Bioorg Khim, 1983 Mar, 9(3), 330 - 42 {Primary structure of the elongation factor G from Escherichia coli . VIII . Structure of tryptic peptides comprising the T4 fragment of limited trypsinolysis of the G-factor}; Alakhov IuB et al.; Products of tryptic hydrolysis of the maleic anhydride modified fragment Th3 from limited thermolytic hydrolysis of the G-factor have been studied . Some short peptides which result from the trypsin action on the native G-factor molecule and belong to the fragment T4 obtained on limited trypsinolysis of the G-factor have been separated and their structure has been studied . As a result amino acid sequence has been determined by tryptic peptides containing 322 amino acid residues of the fragment T4 which makes up about 94% of its polypeptide chain. Biochem Int, 1983 Mar, 6(3), 403 - 8 Hydroxyurea inhibits also the syntheses of thymine nucleotides and methionine in the cells of Escherichia coli; Heinonen J et al.; Hydroxyurea not only inhibited the reduction of ribonucleotides, but also gave rise to need of exogenous methionine and thymidine for full growth in the cultures of Escherichia coli K12 . In vitro the drug inhibited the formation of 5,10-methylenetetrahydrofolate by serine hydroxymethyltransferase in the cell extracts, which suggests that this reaction is a secondary target of hydroxyurea in the cells of E . coli . The syntheses of thymine nucleotides and methionine were also the most sensitive targets of hydroxylamine in the cultures of E . coli. Biochem Int, 1983 Mar, 6(3), 339 - 48 Interaction of purified rat liver glucocorticoid-receptor complexes with polynucleotides: strong base composition dependence; Romanov GA et al.; The binding of 10(3)-fold purified rat liver glucocorticoid receptors to natural and synthetic polynucleotides has been studied . The receptors may bind to RNA as well as to DNA . The binding effectiveness of nucleic acids is strongly base composition dependent, with natural DNAs (double-stranded or denatured) greater than E . coli rRNA greater than poly(dA).poly(dT) greater than or equal to poly d(A,T,G)7 greater than or equal to poly(U) greater than poly(A).poly(U) greater than poly(A) approximately poly(C) . The equilibrium (apparent) constants of receptor binding to a number of polynucleotides were calculated by a method proposed. Zentralbl Bakteriol Mikrobiol Hyg {A}, 1983 Mar, 254(1), 64 - 8 Colistin inhibition of mannose-resistant haemagglutination by K88-positive and K99-positive escherichia coli strains . A preliminary report; Sogaard H et al.; Two enteropathogenic E . coli strains isolated from a calf and a piglet succumbed to diarrhoea were studied . The bovine strain carried K99-antigen and the porcine strain was K88-positive . Both strains agglutinated pig erythrocytes in the presence of D-mannose . In the test a bacterial cell density of 3 x 10(9) per ml and doubling dilutions hereof were used . The haemagglutination titres were 16 and 128, respectively . When the bacteria were exposed to colistin before mixing with the red cells, haemagglutination was inhibited completely with 1.0 and 0.5 microgram/mg of colistin . At a colistin concentration of 0.25 microgram/ml (1/4-1/2 of the MIC's) the titres were lowered by a factor of 16-32. Scand J Gastroenterol, 1983 Mar, 18(2), 305 - 11 Studies of antibodies to lipid A and Tamm-Horsfall in patients with inflammatory bowel disease; Mattsby-Baltzer I et al.; Sera from patients with inflammatory bowel disease, ulcerative colitis (UC), or Crohn's disease (MC) were analysed for antibodies to lipid A and Tamm-Horsfall protein (TH), Escherichia coli O antigens, and food antigens, using enzyme-linked immunosorbent assay, indirect haemagglutination, and thin-layer immunoassay, respectively . C-reactive protein (CRP), an indicator of acute inflammation, was also studied . The MC and UC groups were not separated by any of the factors tested . The extent of the disease, however, seemed to influence the IgG antibody response to lipid A and TH . Patients with extended inflammatory areas in the intestine had decreased anti-lipid-A levels during the active phase of the disease compared with patients with less inflammation . In contrast, the IgG anti-TH levels were increased in these patients during both the active and inactive phases compared with less affected patients . Compared with healthy individuals, MC or