homologous and that the tRNA synthesis locus does code for the mitochondrial RNase P RNA subunit . The smallest and most abundant transcript of the tRNA synthesis locus is 490 nucleotides long . However, during purification of the holoenzyme, RNA is degraded and pieces of the original RNA are sufficient to support RNase P activity in vitro. FEBS Lett, 1989 Sep 11, 255(1), 15 - 20 Recombinant proricin binds galactose but does not depurinate 28 S ribosomal RNA; Richardson PT et al.; Preproricin transcripts microinjected into Xenopus oocytes were expressed and the product was segregated by the oocyte endoplasmic reticulum and core glycosylated . Recombinant proricin was soluble, stabilised by intramolecular disulfide bonds and biologically active in that it could bind to immobilized lactose (selectin 2) or immobilized asialofetuin . Affinity-purified proricin did not catalyse the depurination of 28 S ribosomal RNA unless it was reduced, when slight but significant activity was observed . Gel filtration of the reduced proricin fraction showed that this depurination activity was not associated with proricin . The activity was apparently due to ricin A chain released by reduction from mature ricin which was, in turn, generated from proricin, presumably via endogenous oocyte endoprotease activity. Science, 1989 Sep 8, 245(4922), 1104 - 7 Generation of a catalytic antibody by site-directed mutagenesis; Baldwin E et al.; A hybrid Fv fragment of the dinitrophenyl-binding immunoglobulin A (IgA), MPOC315, has been generated by reconstituting a recombinant variable light chain (VL) produced in Escherichia coli with a variable heavy chain (VH) derived from the antibody . The Tyr34 residue of VL was substituted by His in order to introduce a catalytic imidazole into the combining site for the ester hydrolysis . The His mutant Fv accelerated the hydrolysis of the 7-hydroxycoumarin ester of 5-(2,4-dinitrophenyl)-aminopentanoic acid 90,000-fold compared to the reaction with 4-methyl imidazole at pH 6.8 and had an initial rate that was 45 times as great as that for the wild-type Fv . The hydrolyses of aminopropanoic and aminohexanoic homologs were not significantly accelerated . Thus a single deliberate amino acid change can introduce significant catalytic activity into an antibody-combining site, and chemical modification data can be used to locate potential sites for the introduction of catalytic residues. J Biol Chem, 1989 Sep 5, 264(25), 14741 - 7 Purification and characterization of pituitary bovine somatotropin; Wood DC et al.; Bovine somatotropin (bST) has been isolated from pituitary glands and compared in a variety of chemical analyses and bioassays with somatotropin derived from recombinant Escherichia coli . Comparison of pituitary extracts and purified bST by Western blot analysis of two-dimensional gels suggested that the immunoreactive somatotropin species present in the extract were also present in the purified material, with no significant losses or degradation as a result of the purification method . NH2-terminal sequence analysis indicated the presence of equal quantities of Ala-Phe-Pro-Ala-Met-Ser-Leu-Ser- and Phe-Pro-Ala-Met-Ser-Leu-Ser- sequences . The Met-Ser-Leu-Ser-NH2-terminal sequence, a degradation product observed in NIH standard lots, was not detected . Assay of bioactivity in a bovine liver receptor-binding assay and in a female rat growth assay showed pituitary bST and recombinant methionyl-bovine somatotropin to be equipotent . Tryptic maps and sequence analysis of pituitary-derived somatotropin suggest the presence of isoaspartate derivatization at Asp128. J Biol Chem, 1989 Sep 5, 264(25), 15130 - 5 Conversion of monofunctional DNA adducts of cis-diamminedichloroplatinum (II) to bifunctional lesions . Effect on the in vitro replication of single-stranded DNA by Escherichia coli DNA polymerase I and eukaryotic DNA polymerases alpha; Hoffmann JS et al.; Reaction of cis-diamminedichloroplatinum (II) with single-stranded M13 phage DNA in vitro produced monofunctional platinum-DNA adducts on guanine and bifunctional lesions with either two guanine bases (GG) or one adenine and one guanine (AG) . When DNA containing a majority of monofunctional platinum-DNA lesions was dialyzed against 10 mM NaCIO4 at 37 degrees C, conversion of monoadducts to bifunctional lesions was observed . We examined the effect of post-treatment formation of bifunctional lesions on DNA synthesis by Escherichia coli DNA polymerase I and highly purified eukaryotic DNA polymerase alpha from Drosophila melanogaster and calf thymus . Arrest sites on the platinated template were determined by polyacrylamide gel electrophoresis . Monofunctional lesions did not appear to block DNA synthesis . Inhibition of replication increased as bifunctional platinum-DNA lesions formed during post-treatment incubation; GG adducts inhibited replication more than AG . These results suggest that bifunctional GG platinum-DNA adducts may be the major toxic damage of cisplatin. J Biol Chem, 1989 Sep 5, 264(25), 14902 - 8 Comparison of the human immunodeficiency virus type 1 and 2 proteases by hybrid gene construction and trans-complementation; Le Grice SF et al.; To determine the cleavage specificity of the proteases of the type 1 and 2 human immunodeficiency viruses (HIV-1, HIV-2), we interchanged this domain of the polymerase (pol) genes and analyzed the maturation programs of the chimeric polyproteins in an Escherichia coli expression system . In both cases, release of reverse transcriptase and integrase was observed, together with the respective 10-kDa protease form resulting from autocatalysis, although the maturation proceeded less efficiently compared to the homologous system . In further experiments, the ability of both HIV-1 and HIV-2 proteases to release in vivo gag p24 from an in-frame fusion of the full length gag and protease precursors was analyzed . In either case, p24 was released, albeit with greater efficiency in the heterologous gene construction . In vitro mixed lysate experiments with the HIV-1 gag precursor furthermore demonstrate that both enzymes respond to the aspartyl proteinase inhibitor pepstatin A . Taken together, these results illustrate that although different cleavage recognition sequences exist for HIV-1 and -2, they are amenable to the proteases of both viruses, but additionally that subtle differences in the mode of action of both enzymes are observable. J Biol Chem, 1989 Sep 5, 264(25), 14698 - 703 Isolation and characterization of lactose permease mutants with an enhanced recognition of maltose and diminished recognition of cellobiose; Collins JC et al.; In the present study, lactose permease mutants were isolated which have an enhanced recognition toward maltose (an alpha-glucoside) and diminished recognition for cellobiose (a beta-glucoside) . Nine mutants were isolated from a strain encoding a wild-type permease (pTE18) and nine from a strain encoding a mutant permease which recognizes maltose (pB15) . All 18 mutants were subjected to DNA sequencing, and it was found that all mutations are single base substitutions within the lac Y gene effecting single amino acid substitutions within the protein . From the pTE18 parent, substitutions involved Tyr-236 to Phe or His; Ser-306 to Thr; and six independent mutants in which Ala-389 was changed to Pro . From pB15, Tyr-236 was changed to Phe or Asn, Ser-306 to Thr or Leu, Lys-319 to Asn, and His-322 to Tyr, Asn, or Gln . All 18 mutants exhibited enhanced recognition for maltose (compared with the pTE18 strain) and a diminished recognition for cellobiose . In addition, all mutants showed a diminished recognition toward beta-galactosides as well . The Phe-236, His-236, Leu-306, Asn-319, Tyr-322, Asn-322, and Gln-322 mutants were completely defective in the uphill accumulation of methyl-beta-D-thiogalactopyranoside whereas the Asn-236, Thr-306, and Pro-389 mutants could effectively accumulate methyl-beta-D-thiogalactopyranoside against a concentration gradient . The mutants obtained in this study, together with previous lactose permease mutants, tend to be found on transmembrane segments, and those which are on the same transmembrane segment are often found three or four amino acids away from each other . This pattern is consistent with a protein structure in which important amino acid side chains project from several transmembrane segments in such a way as to form a hydrophilic channel for the recognition and transport of H+ and galactosides . It is proposed that the mechanism for H+/lactose cotransport is consistent with a "flanking gate" model in which the protein contains a single recognition site for galactosides within the channel which is flanked on either side by gates. J Biol Chem, 1989 Sep 5, 264(25), 14638 - 45 Attenuation in the regulation of the pyrBI operon in Escherichia coli . In vivo studies of transcriptional termination; Levin HL et al.; The attenuation model for transcriptional regulation of the Escherichia coli pyrBI operon is based on the assumption that transcription terminates upstream of the structural genes at a rho-independent terminator when cells contain high levels of UTP . When, however, the cells are limited for pyrimidines, the presence of ribosomes translating the short leader peptide is presumed to cause an alteration in the secondary structure of the terminator in a way that allows RNA polymerase to transcribe the entire operon . These two premises of transcriptional regulation were tested by using exonuclease protection assays to map the 3' ends of transcripts extracted from cells containing either ample or depleted concentrations of pyrimidines . The results support the model since 99% of the pyrBI transcripts terminated at the (G + C)-rich region of dyad symmetry upstream of the structural genes when cells were grown in excess uracil . In addition, a significant portion (36%) of the pyrBI transcripts extracted from cells containing reduced pyrimidine concentrations extended past the dyad into the structural genes . This observation correlated with the amounts of aspartate transcarbamoylase synthesized in cells under the various conditions . The mapping technique was also used to determine the position of the 5' ends of the transcripts to measure contributions of two potential start sites (P1 and P2) to the pool of pyrBI transcripts . The results show that under all conditions no more than 3% of the total transcripts had 5' ends corresponding to the upstream promoter, P1 . In cells lacking P1 virtually all transcripts from P2 terminated at the (G + C)-rich hairpin when the cellular level of pyrimidines was high . Conversely 57% of the transcripts extended past the terminator when cells were grown in UMP . The S1 nuclease technique also provided a measure of the steady state level of transcripts originating at P2 . In cells depleted of pyrimidines there was a 5-10-fold increase in these transcripts depending on the number of copies of pyrBI . This increase, which is independent of attenuation, is caused by a different regulatory mechanism which as yet has not been identified. J Biol Chem, 1989 Sep 5, 264(25), 14624 - 6 NMR study of human mutant hemoglobins synthesized in Escherichia coli . Consequences of tyrosine alpha 42 substitutions; Ishimori K et al.; The hydroxyl group of Tyr alpha 42 in human hemoglobin forms a hydrogen bond with the carboxylate of Asp beta 99 which is considered to be one of the most important hydrogen bonds for stabilizing the "T-state." However, no spontaneous mutation at position 42 of the alpha subunit has been reported, and the role of the tyrosine has not been tested experimentally . Two artificial human mutant hemoglobins in which Tyr alpha 42 was replaced by phenylalanine or histidine were synthesized in Escherichia coli, and their proton NMR spectra were studied with particular attention to the hyperfine-shifted and hydrogen-bonded proton resonances . The site-directed mutagenesis of the Tyr alpha 42----Phe removes the hydrogen bond described above and prevents transition to the T-state so that the mutant Hb is rather similar to the "R-state" even when deoxygenated . On the other hand, the mutation from tyrosine to histidine causes less drastic structural changes, and its quaternary and tertiary structures are almost the same as native deoxy-Hb A . This may be attributed to the formation of a new hydrogen bond between His alpha 1(42) and Asp beta 2(99) . These observations indicate that the hydrogen bond formed between Tyr alpha 42 and Asp beta 99 is required to convert unliganded Hb to the T-state. J Biol Chem, 1989 Sep 5, 264(25), 14860 - 4 The importance of the link between Glu204 of the catalytic chain and Arg130 of the regulatory chain for the homotropic and heterotropic properties of Escherichia coli aspartate transcarbamoylase; Stebbins JW et al.; Recent x-ray crystallographic studies of aspartate transcarbamoylase bound with CTP have detected molecular asymmetry in the interface between the catalytic and regulatory subunits (Kim, K . H., Pan, Z., Honzatko, R . B., Ke, H.-M., and Lipscomb, W . N . (1987) J . Mol . Biol . 196, 863-875) . In three of the six interfaces, a salt link occurs between Arg130 of the regulatory chain and Glu204 of the catalytic chain; however, these same residues are 15 A apart in the other three interfaces . In order to determine if this is important for the function of the enzyme, two mutant versions of aspartate transcarbamoylase were created by site-specific mutagenesis . Glu204 of the catalytic chain was converted to a glutamine (Glu204c----Gln) and Arg130 of the regulatory chain was converted to a glycine (Arg130r----Gly) . The thermal stability of the Arg130r----Gly enzyme is dramatically reduced, whereas the thermal stability of the Glu204c----Gln enzyme is unaltered compared to the wild-type enzyme . The maximal velocity of both mutant enzymes is identical with that of the wild-type enzyme, however both mutant enzymes have altered substrate affinity and regulatory properties . Based on these studies, the link between Glu204 of the catalytic chain and Arg130 of the regulatory chain is important for the heterotropic properties of the enzyme . Furthermore, the interface between the domain of the regulatory chain which binds zinc and the domain of the catalytic chain which binds aspartate may be more important for CTP inhibition than ATP activation . These data also suggest that heterotropic cooperativity is very sensitive to alterations in the catalytic-regulatory interface . However, no clear relationship has been observed between the structural asymmetry and the function of the enzyme. J Mol Biol, 1989 Sep 5, 209(1), 171 - 5 Densely packed beta-structure at the protein-lipid interface of porin is revealed by high-resolution cryo-electron microscopy; Sass HJ et al.; Porin is an integral membrane protein that forms channels across the outer membrane of Escherichia coli . Electron microscopic studies of negatively stained two-dimensional porin crystals have shown three stain accumulations per porin trimer, revealing the locations of pores spanning the membrane . In this study, reconstituted porin lattices embedded in glucose were investigated using the low-dose technique on a cryo-electron microscope equipped with a helium-cooled superconducting objective lens . The specimen temperature was maintained at 5 K to yield an improved microscopic and specimen stability . Under these conditions, we obtained for the first time electron diffraction patterns from porin lattices to a resolution of 3.2 A and images showing optical diffraction up to a resolution of 4.9 A . Applying correlation averaging techniques to the digitized micrographs, we were able to reconstruct projected images of the porin trimer to a resolution of up to 3.5 A . In the final projection maps, amplitudes from electron diffraction and phases from these images were combined . The predominant feature is a high-density narrow band (about 6 A in thickness) that delineates the outer perimeter of the trimer . Since the molecule consists of almost exclusively beta-sheet structure, as revealed by spectroscopic data, we conclude that this band is a cylindrical beta-pleated sheet crossing the membrane nearly perpendicularly to its plane . Another intriguing finding is a low-density area (about 70 A2) situated in the centre of the trimer. J Biol Chem, 1989 Sep 5, 264(25), 15144 - 50 Myosin heavy chain kinase from developed Dictyostelium cells . Purification and characterization; Ravid S et al.; We purified to homogeneity the Dictyostelium discoideum myosin heavy chain kinase that is implicated in the heavy chain phosphorylation increases that occur during chemotaxis . The kinase is initially found in the insoluble fraction of developed cells . The major purification step was achieved by affinity chromatography using a tail fragment of Dictyostelium myosin (LMM58) expressed in Escherichia coli (De Lozanne, A., Berlot, C . H., Leinwand, L . A., and Spudich, J . A . (1988) J . Cell Biol . 105, 2990-3005) . The kinase has an apparent molecular weight of 84,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis . The apparent native molecular weight by gel filtration is 240,000 . The kinase catalyzes phosphorylation of myosin heavy chain or LMM58 with similar kinetics, and the extent of phosphorylation for both is 4 mol of phosphate/mol . With both substrates the Vmax is about 18 mumol/min/mg and the Km is 15 microM . The myosin heavy chain kinase is specific to Dictyostelium myosin heavy chain, and the phosphorylated amino acid is threonine . The kinase undergoes autophosphorylation . Each mole of kinase subunit incorporates about 20 mol of phosphates . Phosphorylation of myosin by this kinase inhibits myosin thick filament formation, suggesting that the kinase plays a role in the regulation of myosin assembly. J Biol Chem, 1989 Sep 5, 264(25), 15074 - 82 Characterization of the spoT gene of Escherichia coli; Sarubbi E et al.; The Escherichia coli spoT gene encodes a guanosine-3',5'-bispyrophosphate (ppGpp) 3'-pyrophosphohydrolase known to be responsible for cellular (ppGpp) degradation . The DNA sequence of the spoT region is presented . The spoT gene is deduced to be 702 codons long, with a probable UUG initiation codon, and a deduced mass of 79,342 daltons . Two spoT mutations (spoT202 and spoT203) have been localized to an open reading frame by complementation of function as well as by genetic marker rescue . The ability to overexpress the spoT gene is limited, but enough ppGppase activity can be made to reverse ppGpp accumulation during the stringent response to amino acid starvation . The spoT gene is located within a larger spo operon and is flanked by two smaller genes . The first gene in the operon encodes omega, a protein that copurifies with RNA polymerase (Gentry, D . R., and Burgess, R . R . (1986) Gene (Amst.) 48, 33-40) . The spoT gene is the second gene in the operon; it is followed by a third open reading frame deduced to encode a protein with a mass of 25,343 daltons . Insertion of a kanamycin resistance gene in the omega gene reduces spoT gene expression as judged by lowered ppGppase activity, relA-dependent reduction of growth rate, and abolition of spoT mutant complementation activity . These effects are reversed by expression of the spoT gene, but not the omega gene, in trans . Transcription of the spo operon occurs in a clockwise direction on the E . coli chromosome and is probably directed by at least two promoters. J Biol Chem, 1989 Sep 5, 264(25), 15012 - 21 Adenosine-5'-phosphosulfate kinase from Escherichia coli K12 . Purification, characterization, and identification of a phosphorylated enzyme intermediate; Satishchandran C et al.; Adenosine-5'-phosphosulfate kinase (ATP:adenylylsulfate 3'-phosphotransferase), the second enzyme in the pathway of sulfate activation, has been purified (approximately 300-fold) to homogeneity from an Escherichia coli K12 strain, which overproduces the enzyme activity (approximately 100-fold) . The purified enzyme has a specific activity of 153 mumol of 3'-phosphoadenosine 5'-phosphosulfate (PAPS) formed/min/mg of protein at 25 degrees C . The enzyme is remarkably efficient with a Vmax/Km(APS) of greater than 10(8) M-1 s-1, indicating that at physiologically low substrate concentrations the reaction is essentially diffusion limited . Upon incubation with MgATP a phosphorylated enzyme is formed; the isolated phosphorylated enzyme can transfer its phosphoryl group to adenosine 5'-phosphosulfate (APS) to form PAPS or to ADP to form ATP . The phosphorylated enzyme exists as a dimer of identical 21-kilodalton subunits, while the dephosphorylated form primarily exists as a tetramer . Divalent cations are required for activity with Mg(II), Mn(II), Co(II), and Cd(II) activating . Studies of the divalent metal-dependent stereoselectivity for the alpha- and beta-phosphorothioate derivatives of ATP indicate metal coordination to at least the alpha-phosphoryl group of the nucleotide . Steady state kinetic studies of the reverse reaction indicate a sequential mechanism, with a rapid equilibrium ordered binding of MgADP before PAPS . In the forward direction APS is a potent substrate inhibitor, competitive with ATP, complicating kinetic studies . The primary kinetic mechanism in the forward direction is sequential . Product inhibition studies at high concentrations of APS suggest an ordered kinetic mechanism with MgATP binding before APS . At submicromolar concentrations of APS, product inhibition by both MgADP and PAPS is more complex and is not consistent with a solely ordered sequential mechanism . The formation of a phosphorylated enzyme capable of transferring its phosphoryl group to APS or to MgADP suggests that a ping-pong pathway in which the rate of MgADP dissociation is comparable to the rate of APS binding might contribute at very low concentrations of APS . The substrate inhibition by APS is consistent with APS binding to the enzyme, to form a dead-end E.APS complex. J Biol Chem, 1989 Sep 5, 264(25), 14806 - 11 Purification and characterization of recombinant human parathyroid hormone-related protein; Hammonds RG Jr et al.; Full-length human parathyroid hormone-related protein (PTHrP-(1-141} as well as a carboxyl-terminal shortened form (PTHrP-(1-108} have been expressed from recombinant DNA-derived clones . These proteins were expressed in Escherichia coli as fusion proteins so that cyanogen bromide cleavage yields the desired product . Both proteins were purified and then characterized by sodium dodecyl sulfate gel electrophoresis, amino-terminal amino acid sequencing, peptide mapping, and mass spectral analysis . Recombinant PTHrP-(1-141), PTHrP-(1-108), synthetic PTHrP-(1-34), and naturally derived PTHrP are all equipotent in the stimulation of cyclic AMP levels in the osteoblast-like cell line UMR 106-01 . However, PTHrP-(1-141) and -(1-108) are two to four times more active than PTHrP-(1-34) in the stimulation of plasminogen activator activity from this cell line . PTHrP-(1-141) reacts equipotently with PTHrP-(1-34) in a radioimmunoassay using an antiserum prepared against PTHrP-(1-34) . PTHrP-(1-141), -(1-108), and -(1-84) were used as PTHrP-specific mobility standards on sodium dodecyl sulfate gel electrophoresis to determine the approximate length of two forms of naturally derived PTHrP . The data show that PTHrP purified from the lung tumor cell line BEN contains a major form of about 108 amino acids and another form of about 141 amino acids. J Biol Chem, 1989 Sep 5, 264(25), 14769 - 74 Sequence of the cloned Escherichia coli K1 CMP-N-acetylneuraminic acid synthetase gene; Zapata G et al.; The Escherichia coli CMP-N-acetylneuraminic acid (CMP-NeuAc) synthetase gene is located on a 3.3-kilobase (kb) HindIII fragment of the plasmid pSR23 which contains the genes for K1 capsule production (Vann, W . F., Silver, R . P., Abeijon, C., Chang, K., Aaronson, W., Sutton, A., Finn, C . W., Lindner, W., and Kotsatos, M . (1987) J . Biol . Chem . 