UC patients showed decreased IgG anti-lipid-A levels . Both IgG and IgA anti-TH and total anti-O levels were increased compared with controls . Antibodies to cow's milk were lower in patients with inflammatory bowel disease than in the control group . CRP was increased in patients with active inflammatory bowel disease compared with inactive. J Gen Microbiol, 1983 Mar, 129 (Pt 3), 681 - 6 Control of recA dependent activities in Escherichia coli: a possible role for the recF product; Thomas A et al.; DNA repair and genetic recombination have been studied in recF143 mutants of Escherichia coli in which expression of inducible, SOS repair activities was altered by additional mutations either in lexA or recA . recF143 and lexA3 appeared to act additively to increase sensitivity to UV irradiation and to reduce recombination proficiency . The recF defect was suppressed in strains carrying the tif-1 allele of recA but not in strains carrying mutations that increased synthesis of recA+ protein or which directly inactivated lexA repressor . These and other data are interpreted to suggest that the recF defect is related to a reduced activity of recA protein . The implications of these findings are discussed in relation to the control of SOS activities and cell division during normal growth. J Cell Sci, 1983 Mar, 60, 67 - 87 Effects of endotoxin treatment on the differentiation of guinea pig megakaryocytes to blood platelets; Raha S et al.; The effect of endotoxin treatment (30 micrograms/kg body weight) on megakaryocyte development and platelet production in guinea pigs was investigated . A moderate thrombocytopenia was noted at 24 h after endotoxin administration . Recovery was observed at 48 h when the platelet count had almost reached the normal level . A significant increase in platelet production was obtained at 48 h, indicated by an enhancement in incorporation of labelled sulphate into platelets and also by an increase in the number of circulating heavier platelets . Megakaryocytes were classified into different developmental stages - megakaryoblast, promegakaryocyte, megakaryocyte - using standard morphological criteria (size, shape of nucleus, nucleus/cytoplasm ratio) . The number of mature megakaryocytes increased at 24 and 48 h after endotoxin treatment . It was possible to distinguish between two different types of mature megakaryocytes - early mature and mature megakaryocytes - by the presence of a methanol-sensitive acid phosphatase . This enzyme is found mainly in mature, presumably platelet-forming, megakaryocytes and in platelets . The increased number of mature megakaryocytes at 24 h was due to an enhancement in the number of early mature megakaryocytes, whereas the change at 48 h was due largely to an increased number of mature, platelet-forming megakaryocytes . Megakaryocytes were isolated from bone marrow of guinea pigs by Percoll density-gradient centrifugation and incubated with radioactively labelled leucine . Compared with the untreated controls megakaryocytes isolated 24 h after endotoxin treatment took up and incorporated higher amounts of leucine into protein, indicating a higher metabolic capacity of the early mature megakaryocytes . No alteration in the ploidy distribution of megakaryocytes was noted after endotoxin treatment. J Biochem (Tokyo), 1983 Mar, 93(3), 927 - 30 Construction of plasmid vectors for cloning of promoters using the dihydrofolate reductase gene of Escherichia coli K-12; Iwakura M et al.; Vectors for cloning promoter-DNA fragments were derived from pTP 30-5 which was constructed in our previous work (M . Iwakura et al . (1982) J . Biochem . 91, 1205) . The selection was based on the expression of trimethoprim resistance of transformed bacteria in which the enhancement of dihydrofolate reductase production was directed by the cloned promoter . A linear relationship between the content of dihydrofolate reductase and the strength of trimethoprim resistance was observed . It is suggested that the promoter activities can be estimated by trimethoprim resistance without measuring the enzyme activities. Z Naturforsch {C}, 1983 Mar-Apr, 38(3-4), 294 - 6 Circular permutation analysis of phage T4 DNA by electron microscopy; Grossi GF et al.; Phage T4 is known to have a linear duplex chromosome that is circularly permuted and terminally repeated . We found, by denaturation and