262, 17556-17562) . The CMP-NeuAc synthetase gene expression was increased 10-30-fold by cloning of a 2.7-kb EcoRI-HindIII fragment onto the vector pKK223-3 containing the tac promoter . The complete nucleotide sequence of the gene encoding CMP-NeuAc synthetase was determined from progressive deletions generated by selective digestion of M13 clones containing the 2.7-kb fragment . CMP-NeuAc synthetase is located near the EcoRI site on this fragment as indicated by the detection of an open reading frame encoding a 49,000-dalton polypeptide . The amino- and carboxyl-terminal sequences of the encoded protein were confirmed by sequencing of peptides cleaved from both ends of the purified enzyme . The nucleotide deduced amino acid sequence was confirmed by sequencing several tryptic peptides of purified enzyme . The molecular weight is consistent with that determined from sodium dodecyl sulfate-gel electrophoresis . Gel filtration and ultracentrifugation experiments under nondenaturing conditions suggest that the enzyme is active as a 49,000-dalton monomer but may form aggregates. Biochemistry, 1989 Sep 5, 28(18), 7313 - 8 Lysine-60 in the regulatory chain of Escherichia coli aspartate transcarbamoylase is important for the discrimination between CTP and ATP; Zhang Y et al.; Lysine-60 in the regulatory chain of aspartate transcarbamoylase has been changed to an alanine by site-specific mutagenesis . The resulting enzyme exhibits activity and homotropic cooperativity identical with those of the wild-type enzyme . The substrate concentration at half the maximal observed specific activity decreases from 13.3 mM for the wild-type enzyme to 9.6 mM for the mutant enzyme . ATP activates the mutant enzyme to the same extent that it does the wild-type enzyme, but the concentration of ATP required to reach half of the maximal activation is reduced approximately 5-fold for the mutant enzyme . CTP at a concentration of 10 mM does not inhibit the mutant enzyme, while under the same conditions CTP at concentrations less than 1 mM will inhibit the wild-type enzyme to the maximal extent . Higher concentrations of CTP result in some inhibition of the mutant enzyme that may be due either to hetertropic effects at the regulatory site or to competitive binding at the active site . UTP alone or in the presence of CTP has no effect on the mutant enzyme . Kinetic competition experiments indicate that CTP is still able to displace ATP from the regulatory sites of the mutant enzyme . Binding measurements by equilibrium dialysis were used to estimate a lower limit on the dissociation constant for CTP binding to the mutant enzyme (greater than 1 x 10(-3) M) . Equilibrium competition binding experiments between ATP and CTP verified that CTP still can bind to the regulatory site of the enzyme . For the mutant enzyme, CTP affinity is reduced approximately 100-fold, while ATP affinity is increased by 5-fold.(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1989 Sep 5, 28(18), 7289 - 97 Crystallographic studies of the mechanism of xylose isomerase; Farber GK et al.; The mechanism of xylose isomerase (EC 5.3.1.5) has been studied with X-ray crystallography . Four refined crystal structures are reported at 3-A resolution: native enzyme, enzyme + glucose, enzyme + glucose + Mg2+, and enzyme + glucose + Mn2+ . One of these structures (E.G.Mg) was determined in a crystal mounted in a flow cell . The other structures were equilibrium experiments carried out by soaking crystals in substrate containing solution . These structures and other studies suggest that, contrary to expectation, xylose isomerase may not use the generally expected base-catalyzed enolization mechanism . A mechanism involving a hydride shift is consistent with the structures presented here and warrants further investigation . Additional evidence in support of a hydride shift comes from comparing xylose isomerase with triosephosphate isomerase which is known to catalyze an analogous reaction via an enediol intermediate . Evidence is presented that suggests that aldose-ketose isomerases can be divided into two groups . Phospho sugar isomerases generally do not require a metal ion for activity and show exchange of substrate protons with solvent . In contrast, simple sugar isomerases all require a metal ion and show very low solvent exchange . These observations are rationalized on the basis of the need for stereospecific sugar binding. J Biol Chem, 1989 Sep 5, 264(25), 14591 - 3 Putative metal finger structure of the human immunodeficiency virus type 1 enhancer binding protein HIV-EP1; Maekawa T et al.; The region containing two copies of the sequence GGGACTTTCC in the human immunodeficiency virus type 1 (HIV-1) long terminal repeat, that is an NF-kappa B binding site, functions as an enhancer element for HIV transcriptional regulation . By a Southwestern method we have isolated a cDNA encoding the HIV-1 enhancer binding protein (HIV-EP1) from a human B-cell lambda gt11 library . DNase I footprinting analysis using the HIV-EP1 protein expressed in Escherichia coli showed that HIV-EP1 specifically bound to the HIV-1 enhancer . HIV-EP1 protein contains a domain with two tandem "zinc finger" sequences initially described in the Xenopus transcription factor IIIA . This represents the first demonstration of the structural feature of the protein that binds to the HIV-1 enhancer. Biochemistry, 1989 Sep 5, 28(18), 7401 - 8 Site-directed mutagenesis of histidine-13 and histidine-114 of human angiogenin . Alanine derivatives inhibit angiogenin-induced angiogenesis; Shapiro R et al.; The roles of His-13 and His-114 in the ribonucleolytic and angiogenic activities of human angiogenin have been investigated by site-directed mutagenesis . Replacement of either residue by alanine (H13A and H114A) decreases enzymatic activity toward tRNA by at least 10,000-fold and virtually abolishes 10,000-fold and virtually abolishes angiogenic activity in the chick embryo chorioallantoic membrane assay . Both the H13A and H114A mutant proteins compete effectively with angiogenin in the latter assay; only a 5-fold molar excess of H13A over unmodified protein is required for complete inhibition . The His----Ala substitutions, however, do not have any significant effect on the interaction of angiogenin with human placental ribonuclease inhibitor, an extremely potent inhibitor of angiogenin (Ki approximately 7 x 10(-16 M) previously shown to interact with another active-site residue, Lys-40 . The effects of more conservative replacements-glutamine at position 13 and asparagine at position 114--were also examined . While the enzymatic activity of the H114N mutant was at least 3300-fold less than for the unmodified protein, the H13Q derivative had only 300-fold reduced activity toward tRNA and cytidylyl(3'----5') adenosine . Both substitutions substantially decreased angiogenic activity . The parallel effects on ribonucleolytic and biological activities observed with all four mutant proteins provide strong evidence that the latter activity of angiogenin is dependent on a functional enzymatic active site . The capacity of the H13A and H114A derivatives to compete with angiogenin in the chorioallantoic membrane assay suggests several additional features of the biological mode of action of this protein. J Mol Biol, 1989 Sep 5, 209(1), 65 - 77 Rates of aminoacyl-tRNA selection at 29 sense codons in vivo; Curran JF et al.; We have placed aminoacyl-tRNA selection at individual codons in competition with a frameshift that is assumed to have a uniform rate . By assaying a reporter in the shifted frame, relative rates for association of the 29 YNN codons and their cognate aminoacyl-tRNAs were obtained during logarithmic growth in Escherichia coli . For five codons, three beginning with C and two with U, these relative rates agree with relative in vitro rates for elongation factor Tu-mediated aminoacyl-tRNA binding to ribosomes and subsequent GTP hydrolysis . Therefore, the frameshift assay probably measures this process in vivo . Observed rates for aminoacyl-tRNA selection span a 25-fold range . Therefore, the time required to transit different codons in vivo probably differs substantially . Codons very frequently used in highly expressed genes generally select aminoacyl-tRNAs more quickly than do rarely used codons . This suggests that speed of aminoacyl-tRNA selection is a significant factor determining biased use of synonymous codons . However, the preferential use of codons appears to be marked only for codons with the highest rates of aminoacyl-tRNA selection . Rapid selection in vivo is usually effected by elevation of the tRNA concentration for codons with moderate intrinsic speed (rate constant), not by choosing intrinsically fast codons . Despite a preference for high rate, there are quickly translated codons that are not commonly used, and common codons that are translated relatively slowly . Other factors are therefore more important than speed for some codons . Strong preference for rapid aminoacyl-tRNA selection is not observed in weakly expressed genes . Instead, there is a slight preference for slower aminoacyl-tRNA selection . The rate of aminoacyl-tRNA selection by a YNC codon is always greater than the rate of the corresponding YNU codon even though in many YNC/U pairs both codons react with the same elongation factor Tu/GTP/aminoacyl-tRNA complex . Thus, for these tRNAs, the differences between in vivo rate constants of tRNAs are dependent on the nature of anticodon base-pairing . However, no more general relationship is evident between codon/anticodon composition and rate of aminoacyl-tRNA selection . The frameshift method can be extended to all codons. J Biol Chem, 1989 Sep 5, 264(25), 15120 - 9 Transcriptional activation of pBR322 DNA can lead to duplex DNA unwinding catalyzed by the Escherichia coli preprimosome; Parada CA et al.; Mechanisms that could operate to initiate pBR322 DNA replication in the absence of RNase H and DNA polymerase I are described . Two different pathways leading to extensive unwinding of pBR322 DNA have been observed under DNA replication reaction conditions in vitro . In the presence of RNA polymerase and DNA gyrase, specifically initiated RNA II (the leading-strand primer precursor) can form an RNA-DNA hybrid with the template that starts just upstream of the origin of DNA replication and continues for about 3 kilobases . Subsequent digestion of the RNA in this RNA-pBR322 DNA hybrid results in the formation of a highly unwound DNA termed form I . If DNA gyrase is absent during the RNA polymerase-catalyzed elongation of RNA II, a stable RNA-pBR322 DNA hybrid can still form that is localized to the origin region of the genome . Formation of this hybrid activates the primosome assembly site present on the lagging-strand DNA template, by displacing it to a single-stranded conformation, thereby allowing preprimosome assembly . Once assembled, the DNA helicase activity of the preprimosome, in the presence of the single-stranded DNA binding protein and DNA gyrase but in the absence of any further transcription, can also result in extensive unwinding of pBR322 DNA . The product of this reaction, form I DNA, is more unwound than form I DNA . The formation of both form I and form I DNA is inhibited by the presence of excess RNA I, as well as by RNase H at concentrations sufficient to catalyze the normal processing of RNA II required for initiation of leading-strand DNA synthesis . These results suggest that RNA II-pBR322 DNA hybrid formation is essential to permit preprimosome assembly during pBR322 DNA replication under conditions where both RNase H and DNA polymerase I are absent. Biochim Biophys Acta, 1989 Sep 4, 984(2), 188 - 92 How many Na+-dependent carriers for L-alanine and L-proline in the eel intestine? Studies with brush-border membrane vesicles; Vilella S et al.; Using brush-border membrane (BBM) vesicles prepared from the intestine of the European eel, the specificity of L-alanine and L-proline Na+-dependent transport was investigated by measuring the uptake of isotopically labelled substrates . In the presence of Na+ ions, cross-inhibition between alanine and proline transports was observed; in addition alpha-(methylamino)isobutyric acid (MeAIB) inhibited proline but had no effect on alanine uptake . These results can be explained by the presence, in eel intestinal BBM vesicles, of at least two distinct agencies for Na+-dependent proline and alanine translocation . The first system is specific for alanine and short-chain neutral amino acids; the second system, specific for imino acids and the N-methylated analogues, is regulated by alanine concentration. Biochim Biophys Acta, 1989 Sep 4, 1013(1), 21 - 7 The assessment of relative surface hydrophobicity as a factor involved in the activation of human polymorphonuclear leukocytes by uropathogenic strains of Escherichia coli; Steadman R et al.; The initiation of the respiratory burst and the degranulation of human polymorphonuclear leukocytes (PMN) in response to stimulation by uropathogenic strains of Escherichia coli is dependent on the expression of Type 1 fimbriae by those strains . These PMN responses correlate with an increasing tendency of the interacting E . coli strain to be retained on hydrophobic columns . The present work assessed the measurement of relative surface hydrophobicity in relation to PMN activation . Type 1 fimbriate organisms bound most readily to Octyl-Sepharose columns and were strongly agglutinated in the salt aggregation test . In contrast, the same organisms partitioned to the dextran-rich (hydrophilic) phase of aqueous two-polymer phase systems . Electron microscopic observation of the organisms eluted from the Octyl-Sepharose columns and of the organisms recovered from both phases of the aqueous two-phase systems demonstrated, however, that both Type 1 and P-fimbriate organisms were retained on the columns and partitioned into the dextran-rich phase as a consequence of their being fimbriate and failed to identify this as a major factor in the activation of PMN . In addition electron microscopy demonstrated that each P-fimbriate population had fewer organisms expressing fimbriae than did Type 1 fimbriate populations, confirming the importance of phase variation as a factor affecting the physicochemical characteristics of a bacterial population. Rev Esp Enferm Apar Dig, 1989 Sep, 76(3), 203 - 9 {Immunosuppression with cyclosporin after orthoptic liver transplant in pigs}; Arias J et al.; The immunosuppressive effect of cyclosporine A (CsA) was studied in six pigs that underwent orthotopic liver transplant (OLT) . The drug was administered i.v . in low doses (1.5-4 mg/Kg-1/12 h-1) and associated with wide spectrum antibiotics . The mean survival of the animals was 23.4 +/- 11.2 days . All animals had fever, jaundice and hypertrichosis, and the preoperative corporal weight remained constant . Liver abscesses by E . coli were common autopsy findings . Livers also presented biliary regeneration and cholestasis with a slight lymphocytic infiltration . OLT in pigs without immunosuppressive treatment causes and elevation in serum gamma-GTP, GOT, LDH, alkaline phosphatase and bilirubin . In the post-mortem findings, the livers did not present abscesses but evidenced intense atrophy with hepatocellular necrosis and abundant lymphocytic infiltration. Masui, 1989 Sep, 38(9), 1188 - 94 {The effects of ONO 3708, a new thromboxane receptor antagonist, on cardiovascular response during the early phases of endotoxin shock in anesthetized dogs}; Sasao J et al.; The effects of ONO 3708, a new thromboxane A2 receptor antagonist, on cardiovascular and airway responses, at an early phase during endotoxin shock were investigated in anesthetized dogs . The i.v . infusion (1mg.kg-1) of E.coli endotoxin caused an increase in mean pulmonary artery pressure (MPAP) from 9.9 +/- 1.0 to 19.1 +/- 2.3 mmHg at 5 min, and at 15 min after infusion, elevated MPAP returned toward the control level . Pretreatment with ONO 3708 abolished these effects of endotoxin on pulmonary artery pressure at an early phase . The change in airway pressure reached a maximum of 14.4 +/- 1.7 cmH2O from 10.0 +/- 1.9 at 5 min, followed by a gradual decline toward a baseline value at 30 min in the control group . ONO 3708 significantly attenuated increase in airway pressure induced by E . coli endotoxin . But pretreatment with ONO 3708 could not prevent decrease in systemic arterial pressure and cardiac output induced by endotoxin . These results suggest that role of thromboxane A2 on the cardiovascular response during endotoxin shock is played only on pulmonary vascular changes, and ONO 3708 has a beneficial effect at least during the early phase of endotoxin shock. J Parenter Sci Technol, 1989 Sep-Oct, 43(5), 246 - 9 Pyrogens in small-volume parenterals prepared in hospital pharmacy; Lacasa C et al.; Three pyrogen assay methods {the rabbit method, Limulus Amebocyte Lysate (LAL) gelation and chromogenic substrate, and bioburden prior to sterilization} were evaluated in three standard batches of small-volume parenteral preparations; i.e., water for injection, sodium chloride 0.9% injection, and sodium bicarbonate 8.4% injection . The same preparations were also assayed after contamination with Escherichia coli followed by sterilization . All methods gave the same results with the water for injection and the sodium chloride 0.9% . With sodium bicarbonate 8.4%, only the rabbit method was valid . For sodium chloride 0.9%, the chromogenic substrate method was valid; the gelation method was not valid . Endotoxins from the manufacturing plant were less pyrogenic in rabbits than were standard endotoxins or those from hospital E . coli. J Interferon Res, 1989 Sep, 9 Suppl 1, S61 - 5 High levels of circulating neutralizing antibody in normal animals to recombinant mouse interferon-beta produced in yeast; Sedmak JJ et al.; Plasma from normal outbred Swiss mice not previously given interferon (IFN) neutralized a glycosylated (high-mannose) recombinant murine IFN-beta made in yeast (rMuIFN-beta y) . The neutralization antibody titers were as high as 1:6,000 versus rMuIFN-beta y, whereas the titers obtained with native murine IFN-beta (MuIFN-beta) were 100-fold less; a recombinant murine IFN-beta make in Escherichia coli (rMuIFN-beta ec) was not neutralized at 1:10 dilution of plasma . An ELISA using rMuIFN-beta y demonstrated that it is gamma immunoglobulins in normal mouse plasma that bind rMuIFN-beta y . Normal rabbit serum also had very high endogenous neutralizing activity (1:5,300) against rMuIFN-beta y . Significant neutralizing activity also was detected in newborn bovine and normal human serum as well as pooled human immune serum globulin . Thus, endogenous neutralizing activity directed presumably at the carbohydrate residues of highly glycosylated recombinant proteins produced in yeast may limit their clinical utility. J Appl Physiol, 1989 Sep, 67(3), 963 - 9 Blunted febrile response to intravenous endotoxin in starved rats; Shido O et al.; The effects of fasting on the febrile responses to intravenous injection of bacterial lipopolysaccharide (LPS; endotoxin) of Escherichia coli were investigated in rats . Ad libitum-fed rats (C) produced a biphasic fever with an increase in the temperature difference between brown adipose tissue and colon and shivering activity (SA) . Measurement by a direct calorimeter showed no particular changes in heat loss . Rats starved for 4 days (F4) responded to intravenous LPS with a monophasic fever accompanied by an increase in SA only . However the maximal rise in colonic temperature (Tco) did not differ from C rats . Subsequent 2-day fasting reduced SA and the maximal fever height . Endogenous pyrogen (EP) injected intravenously produced a prompt rise in Tco followed by prolonged hyperthermia in C rats . In the F4 rats, there was no such sustained rise in Tco as a result of intravenous EP . The response in Tco to intravenous prostaglandin E2 (PGE2) was the same in fed and starved rats . The administration of LPS, EP, and PGE2 into the lateral ventricle evoked a similar extent of hyperthermia in C and F4 rats . Because the second phase of fever has been shown to occur after pyrogens are translated into a febrile stimulus within the blood-brain barrier, it is assumed that the functional changes of the blood-brain barrier such as in the permeability of pyrogens or in the sensitivity of pyrogen receptors resulted in the absence of the second phase of fever in starved rats. Eur J Immunol, 1989 Sep, 19(9), 1619 - 24 Visualizing interleukin 2 gene expression at the single cell level; Emilie D et al.; To analyze the expression of the interleukin (IL)2 gene at the single cell level, we have constructed a chimeric gene in which the regulatory sequences from the mouse IL2 gene were fused 5' proximal to the coding region of the Escherichia coli beta-galactosidase (lacZ) gene . Once stably introduced into a T cell hybridoma, the IL 2-lacZ reporter gene was shown to display a pattern of