self-reannealing experiments, that circular permutation in T4 native DNA is not random . Their multimodal distribution of permutation is compatible with the "headful packaging" model with the additional specifications that the encapsulation of DNA starts at several sites and these are not random distributed. Plasmid, 1983 Mar, 9(2), 218 - 21 Replication of plasmid R1: Meselson-Stahl density shift experiments revisited; Nordstrom K; Meselson-Stahl density shift experiments have been used extensively to study selection and timing of plasmid replication . Experiments with plasmid R1 were previously performed and the conclusion was that this plasmid replicates one copy at a time and that there is an eclipse period after each replication during which no further replications can take place in the cell (Nordstrom et al., Plasmid 1, 187-203 (1978)) . However, this interpretation is in conflict with other data, mainly with those obtained in copy number shift experiments (Gustafsson and Nordstrom, J . Bacteriol . 141, 106-110 (1980)) . However, the density shift experiments have now been reinterpreted such that there no longer is any conflict with the copy number shift experiments . There does not seem to be any such eclipse period, but newly replicated plasmid molecules are not available for a second replication for about 20% of a generation time. Pathol Res Pract, 1983 Mar, 176(2-4), 185 - 95 Epithelial abnormalities in the renal pelvis in experimental hydronephrosis and pyelonephritis; Baumgart P et al.; Unilateral hydronephrosis was induced by temporary ligature of the left ureter in 29 rabbits . In 21 animals so treated, chronic pyelonephritis was simultaneously induced by intravenous application of a suspension of E . coli . Histologic examination of renal pelvic epithelia in animals killed four weeks after the surgical intervention, revealed the following features: 1 . Simple hyperplasia of urothelium in 12 cases, 2 . atypical hyperplasia - (dysplasia) of urothelium in 10 cases, 3 . v . Brunn's nests in 20 renal pelvises; 11 cases of cystic pyelitis, all combined with Brunn's nests, 4 . metaplastic transformation of visceral mono- or bilayered epithelium into multi-layered urothelium-like structures in 19 renal pelvises . These changes are observed almost exclusively in the left renal pelvis of animals subjected to temporary ureteral ligature . Atypical urothelial hyperplasia is found only together with chronic pyelonephritis . Hyperplastic and dysplastic epithelial changes in the renal pelvis, the formation of Brunn's nests, and cystic pyelitis are interpreted as sequelae of postrenal obstruction or concomitant chronic inflammation. Gene, 1983 Mar, 21(3), 273 - 84 Immune response to human influenza virus hemagglutinin expressed in Escherichia coli; Davis AR et al.; Cloned DNA fragments coding for parts of strain WSN (H1N1) influenza virus hemagglutinin (HA) were fused to a bacterial leader DNA derived from the Escherichia coli trp operon . Fusion proteins produced consisted of 190 amino acids of trpLE' protein at the amino terminus, and HA amino acids, either 1-308, 1-396, or 1-548 (complete HA), at the carboxyl terminus . These proteins were expressed at high levels (10-20% of total protein) in E . coli starved for tryptophan . A CNBr fragment (HA1-211) was derived from HA-308 . Each of the proteins was purified and used for immunizing mice and rabbits . The antibody produced was shown to bind to (i) the HA fusion proteins, (ii) detergent-treated viral HA, (iii) HA, on intact virions, and (iv) the HA on the surface of cells infected with influenza virus . This shows that the HA fusion proteins expressed in bacteria can elicit antibodies that recognize at least some determinants of the native viral HA, and probably could lead to development of an anti-influenza vaccine. Genetika, 1983 Mar, 19(3), 435 - 9 {Coupling of transcription and translation as the factor regulating the transcription of genes rpoBC in Escherichia coli cells}; Lideman LF et al.; The natural transcription polarity of proximal and distal elements of the rplJL-rpoBC operon is increased when translation is inhibited in Escherichia coli cells . It is shown that transcription uncoupling to translation terminates within EcoRI-2,6 fragment of the operon which contains the transcription attenuator . We suggest that transcription attenuation in the rplJL-rpoBC