induction similar to the one observed for the resident IL 2 gene . Two points emerged from monitoring IL 2 expression for the resident IL2 gene . Two points emerged from monitoring IL 2 expression at the single cell level . First, upon activation the expression of the IL2-lacZ gene appears asynchronous . Second, at the peak of induction the percentage of beta-galactosidase+ cells never reached 100% and the level of beta-galactosidase reached among positive cells was highly heterogeneous within a cloned population of T cells . In combination with the possibility to sort viable lymphocytes according to lacZ expression, the IL2-lacZ reporter gene described herein should provide a way to isolate somatic cell mutants deficient in signal transduction by the T cell antigen receptor complex. J Bacteriol, 1989 Sep, 171(9), 5148 - 54 Binding-protein-dependent alanine transport in Rhodobacter sphaeroides is regulated by the internal pH; Abee T et al.; The properties of an L-alanine uptake system in Rhodobacter sphaeroides were studied and compared with those of H+/lactose symport in R . sphaeroides 4P1, a strain in which the lactose carrier of Escherichia coli has been cloned and functionally expressed (F . E . Nano, Ph.D . thesis, University of Illinois, Urbana, 1984) . Previous studies indicated that both transport systems were active only when electron transfer took place in the respiratory or cyclic electron transfer chain, while uptake of L-alanine also required the presence of K+ (M . G . L . Elferink, Ph.D . thesis, University of Groningen, Groningen, The Netherlands, 1986) . The results presented in this paper offer an explanation for these findings . Transport of the nonmetabolizable L-alanine analog 2-alpha-aminoisobutyric acid (AIB) is mediated by a shock-sensitive transport system . The apparently unidirectional uptake of AIB results in accumulation levels which exceed 7 x 10(3) . The finding of L-alanine-binding activity in the concentrated crude shock fluid indicates that L-alanine is taken up by a binding-protein-dependent transport system . Transport of the nonmetabolizable lactose analog methyl-beta-D-thiogalactopyranoside (TMG) by the lactose carrier under anaerobic conditions in the dark was observed in cells and membrane vesicles . This indicates that the H+/lactose symport system is active without electron transfer . Uptake of AIB, but not that of TMG, is inhibited by vanadate with a 50% inhibitory concentration of 50 microM, which suggests a role of a phosphorylated intermediate in AIB transport . Uptake of TMG and AIB is regulated by the internal pH . The initial rates of uptake increased with the internal pH, and and pKa values of 7.2 for TMG and 7.8 for AIB . At an internal pH of 7, no AIB uptake occurred, and the rate of TMG uptake was only 30% of the rate at an internal pH of 8 . In a previous study, we found that K+ plays an essential role in regulating the internal pH (T . Abee, K . J . Hellingwerf, and W . N . Konings, J . Bacteriol . 170:5647-5653, 1988) . The dependence of solute transport in R . sphaeroides on both K+ and activity of an electron transfer chain can be explained by an effect of the internal pH, which subsequently influences the activities of the lactose-and binding-protein-dependent L-alanine transport system. Virology, 1989 Sep, 172(1), 370 - 3 Evidence that nucleocapsid disassembly and a later step in virus replication are inhibited in transgenic tobacco protoplasts expressing TMV coat protein; Osbourn JK et al.; Tobacco mosaic virus (TMV)-like pseudovirus particles containing mRNA for Escherichia coli beta-glucuronidase (GUS) were electroporated into mesophyll protoplasts from control or TMV coat protein (CP)-transgenic tobacco (Nicotiana tabacum cv . Xanthi) . GUS-particles were expressed 100-fold less efficiently in CP-transformed than in control protoplasts whereas unencapsidated GUS mRNA was expressed only 2.8-fold less efficiently . Lower transient expression of packaged GUS mRNA is probably due to inhibited disassembly of nucleocapsids in CP-transgenic protoplasts . Control and U1 CP-transformed protoplasts are equally susceptible to infection by cowpea strain TMV (Cc), as well as unencapsidated Cc or U1 RNA . In contrast, native or in vitro reconstituted U1 TMV particles result in 5- to 6-fold fewer infected CP-transgenic than control protoplasts . When Cc RNA was transcapsidated in U1 CP in vitro, the hybrid virions were equally infectious in both classes of protoplasts . We conclude that although compatible U1 protein-protein interactions significantly inhibit (GUS) nucleocapsid disassembly in CP-transgenic protoplasts, the endogenous CP must also interfere with a later stage of infection involving the homologous viral RNA. Mikrobiologiia, 1989 Sep-Oct, 58(5), 746 - 50 {The effect of substrates and irradiation with low intensity visible light on the rate of division of Escherichia coli}; Tiflova OA et al.; The effect of He--Ne laser irradiation (lambda = 632.8 nm, D = 4.10(3) J/m2) on the growth of Escherichia coli cultures was studied in a minimal medium containing glucose, glycerol of arabinose . The irradiated cultures immediately started to divide with a virtually identical specific growth rate (kappa = 0.78, 0.8 and 0.8 h-1) whatever the growth rate and the latent period of the parent cultures were . The ratio between cell numbers in the irradiated and non-irradiated cultures was highest 1 h after the irradiation: 1.25, 1.3 and 1.5 for the glucose, glycerol and arabinose cultures, respectively . Apparently, the irradiation with visible light of a low intensity creates an additional proton gradient and thus stimulates a new replication and division cycle in the population of cells whose membranes do not have delta pH necessary for the initiation of these processes . The system under study can serve as a model for studying the delta pH-dependent stages in the regulation of cell division. Mikrobiologiia, 1989 Sep-Oct, 58(5), 709 - 15 {The role of intracellular pool of polyamines in the regulation of metabolism in Escherichia coli during aerobic-anaerobic transitions}; Tkachenko AG et al.; The transition from aerobic to anaerobic conditions of Escherichia coli cultivation is accompanied by a drop in the free pool of putrescine in the cell as well as in the medium and by a decrease in the intracellular concentration of spermidine . This process stems from at least two facts: (1) the activity of ornithine decarboxylase, a key enzyme in the synthesis of polyamines, falls down due to a decrease in the level of ATP in the cell; (2) the free pool of polyamines is bound to cell compartments, in particular, to nucleoid DNA . The results make it possible to consider the system of polyamine synthesis as a point in the regulatory interaction between the energetic and constructive types of E . coli metabolism. Biotechniques, 1989 Sep, 7(8), 840 - 50 Automated methods for single-stranded DNA isolation and dideoxynucleotide DNA sequencing reactions on a robotic workstation; Mardis ER et al.; Automated procedures have been developed for both the simultaneous isolation of 96 single-stranded M13 chimeric template DNAs in less than two hours, and for simultaneously pipetting 24 dideoxynucleotide sequencing reactions on a commercially available laboratory workstation . The DNA sequencing results obtained by either radiolabeled or fluorescent methods are consistent with the premise that automation of these portions of DNA sequencing projects will improve the reproducibility of the DNA isolation and the procedures for these normally labor-intensive steps provides an approach for rapid acquisition of large amounts of high quality, reproducible DNA sequence data. J Gen Microbiol, 1989 Sep, 135 ( Pt 9), 2365 - 78 Cloning and expression of Treponema pallidum antigens in Escherichia coli; Bailey MJ et al.; A library of Treponema pallidum genomic DNA fragments produced by partial Sau3A digestion was established in Escherichia coli K12 using the plasmid vector pAT153 . The library was screened using immune syphilitic rabbit serum and six recombinant phenotypes expressing eight treponemal polypeptides were detected . With two exceptions, all the recombinant gene products were the same size as polypeptides detected on Western immunoblots of T . pallidum . The genes encoding three novel gene products, with molecular masses in SDS-PAGE of 42, 17 and 15.5 kDa, which had not been cloned previously from T . pallidum were also identified . Monoclonal antibodies which reacted with four of the eight recombinant polypeptides were generated. Plasmid, 1989 Sep, 22(2), 132 - 42 Shuttle plasmids constructed by the transformation of an Escherichia coli cloning vector into two Deinococcus radiodurans plasmids; Smith MD et al.; An Escherichia coli plasmid that confers kanamycin resistance (Kmr) was inserted into the large Deinococcus radiodurans cryptic plasmids pUE10 and pUE11, yielding pS28 and pS19 . The method of insertion involved both in vitro splicing and the natural transformation of D . radiodurans and yielded full-length clones in E . coli of pUE10 and pUE11 . Both pS28 and pS19 replicated and expressed Kmr in E . coli and D . radiodurans . In both pS28 and pS19, D . radiodurans plasmid sequences were immediately upstream from the Kmr determinant . Transformation experiments suggested that Kmr expression in D . radiodurans was initiated in upstream D . radiodurans sequences . Restriction maps of pS28 and pS19 showed that each plasmid contained three MraI sites . Both pS28 and pS19 transformed the MraI-producing D . radiodurans strain R1 at low frequencies . D . radiodurans strain Sark, which naturally contains pUE10 and pUE11, was transformed by pS28 and pS19 at much higher frequencies . A Sark derivative that was cured for pUE10 was isolated by screening Sark/pS28 subisolates for loss of kanamycin resistance. Physiol Behav, 1989 Sep, 46(3), 535 - 9 Verapamil and indomethacin attenuate endotoxin-induced anorexia; Langhans W et al.; To characterize the mechanism of the anorexia during infection, we investigated the effect of E . coli lipopolysaccharide (LPS) on feeding in rats under various conditions: LPS (125, 100, 75, and 50 micrograms/kg body weight = b . wt.) injected intraperitoneally (IP) reduced food intake by decreasing meal frequency without affecting meal size . The Ca++-channel blocker verapamil (5 mg/kg b . wt., IP) or the antipyretic and antiphlogistic drug indomethacin (2.5 mg/kg b . wt., IP), but not combined alpha- and beta-adrenergic receptor blockade by IP phentolamine plus propranolol (500 micrograms/kg b . wt., each) attenuated the anorectic effect of LPS (125 or 100 micrograms/kg b . wt.) . The results suggest that a phospholipase A2-sensitive mechanism contributes to the anorexia during injection. Biull Eksp Biol Med, 1989 Sep, 108(9), 369 - 72 {Morphofunctional characteristics of the duodenum in the early stages of experimental escherichiosis}; Parkhomenko IuG et al.; Using the model of experimental colibacillosis in mice morphological, immunomorphological and morphometrical studies of duodenum 1 to 24 hours after inoculation were performed . Typical dynamics of cellular composition in the intestinal wall, accompanied with increased lymphoplasmacellular infiltration, and dystrophic changes in cells of submucosal and intermuscular neuroplexuses were found . The data obtained testify, that the infectious process at the acute state of experimental colibacillosis has an immune character (immediate hypersensitivity of local type), characterised by increasing in immunoglobulin-containing cells and including in this process of mast and endocrine cells. Mol Biol (Mosk), 1989 Sep-Oct, 23(5), 1340 - 9 {The effect of supercoiling of DNA from colicinogenic plasmids on the expression of col, imm and lys genes}; Panchenko IuA et al.; The expression of colicin genes is controlled by the SOS-system (Lex A repressor) and the adenylate-cyclase system (cAMP-CAP complex) . The effect of plasmid DNA supercoiling on the expression of the operons of colicins E1, E2, and E3 has been studied by using E . coli minicells . It has been shown for the colicin E1 operon that it is the promoter that is influenced by supercoiling: an increase in negative supercoiling elevates the expression and, vice versa, DNA relaxation reduces the expression . The effect of supercoiling on gene activity of the colicin E1 immunity protein has not been observed, which may be due to the specific orientation of this gene . With the two other colicins supercoiling affects the expression of all genes which constitute the operon . The regulation of the colicin operon expression has been confirmed to occur at three levels: by the LexA protein, by the cAMP-CAP complex, and by the plasmid DNA supercoiling. Mol Biol (Mosk), 1989 Sep-Oct, 23(5), 1248 - 62 {Pattern recognition in the computer analysis of nucleotide sequences}; Aleksandrov NN et al.; We have used an algorithm from the pattern recognition theory "generalized portrait" to find a distinguishing vector for Escherichia coli promoters . We have made an attempt to solve closely linked problems for choosing significant signs of that signal, multiple alignment and for calculation of the recognition vector (matrix) . The promoters with known strength have been ranged with this vector . The analysis of the occurrence of predicted promoters has been carried out . The promoters search program for IBM-compatible computers is available from the authors. J Biochem (Tokyo), 1989 Sep, 106(3), 460 - 7 Purification and characterization of the active serine: pyruvate aminotransferase of rat liver mitochondria expressed in Escherichia coli; Oda T et al.; In the previous study (Oda, T., et al . (1985) Eur . J . Biochem . 150, 415-421), we isolated a cDNA clone which expressed in Escherichia coli a specific size of product having the activity of rat liver serine:pyruvate aminotransferase (SPTm) . This specific product (SPT10) was purified to homogeneity through three different column chromatographies . The amino acid composition and N-terminal amino acid sequence of the purified enzyme agreed with those predicted from the nucleotide sequence of cDNA and showed that SPT10 consists of the whole amino acid sequence of mature SPTm and several extra amino acid residues at the N-terminus . The catalytic and physical properties of SPT10, such as substrate specificity, Km for alpha-keto acids, electric charge, and quaternary structure, were all very similar to those of SPTm . Using several cDNA clones which lack a 5'-terminal sequence corresponding to a portion of the N-terminal amino acid sequence of SPTm, we examined the expression profile of the specific product in bacteria transformed with each cDNA clone . The products encoded by these cDNAs were segregated into inclusion bodies and were neither catalytically active nor easily solubilized by sonication . In contrast, the inclusion bodies were not formed in the bacteria transformed with the cDNA clone for SPT10. J Biochem (Tokyo), 1989 Sep, 106(3), 436 - 41 Production and characterization of recombinant human neutrophil chemotactic factor; Furuta R et al.; A putative mature human neutrophil chemotactic factor (NCF) corresponding to the C-terminal 72 amino acids of its precursor was directly produced in Escherichia coli by recombinant DNA technology . Human NCF was present in both the soluble and insoluble protein fractions of the homogenate of host cells, and it was partially purified as a water-soluble polypeptide from both fractions, separately . The partially purified NCF preparation was highly purified to an endotoxin-free homogeneous polypeptide by means of CM-Sepharose CL-6B column chromatography and gel filtration on Toyopearl HW-55 . No difference between the human NCF preparations purified from both starting materials could be found concerning purity, primary structure, solubility, molecular weight, and chemotactic activity for human neutrophils . The amino acid sequence of recombinant human NCF was identical to the sequence deduced from the cDNA sequence . A methionine residue due to the translation initiation codon was removed . Recombinant human NCF was found to be biologically active and to exhibit chemotactic activity for human neutrophils in vitro and cause a neutrophil infiltration in vivo in mice. Genes Dev, 1989 Sep, 3(9), 1462 - 71 Identification of the sigma E subunit of Escherichia coli RNA polymerase: a second alternate sigma factor involved in high-temperature gene expression; Erickson JW et al.; The rpoH gene of Escherichia coli encodes sigma 32, the 32-kD sigma-factor responsible for the heat-inducible transcription of the heat shock genes . rpoH is transcribed from at least three promoters . Two of these promoters are recognized by RNA polymerase containing sigma 70, the predominant sigma-factor . We purified the factor responsible for recognizing the third rpoH promoter (rpoH P3) and identified it as RNA polymerase containing a novel sigma-factor with an apparent Mr of 24,000 . This new sigma, which we call sigma E, is distinct from the known sigma factors in molecular weight and promoter specificity . sigma E holoenzyme will not recognize the sigma 70- or sigma 32-controlled promoters we tested, but it does transcribe the htrA gene, which is required for viability at temperatures greater than 42 degrees C . The in vivo role of sigma E is not known . The transcripts from the sigma E-controlled rpoH P3 and htrA promoters are most abundant at very high temperature, suggesting the sigma E holoenzyme may transcribe a second set of heat-inducible genes that are involved in growth at high temperature or in thermotolerance. Genetika, 1989 Sep, 25(9), 1541 - 50 {Analysis of expression of the gene from the BRa puff of Chironomus thummi encoding the low molecular weight secretory protein}; Bogachev SS et al.; The plasmid containing F6.2 gene from the BRa of Chironomus thummi within the pUR 292 vector was constructed . Chimeric protein containing beta-galactosidase-F6.2 polypeptide was produced in Escherichia coli BMH71-18 . The protein obtained has immunological similarity with secretion protein of 67,000 D from Ch . thummi salivary gland . The gene for sp67 is active in main and in special lobe of the gland . Based on the data obtained and on the previous results of the authors, conclusion is made that BRa is a complex locus containing several types of genes. Jpn J Surg, 1989 Sep, 19(5), 556 - 62 Thromboxane as a possible hepatotoxic factor increased by endotoxemia in obstructive jaundice; Hanai T et al.; In a study using rats, we investigated whether liver damage induced by endotoxemia in obstructive jaundice is associated with thromboxane (TX) in order to acertain whether its vasoconstrictive and platelet aggregating properties play a role in reducing liver blood flow . The rats were divided into the following 5 groups; a control group, an endotoxin (Et) group, a bile duct ligation (BDL) group, a bile duct ligation and endotoxin (BDL + Et) group and an OKY046 (Thromboxane synthetase inhibitor) treated bile duct ligation + endotoxin (OKY-BDL + Et) group . The blood TXB2 levels in the Et, BDL and BDL + Et groups were higher than those in the control group . The liver TXB2 levels in the Et and BDL + Et groups were also higher than those in the control group . Liver phospholipids and liver blood flow decreased in the BDL + Et group, whereas in the OKY-BDL + Et group they returned close to the control group levels by decreasing the TXB2 levels in both the liver and blood to normal . These results suggest that the high level of TX in the blood and liver tissue may further aggrevate the liver during endotoxemia in obstructive jaundice by inhibiting liver blood flow. Gig Sanit, 1989 Sep, (9), 26 - 9 {Colicin genotype characteristics of pathogenic Escherichia circulating in the environment}; Bei TV; Colicin identification of pathogenic Escherichia, isolated from the environmental objects, showed that determination of colicinogenicity signs and sensitivity to standard colicins in pathogenic E . to a more considerable degree depended on strain sulfur-group membership than on the amanating objects . The analysis of colicin genotype characteristics of pathogenic E., circulating in environment, provided much information and could be used in identifying a source of infection and ways of escherichiosis pathogen transmission. Z Naturforsch {C}, 1989 Sep-Oct, 44(9-10), 838 - 44 {Physiologic significance of "stringent control" in Escherichia coli under extreme starvation}; Mach H et al.; The viability of three isogenic relA+/relA strain pairs of Escherichia coli (CP78/CP79; NF161/NF162; CP107/CP143) was studied during prolonged starvation for amino acids, glucose or phosphate . After amino acid limitation we found a prolonged viability of all relA+ strains which synthesized ppGpp . We suggest that some ppGpp-mediated pleiotropic effects of the stringent response (e.g . glykogen accumulation, enhanced protein turnover) might be involved in this prolongation of survival . After glucose or phosphate starvation there was no difference in the relA+/relA strains either in the ppGpp content or in the survival. Arzneimittelforschung, 1989 Sep, 39(9), 1073 - 80 Preparation and biological activity of new substituted antimalarial diaminodiphenylsulfones; Pieper H et al.; Starting from 4,4'-diamino-diphenylsulfone (DDS) as a lead structure, new 2-substituted analogues as well as new 2-substituted 4-alkylamino-4'-amino diphenylsulfones have been designed and synthetized in different ways . This has led to compounds the inhibitory activity of which against 7,8-dihydropteroic acid synthase of plasmodia and mycobacteria is clearly superior to that of sulfadoxine and in most cases to that of DDS . Of special interest is 4'-amino-4-n-propylamino-2-methyl-diphenylsulfone . Together with inhibitors of 7,8-dihydrofolate reductase in vitro and in vivo it possesses a marked synergistic inhibitory activity against plasmodia . In contrast to DDS in doses up to 200 mg/kg p.o . (cat) no methemoglobin formation is observed . The compound has been selected for further studies. Mol Gen Genet, 1989 Sep, 218(3), 431 - 6 Purification and characterization of the sopB gene product which is responsible for stable maintenance of mini-F plasmid; Watanabe E et al.; The mini-F plasmid has the trans-acting sopA, sopB genes and the cis-acting sopC DNA which are essential for plasmid partitioning . In this paper, we report the purification of the sopB gene product from extracts of cells harboring a