operon is regulated by the coupling of transcription to translation of the intergenic fragment which overlaps the attenuator sequence. Genetika, 1983 Mar, 19(3), 416 - 24 {Participation of plasmid F in the replication of the chromosome of an Hfr strain of Escherichia coli K-12}; Chernin LS et al.; In the region of plasmid F DNA with coordinates 52,2-55,8 kb, the chr ("chromosome replication") locus has been revealed . A failure in the functioning of this locus in the integrated plasmid, which leads to a temperature-sensitive disturbance in chromosome replication of the Hfr strain and to the changes in its sensitivity to some membranotropic agents . Integration of an F segment containing the chr+ allele into the chromosome of an F-like derivative of such Hfr strain (retaining a mutant part of the F DNA), results in formation of temperature-resistant clones . In these clones, chromosomal replication is controlled by the plasmid replicon at the elevated temperature . It has been concluded that the F plasmid can control chromosome replication of the dna+ HfrC strain of Escherichia coli K-12 and that the product of the chr gene is a membrane protein involved in chromosomal replication. Anal Biochem, 1983 Mar, 129(2), 296 - 304 Determination of cobalamins in biological material . II . The cobalamins in human plasma and erythrocytes after desalting on nonpolar adsorbent material, and separation by one-dimensional thin-layer chromatography; Gimsing P et al.; An improved method for determination of cobalamins in biological materials is described . The cobalamins are extracted with ethanol after preincubation with cadmium acetate, which inhibits nonspecific adsorption of hydroxocobalamin to proteins . The extracts are desalted on Amberlite XAD-2 columns, which at least doubles the capacity of the analysis, as compared to the previous phenol procedure . The cobalamins are separated by one-dimensional thin-layer chromatography . Bioautography with Escherichia coli strain 113-3 is performed after the light-sensitive cobalamins and hydroxocobalamin have been converted to sulfitocobalamin . This is done in order to ensure comparable growth responses to equal amounts of the cobalamins . Reference intervals of plasma and erythrocytes of healthy blood donors for methyl-, 5'-deoxyadenosyl-, cyano-, hydroxo/aquo-, and sulfitocobalamin are presented . The differences between the present and previous results are discussed. Zh Mikrobiol Epidemiol Immunobiol, 1983 Mar, (3), 63 - 5 {Use of the toxin-tissue receptor reaction for detecting toxic substances of the causative agents of acute intestinal diseases}; Bunin KB et al.; The possibility of using erythrocytic ganglioside diagnostic reagents (EGDR) for the detection of V . cholerae, E . coli and S . typhimurium enterotoxins in the passive hemagglutination (PHA) test has been shown . Museum strains and cultures isolated from patients with acute intestinal diseases were tested for the presence of enterotoxins . Cell-free extracts were studied by biological methods and by serological titration in the PHA test with the use of EGDR . The diagnostic reagent was found to interact only with those enterotoxins whose specific receptors were gangliosides GM1. Ukr Biokhim Zh, 1983 Mar-Apr, 55(2), 162 - 8 {Nuclear DNAse from rat brain}; Ivanov VA et al.; Nuclear DNase specific for single-stranded DNA was obtained from the rat brain . The enzyme was isolated from a soluble protein fraction of total cell nuclei using gel filtration and ion-exchange chromatography . Nuclear DNase is Mg2+-dependent exodeoxyribonuclease and hydrolyzes homological, heterological and synthetic substrate at the same rate . It is established that nucleoside-5'-monophosphates are the major product of native and synthetic polydeoxyribonucleotides hydrolysis . It is supposed that the enzyme participates in reparation of neuron DNA. J Dairy Sci, 1983 Mar, 66(3), 556 - 61 Depression of B-lymphocytes by mastitis and treatment with levamisole; Ishikawa H et al.; Proportion of B-lymphocytes in milk and blood decreased simultaneously with development of mastitis induced with endotoxin and recovered with disappearance of clinical signs . However, the change in T-lymphocytes was slight . The reduced percentage of B-lymphocytes was caused by reduction in absolute number of such cells . When used orally, levamisole increased the proportion B-lymphocytes of milk to the same as or more than that of similar cells in peripheral blood . This compound may enhance activity of the bovine mammary immune system and be of value for control of bovine mastitis. J Clin Microbiol, 1983 Mar, 17(3), 419 - 21 Correlation between biochemical and serological characteristics of Escherichia coli and results of the Serény test; Toledo MR et al.; A total of 1,335 Escherichia coli strains isolated from patients with diarrhea were submitted to the Sereny test . Of these strains, 1,035 were also submitted to slide agglutination test, using antisera for invasive E . coli serogroups . Of 325 isolates that were lysine decarboxylase negative, 138 reacted with one of the E . coli antisera used . From this group of 138, 137 were positive in the Sereny test . Strains that were lysine decarboxylase positive (1,010 strains) were all negative in the Sereny test . These results show a close correlation between the biochemical and serological characteristics of E . coli and the results of the Sereny test. J Can Assoc Radiol, 1983 Mar, 34(1), 36 - 8 Ilio-psoas abscess in Crohn's disease; Gray RR et al.; We encountered 12 patients with psoas abscess in a two-year period . Of these, four were due to Crohn's disease making this the most important single cause of such abscesses in our series . Psoas abscess may be the presenting complaint in Crohn's disease and it should be considered as a possible cause of any psoas abscess from which enteric organisms are cultured. Infect Immun, 1983 Mar, 39(3), 1300 - 6 Partial purification and characterization of an escherichia coli toxic factor that induces morphological cell alterations; Caprioli A et al.; A factor produced by several strains of Escherichia coli isolated from enteritis-affected children has been shown to produce both a necrotizing effect on rabbit skin and striking morphological alterations on CHO, Vero, and HeLa cells . The same strains were found to have hemolytic activity on sheep erythrocytes . The toxic, cell-altering factor was demonstrated to be different from both heat-labile and heat-stable enterotoxins and from Vero toxin . The main effect induced by the isolated factor on cultured cells was the formation of large multinucleated cells . The partial purification achieved suggests that the same factor (most likely a protein with a molecular weight of 70,000 to 80,000) is responsible for toxic and cell-altering activities, whereas a different molecular species is responsible for hemolytic activity. Infect Immun, 1983 Mar, 39(3), 1167 - 74 Isolation and nucleotide sequence determination of a gene encoding a heat-stable enterotoxin of Escherichia coli; Moseley SL et al.; A gene encoding a heat-stable enterotoxin (ST) from an Escherichia coli strain isolated from a human with diarrhea was cloned and characterized by nucleotide sequence analysis . The gene was found to be partially homologous to a previously characterized ST gene from an E . coli strain of bovine origin . Hybridization studies showed that most ST-producing strains of E . coli isolated from humans with diarrhea possess genes highly homologous to either the ST gene from the bovine strain or the ST gene characterized in the present study. Biophys Chem, 1983 Mar, 17(2), 165 - 9 Correlation between biological activity of 30 S subunits of Escherichia coli ribosomes and their conformation changes revealed by optical mixing spectroscopy; Dobitchin PD et al.; Spectral analysis of light scattered from solutions of 30 S subunits was performed by the method of regularization of the inverse spectral problem . The subunits observed under ionic conditions which preserved their biological activity (200 mM NH4Cl at 1 mM MgCl2) revealed a monodisperse pattern of scattering with diffusion constant D = (1.83 +/- 0.10) X 10(-7) cm2/s . The polydispersity and compaction of 30 S subunits were observed under inactivation ionic conditions (30 mM NH4Cl at 1 mM MgCl2) . The number of compacted particles correlates with the irreversible loss of biological activity, the ability of 30 S subunits to bind specific tRNA. Biochemistry, 1983 Mar 1, 22(5), 1229 - 36 A stereochemical and positional isotope exchange study of the mechanism of activation of isoleucine by isoleucyl-tRNA synthetase from Escherichia coli; Lowe G et al.; Isoleucyl-tRNA synthetase from Escherichia coli catalyzes the activation