pBR322 derivative carrying the sopB gene . The purity of the final preparation was more than 95%, as determined by densitometry . The amino acid sequence of the amino-terminal region of the protein for the 17 residues identified was identical to that predicted from the DNA sequence of the sopB gene . Therefore, it was concluded that the protein was the sopB gene product . Using anti-SopB serum, the SopB protein was detected in the cell lysates of F+, F', and Hfr strains . The SopB protein bound to the plasmid DNA of a pBR322 derivative carrying the sopC DNA segment, but not to the vector plasmid pBR322. Mol Gen Genet, 1989 Sep, 218(3), 397 - 401 Context specific misreading of phenylalanine codons; Precup J et al.; It has previously been shown that the phenylalanine codon UUC encoding residue 8 of the Escherichia coli argI gene product, ornithine transcarbamylase, is misread as leucine at a high frequency during phenylalanine starvation . However, no misreading of the UUU encoding residue 3 was observed under these conditions . Using oligonucleotide-directed, site-specific mutagenesis, we have constructed mutants where these codons have been changed . Using these mutant argI genes we see a high level of mistranslation at position 8 during phenylalanine starvation whether the codon is UUU or UUC . With either codon at position 3 we see no leucine substitution . We also constructed a gene with a leucine codon at position 3 . The product of this latter mutated gene is stable and active, indicating that preferential turnover of mistranslated protein is not obscuring an otherwise high rate of misreading . This would seem to indicate that it is the context rather than the particular phenylalanine codon which is important in determining these misreading levels. Biochem Int, 1989 Sep, 19(3), 593 - 601 Potentiation of cysteine proteinase-inhibitor activity of full-length Ha-ras oncogene products by denaturation-renaturation; Hiwasa T et al.; We have investigated protease-inhibitory activity against cathepsin L of human and murine Ha-ras oncogene products (p21s) produced by Escherichia coli . The inhibitory activity of full-length p21s was much weaker than that of truncated p21s, however, the inhibitory activity was potentiated after denaturation with 8 M urea and 2 M NaCl followed by renaturation . These suggest that p21 can be folded into multiple three-dimensional conformations which have different protease-inhibitory activities. Anal Biochem, 1989 Sep, 181(2), 336 - 40 Purification of plasmid-expressed proteins which lack functional assay systems; Marvel CC et al.; A general method for the purification of proteins whose genes are cloned into plasmid vectors, but whose biochemical and functional characteristics are unknown, is described . A cell-free transcription-translation system from Escherichia coli K-12 is used to synthesize in vitro radiolabeled protein expressed from recombinant plasmid vectors . The radiolabeled proteins are then fractionated and used as markers for the purification of nonradiolabeled proteins without recourse to functional assays . Biochemical analysis of the purified proteins can reveal information about their cellular localization, binding parameters, and physical, enzymatic, or regulatory properties . This information complements in vivo genetic analysis with the goal of identifying the gene and the function of its protein product . An example using this technique in which the product of the usg gene in the hisT operon of E . coli has been purified and biochemically characterized is described. Onderstepoort J Vet Res, 1989 Sep, 56(3), 207 - 9 Adverse effects of a proposed equine sublethal endotoxin model; Stadler P et al.; Commercially available Escherichia coli 055: B5 lipopolysaccharide was administered intravenously experimentally at a dosage of 10 micrograms/kg to 2 horses . Various clinical and clinico-pathological parameters were monitored before and after the endotoxin administration . Because of a hopeless prognosis, and for humane reasons, euthanasia was applied on both horses 6 h after administration . Values recorded for the different parameters, including the blood lactate level, were consistent with a lethal condition . It would appear that an intravenous dose of 10 micrograms/kg of endotoxin is potentially lethal to horses. Klin Wochenschr, 1989 Sep 1, 67(17), 843 - 6 Molecular genetics of the human Na+/glucose cotransporter; Hediger MA et al.; Recent success in expression cloning has revealed the primary structure of the Na+/glucose cotransporter from rabbit small intestine, and this has subsequently led to the cloning of the Na+/glucose cotransporters from human small intestine and human kidney . Close homology is evident between the rabbit and human intestinal Na+/glucose cotransporters at the DNA level, and the predicted amino acid and secondary structure levels . The Na+/glucose cotransporter amino acid sequence from human kidney is 57% identical with that from human small intestine . Significant homology also exists between these Na+/glucose cotransporters and the E . coli Na+/proline cotransporter (putP) . The rabbit intestinal Na+/glucose cotransporter has 11 potential membrane spanning regions and 2 hydrophilic regions containing highly charged residues . The amino acid sequence shows two potential N-glycosylation sites (N-X-T/S) . Using an in vitro translation approach we were able to determine that only one of these (Asn 248) is glycosylated . Expression experiments with Xenopus oocytes using the N-glycosylation inhibitor tunicamycin indicate that glycosylation of Asn 248 is required for functional expression of the transporter . The N-X-T/S sequence at Asn 248 is conserved in the human intestinal and the human renal Na+/glucose cotransporter . Chromosomal localization studies map the human intestinal Na+/glucose cotransporter gene (SGLT1) to the q11.2----qter region of chromosome 22 and the human renal Na+/glucose cotransporter gene (SGLT2) to the q-arm of chromosome 16.(ABSTRACT TRUNCATED AT 250 WORDS) J Membr Biol, 1989 Sep, 110(3), 227 - 33 Solubilization and reconstitution of the Rickettsia prowazekii ATP/ADP translocase; Plano GV et al.; The ATP/ADP translocase protein of Rickettsia prowazekii, an obligate intracellular parasite that had been grown in the chick yolk sac, was solubilized and reconstituted into liposomes composed of Escherichia coli phospholipid by an octylglucoside dilution procedure . Proteoliposomes prepared from membranes of Renografin-purified R . prowazekii translocated ATP by an obligate exchange mechanism . Influx of extravesicular ATP required intravesicular transportable nucleotide and efflux of intravesicular ATP required transportable extravesicular nucleotide in the medium . The transport activity was insensitive to carboxyatractyloside and bongkrekic acid, inhibitors of mitochondrial ADP/ATP translocation . Proteoliposomes prepared from membranes of standard (non-Renografin-purified) R . prowazekii exhibited both an inhibitor-sensitive mitochondrial translocase activity and an inhibitor-resistant rickettsial translocase activity . Proteoliposomes prepared from uninoculated yolk sac membranes exhibited only the inhibitor-sensitive mitochondrial translocase activity . The substrate specificity of each reconstituted translocase was determined and shown to correspond with that reported for intact mitochondria or rickettsiae . Following influx of ATP the steady-state value for intravesicular labeled ATP was dependent on the concentration of intravesicular nucleotide available for exchange. Gene, 1989 Sep 1, 81(1), 193 - 4 Nucleotide sequence of the Escherichia coli tRNA(3Leu) gene; Wahab SZ et al.; A 300-nucleotide sequence was determined which includes the tRNA(3Leu) coding region and the flanking sequences. Can J Microbiol, 1989 Sep, 35(9), 843 - 9 {Modification of the envelope and the protein content of Escherichia coli surviving in sea water}; Gauthier MJ et al.; A toxigenic strain of Escherichia coli displayed important structural modifications when placed in seawater which naturally lacked nutritive elements, as observed by electron microscopy . These include cell wall and cell body distortion, modification of the membranes, central segregation of the chromosome, and retraction of the cytoplasm . These modifications were accompanied by a decrease in cell protein content of approximately 40% . Certain cytoplasmic membrane proteins were lost, and new ones appeared . The development of these changes was considerably slower in cells that had previously been grown in a seawater medium . This suggests that osmotic regulation mechanisms, which enable E . coli to survive much longer in marine conditions, may have a protective influence. Biochem J, 1989 Sep 1, 262(2), 581 - 9 Mechanism of DNA strand nicking at apurinic/apyrimidinic sites by Escherichia coli {formamidopyrimidine}DNA glycosylase; Bailly V et al.; Escherichia coli {formamidopyrimidine}DNA glycosylase catalyses the nicking of both the phosphodiester bonds 3' and 5' of apurinic or apyrimidinic sites in DNA so that the base-free deoxyribose is replaced by a gap limited by 3'-phosphate and 5'-phosphate ends . The two nickings are not the results of hydrolytic processes; the {formamidopyrimidine}DNA glycosylase rather catalyses a beta-elimination reaction that is immediately followed by a delta-elimination . The enzyme is without action on a 3'-terminal base-free deoxyribose or on a 3'-terminal base-free unsaturated sugar produced by a beta-elimination reaction nicking the DNA strand 3' to an apurinic or apyrimidinic site. Appl Environ Microbiol, 1989 Sep, 55(9), 2424 - 7 Production of 4-methylumbelliferyl heptanoate hydrolase by Escherichia coli exposed to seawater; Fiksdal L et al.; The production of an enzyme, 4-methylumbelliferyl heptanoate hydrolase, in Escherichia coli exposed to enriched and nonenriched seawater was studied . In all media, except for seawater with no or very small amounts of organic material and seawater enriched with peptone, 4-methylumbelliferyl heptanoate hydrolase activity increased by 2 to 3 orders of magnitude within 2 days . Increased enzyme activity was assumed to be related to cells not undergoing lysis but adapting to conditions of nutrient limitation. Appl Environ Microbiol, 1989 Sep, 55(9), 2414 - 5 Convenient preparative synthesis of {14C}trehalose from {14C}glucose by intact Escherichia coli cells; Brand B et al.; At high osmolarity, Escherichia coli synthesizes trehalose intracellularly, irrespective of the nature of the carbon source . Synthesis proceeds via the transfer of UDP-glucose to glucose 6-phosphate, yielding trehalose 6-phosphate, followed by its dephosphorylation to trehalose (H.M . Giaeyer, B.O . Styrvold, I . Kaasen, and A.R . Strom, J . Bacteriol . 