of {18O2}isoleucine by adenosine 5'-{(R)-alpha-17O}triphosphate with inversion of configuration at phosphorus . Moreover, isoleucyl-tRNA synthetase does not catalyze positional isotope exchange in adenosine 5'-{beta-18O2}triphosphate in the absence of isoleucine or in the presence of the competitive inhibitor isoleucinol, which effectively eliminates the possibility of either adenylyl-enzyme or adenosine metaphosphate intermediates being involved . Together, these observations require that isoleucyl-tRNA synthetase catalyzes the activation of isoleucine by associative "in line" nucleotidyl transfer . The synthesis of adenosine 5'-{(R)-alpha-17O}diphosphate and its conversion to adenosine 5'-{(R)-alpha-17O}triphosphate is described and an explanation provided for the reported differences between the treatment of adenosine 5'-{(S)-alpha-thiodiphosphate} with cyanogen bromide and bromine in {18O}water. Biochemistry, 1983 Mar 1, 22(5), 1193 - 200 Composition of the Escherichia coli 70S ribosomal interface: a cross-linking study; Chiam CL et al.; 70S tight-couple ribosomes from Escherichia coli were cross-linked by using the bifunctional reagent phenyl-diglyoxal (PDG) . The reaction was stopped after 4-h incubation while still in the linear range . In comparison with untreated ribosomes, 30% of those treated with PDG were shown, by sucrose gradient experiments, not to be separable into their subunits, but remained as 70S particles . There was no detectable change in the structure of the reacted particles when their sedimentation behavior was compared with that of native 70S controls . When the cross-linking reaction was performed in the presence of tRNAPhe and poly(U), the reacted ribosomes retained 40-50% of their tRNA binding activity . The reaction leads predominantly to the formation of RNA-protein cross-links but protein--protein as well as RNA-RNA cross-links could also be detected . Cross-linked material was extracted, and the individual RNAs were separated into 23S, 16S, and 5S RNAs . Proteins were identified electrophoretically after reversal of the RNA-protein cross-links . Proteins were found to be cross-linked to RNAs within and across the ribosomal subunits; the latter are considered to be close to or at the 70S subunit interface . The arrangement of RNA and protein at the subunit interface is discussed. Arch Biochem Biophys, 1983 Mar, 221(2), 329 - 32 Electrostatic facilitation of the reaction catalyzed by the manganese-containing and the iron-containing superoxide dismutases; Benovic J et al.; Both the iron-containing and the manganese-containing superoxide dismutases from Escherichia coli show diminished activity with increasing ionic strength, indicative of electrostatic facilitation of the catalyzed reaction . Since both enzymes bear a net negative charge at the assay pH, as does the substrate, this suggests a cationic locale in the active site region . Acetylation of the enzymes inverted their response to increasing ionic strength . It thus appears that lysine residues provide the observed electrostatic facilitation . A specific inhibition by large monovalent anions was observed with the iron-containing superoxide dismutase and was taken to indicate the presence of a cationic group, within a hydrophobic crevice, at the active site. Proc Natl Acad Sci U S A, 1983 Mar, 80(6), 1541 - 5 In vitro bypass of UV-induced lesions by Escherichia coli DNA polymerase I: specificity of nucleotide incorporation; Rabkin SD et al.; A variety of DNA polymerases, synthesizing in vitro on an UV-irradiated phi X174 DNA template, terminate synthesis one nucleotide before the 3' pyrimidines of putative dimers on the template . We have devised a system using Escherichia coli DNA polymerase I (Klenow fragment) that can synthesize past at least some of these dimers . The bypass is carried out in a multistep process--first, the incorporation of nucleotides opposite the pyrimidines in the dimer and, then, the addition of nucleotides complementary to the bases distal to the dimer . The insertion of a nucleotide opposite the first (3') pyrimidine of a putative dimer in the presence of Mn2+ occurs in a concentration-dependent fashion with a 3- to 4-fold preference for purine nucleotides over pyrimidine nucleotides . In the presence of Mg2+, insertion is less frequent . Correlation of these results with in vivo mutation data