170:2841-2849, 1988) . This reaction was exploited to preparatively synthesize {14C}trehalose from exogenous {14C}glucose by using intact bacteria of a mutant (DF214) that could not metabolize glucose . The total yield of radiochemically pure trehalose from glucose was routinely more than 50%. Tijdschr Diergeneeskd, 1989 Sep 1, 114(17), 890 - 8 {Escherichia coli mastitis in cattle . II . Pathogenesis and symptomatic therapy}; Lohuis JA et al.; The pathogenesis, course run by the disease, and symptomatic treatment of mastitis due to Escherichia coli in dairy cattle are reviewed in relation to the functioning of cellular and humoral defence mechanisms . The systemic symptoms of disease during mastitis caused by E . coli are attributed to the release and subsequent absorption from the udder of endogenous inflammatory mediators, rather than the direct absorption of endotoxins into the circulation . The course run by the disease during mastitis due to E . coli varies considerably . Reasons for these variations include genetic variation, stage of lactation and the function of humoral and cellular defence mechanisms. Res Vet Sci, 1989 Sep, 47(2), 178 - 84 Effects of allopurinol in experimental endotoxin shock in horses; Lochner F et al.; The effect of allopurinol pretreatment 12 hours before an intraperitoneal challenge with a sublethal dose of Escherichia coli endotoxin (50 micrograms kg-1) was evaluated in 18 horses . The horses were divided among three equal groups: 1-endotoxin alone; 2-5 mg allopurinol kg-1 bodyweight plus endotoxin; and 3-50 mg allopurinol kg-1 bodyweight plus endotoxin . A variety of evaluation parameters were used . No differences among the groups were noted in rectal temperature, heart rate, respiration rate, haematological values, blood PaO2, blood PaCO2, blood pH or blood bicarbonate . Significant (P less than 0.05) differences between the groups were noted as regards the changes in capillary refill time, base excess, blood glucose, blood lactate, blood beta-glucuronidase and recumbency time . The protection afforded by 5 mg allopurinol kg-1 appeared to be superior to that with 50 mg allopurinol kg-1. Mol Biol Evol, 1989 Sep, 6(5), 526 - 38 Statistical tests for detecting gene conversion; Sawyer S; Statistical tests for detecting gene conversion are described for a sample of homologous DNA sequences . The tests are based on imbalances in the distribution of segments on which some pair of sequences agrees . The methods automatically control for variable mutation rates along the genome and do not depend on a priori choices of potentially monophyletic subsets of the sample . The tests show strong evidence for multiple intragenic conversion events at two loci in Escherichia coli . The gnd locus in E . coli shows a highly significant excess of maximal segments of length 70-200 bp, which suggests conversion events of that size . The data also indicate that the rate of these short conversion events might be of the order of neutral mutation rate . There is also evidence for correlated mutation in adjacent codon positions . The same tests applied to a locus in an RNA virus were negative. Dtsch Tierarztl Wochenschr, 1989 Sep, 96(8), 419 - 21 {Verification of the protective effect of a toxoid vaccine against edema disease of weaned piglets in an infection model}; Awad-Masalmeh M et al.; 105 piglets (56 vaccinated and 49 control animals) were utilized in 6 consecutive experiments . Each used litter was divided randomly into vaccine and control animals . One week prior to weaning each of the 56 piglets of the vaccine groups received 5 mg of nonpurified toxin treated with glutaraldehyd subcutaneously whereas to the remaining 49 control animals an extract of apathogenic E . coli was administered . During the first 12 hours post weaning each of the 105 piglets was challenged perorally with 10(10) cfu of edema principle toxin producing germs of E . coli serogroup O 139 . 23 animals of the control groups (46.9%) and one animal of vaccine groups (1.8%) died due to the infection between days four and five post challenge . These control animals showed classical clinical symptoms as well as pathological findings typical for edema disease . In contrast, such findings as mentioned before could not be observed in the vaccinated piglets . The remaining part of the control animals and eight of those vaccinated ones exhibited edema disease symptoms . The vaccinated animals have shed the challenge strain one to three days, while the survivals of control groups shed those germs for two to six days . The vaccinated piglets showed a better growth rate than the remaining control animals . Presented data suggest that our toxoid immunizing procedure can be used successfully against edema disease of swine. Circ Shock, 1989 Sep, 29(1), 1 - 12 Serum amino acids in experimentally induced endotoxic shock: the prognostic significance of hyperalaninemia; Metzler B et al.; Serum amino acid levels were determined in 40 male rabbits before and 2, 6, and 24 hr after shock induction with 50 micrograms kg-1 endotoxin from Escherichia coli 0 111 and compared with amino acid levels of 32 age-matched control animals . Whereas total amino acid concentration in the control group decreased continuously over the 24 hr period due to fasting of the animals, the corresponding concentration in the endotoxin-treated animals remained at the initial level for the first 6 hr after shock induction . During this period, the release of amino acids from peripheral tissues was apparently enhanced, as evidenced by elevated levels of serum phenylalanine and tyrosine . Endotoxin administration exerted a salient effect on serum alanine levels . In all endotoxin-treated rabbits, alanine concentration was increased significantly 2 hr (P less than 0.00001) and 6 hr (P less than 0.00001) after injection of the toxin, whereas alanine levels in the control group remained essentially unchanged . In most animals, peak alanine concentrations were observed at the 6 hr sampling time . Blood glucose measurements indicated that by this time the hyperglycemic period, which is typical of the initial phase of endotoxic shock, was already finished . A high level of alanine following shock induction alluded to the impending death of the animal . In all rabbits in which alanine concentration rose by more than 600 mumols l-1 during the first 2 hr after endotoxin administration, the increase correlated significantly with the rate of lethality (P less than 0.0001) . In contrast, alanine levels of surviving animals increased only moderately . It was concluded from these results that monitoring serum alanine levels might be a valuable and practical tool to assess the severity and outcome of endotoxic shock. Am J Pathol, 1989 Sep, 135(3), 427 - 33 Interleukin-6 immunoreactivity in human tumors; Tabibzadeh SS et al.; The cytokine, interleukin-6 (IL-6), has emerged as a likely mediator of many of the systemic alterations observed in patients with cancer (fever, increased erythrocyte sedimentation rate, and alterations in plasma protein composition) and may also mediate local effects such as alteration in proliferation of tumor cells, increased tumor cell motility, and decreased intercellular adhesions between tumor cells . The distribution of IL-6 immunoreactivity in different human tumors was studied . IL-6 immunoreactivity was detected by the avidin-biotin-complex (ABC) procedure using a polyclonal rabbit antiserum raised against an E coli-derived human IL-6 (rIL-6) . Preimmune rabbit serum used as a control did not yield specific staining and preadsorption of the IL-6 antiserum with rIL-6 abolished specific staining . Strong-to-moderate IL-6 immunoreactivity was observed in the neoplastic elements present in primary squamous cell carcinomas, in adenocarcinomas of mammary, colonic, ovarian, and endometrial origin, in various adenocarcinomas metastatic to lymph nodes, and in soft tissue tumors including leiomyosarcoma and neurofibrosarcoma . Weak-to-moderate IL-6 immunostaining was observed in Hodgkin's and non-Hodgkin's lymphomas . This study demonstrates that most human tumors stain positively for IL-6, adding weight to the hypothesis that IL-6 is a key cytokine that participates in the host-tumor interaction. Proc Natl Acad Sci U S A, 1989 Sep, 86(18), 6888 - 92 Codon choice and gene expression: synonymous codons differ in translational accuracy; Dix DB et al.; Ribosomes programmed by different synonymous codons also differ in discriminating among near-cognate aminoacylated tRNAs . In the initial step of the recognition reaction ribosomes programmed by UUC discriminate less well than ribosomes programmed by UUU against ternary complexes containing three types of Leu-tRNA, and ribosomes programmed by CUC discriminate less well than ribosomes programmed by CUU against ternary complexes containing Phe-tRNA . Furthermore, in the proofreading step ribosomes programmed by UUC discriminate less well than ribosomes programmed by UUU against two of three near-cognate Leu-tRNAs, and ribosomes programmed by CUC discriminate less well than ribosomes programmed by CUU against near-cognate Phe-tRNA . The codon-induced change in reaction rate with near-cognate ternary complexes is greater than that with cognate ternary complexes: the most efficient codon is, therefore, the least accurate . Because the efficient, but inaccurate, codon UUC is used preferentially in highly expressed mRNAs of Escherichia coli, maximization of translational accuracy apparently has not been significant in the evolution of this particular biased codon choice in E . coli. Proc Natl Acad Sci U S A, 1989 Sep, 86(18), 6873 - 7 Codon discrimination and anticodon structural context; Lustig F et al.; Site-directed mutagenesis has been used to change the nucleotide C in the wobble position of tRNA(1Gly) (CCC) to U . The mutated tRNA was tested for its ability to read glycine codons in an in vitro protein-synthesizing system programmed with the phage message MS2-RNA that had been modified by site-directed mutagenesis so as to make it possible to monitor conveniently the reading of all four glycine codons . The results showed that while the efficiency of tRNA(1Gly) (UCC) was comparable to that of mycoplasma tRNA(Gly) (UCC) in the reading of the codon GGA, the mycoplasma tRNA(Gly) was far more efficient than the tRNA(1Gly) (UCC) in the reading of the codons GGU and GGC . Thus, the anticodon UCC, when present in the structural context of the tRNA(1Gly) molecule, behaved as predicted by the wobble rules while in the structural context of the mycoplasma tRNA(Gly) it read without discrimination between the nucleotides in the third codon position, in violation of the wobble restrictions . The result with the codon GGG showed that the anticodon UCC, when present in tRNA(1Gly), was considerably less efficient in reading this codon than it was in the structural context of the mycoplasma tRNA(Gly) . It would therefore seem that the anticodon UCC, when present in a certain tRNA, can be an efficient wobbler, while in the molecular environment of another tRNA it is markedly restricted in its ability to wobble. Eur J Biochem, 1989 Sep 1, 184(1), 47 - 52 Reactivity of the P-site-bound donor in ribosomal peptide-bond formation; Synetos D et al.; The puromycin reaction, catalyzed by the ribosomal peptidyltransferase, has been carried out so as to make the definition of two distinct parameters of this reaction possible . These are (a) the final degree of the reaction which gives the proportion of peptidyl (P)-site binding of the donor and (b) the reactivity of the donor substrate expressed as an apparent rate constant (kobs) . This kobs varies with the concentration of puromycin; the maximal value (k3) of the kobs, at saturating concen