suggests a role for the polymerase in determining the spectrum of base substitution mutagenesis in SOS induced cells. Mutat Res, 1983 Mar, 116(3-4), 317 - 22 Protoanemonin, an antimutagen isolated from plants; Minakata H et al.; Protoanemonin was identified as the factor responsible for the antimutagenicity of Ranunculus and Anemone plants against the strain E . coli B/r WP2 trp . The specific activities found were AD50 = 30 and 47 microgram/plate, respectively, for UV- and N-methyl-N'-nitro-N-nitrosoguanidine-induced mutations . Quantitative analyses of protoanemonin in the crude extracts of Ranunculus and Anemone plants were performed by reversed-phase high-pressure liquid chromatography. J Surg Res, 1983 Mar, 34(3), 246 - 53 Effects of glucose-insulin-potassium (GIK) on the position of the oxyhemoglobin dissociation curve, 2.3-diphosphoglycerate, and oxygen consumption in canine endotoxin shock; Tuynman HA et al.; The effects of glucose-insulin-potassium (GIK) on hemodynamics, oxygen transport, P50, 2,3-diphosphoglycerate (2.3-DPG), and adenosine triphosphate (ATP) were evaluated in canine endotoxin shock . Ten dogs were studied under general anesthesia and controlled ventilation . Shock was induced with Escherichia coli endotoxin (1.5 mg/kg body wt) . Thereafter two groups of five dogs each were formed by randomization . The one group received GIK (glucose 50%, 2 g/kg, insulin 3 U/kg, and 10 mmole K) in the period between 90 and 120 min after endotoxin . The other group received an equal amount of NaCl infusion and served as a control group . Observations were completed at 180 min after endotoxin . GIK resulted in a significant increase of cardiac output, stroke volume, mean arterial pressure, and oxygen consumption . Serum phosphate levels decreased . No changes were observed of P50 in vitro (at 37 degrees C and pH 7.40) and of P50 in vivo, nor of 2.3-DPG and ATP in the red cells . The data suggest that the increased oxygen consumption after GIK in canine endotoxin shock is caused only by improvement of cardiac output and oxygen availability and not by an effect on oxygen unloading capacity of hemoglobin. Eur J Biochem, 1983 Mar 1, 131(1), 119 - 27 Maturation of 5-S rRNA: ribonuclease E cleavages and their dependence on precursor sequences; Roy MK et al.; 9-S RNA is a processing intermediate that accumulates in an RNase E- strain of Escherichia coli . It spans from the RNase III cleavage site, after 23-S rRNA, to the 3' end of the transcript and is derived from rRNA genes which do not contain tRNAs distal to 5-S rRNA . Here, we have studied the processing of 9-S RNA with ribonuclease E . RNase E cleaves 9-S RNA in two sites: one of these is three nucleotides upstream from the 5' end of 5-S rRNA, the other downstream from its 3' end . Both cleavages are probably introduced by the same enzyme, since both cleavages are thermolabile when an extract of a temperature-sensitive RNase E mutant was used for processing in vitro . In order to asses the role of 5' and 3' end precursor-specific sequences in the RNase E reaction, we isolated the molecules lacking nucleotides at the 5' or 3' end . Molecules having the 5' end of 9-S RNA but missing nucleotides from the 3' end (called 8-S RNA) were as good a substrate for RNase E as 9-S, RNA itself . However, molecules having the 3' end of 9-S RNA but the 5' end of p5 (called 7-S RNA), were less efficient substrates for RNase E . Finally, the removal of as little as seven nucleotides from the 5' end of 8-S RNA rendered it almost completely unsuitable as a substrate for RNase E. Biull Eksp Biol Med, 1983 Mar, 95(3), 56 - 9 {Mechanism of action of an exopolysaccharide from Mycobacterium cyaneum on the local cellular reaction in experimental coli infection}; Oleinikova EA et al.; A study was made of the effect of the exopolysaccharide (PS) M . cyaneum B-646 on cellular reaction in peritoneal exudate of white mice infected with E . coli . PS intensified the migration of phagocytic cells to the focus of infection, accelerated neutrophil maturation, promoted an earlier and more active involvement of neutrophils, particularly of macrophages, into the phagocytic process . In this case, there was a marked correlation between the magnitude of phagocyte population (of macrophage population to a greater degree) in peritoneal exudate and absorption capacity . PS activated macrophagal lysosomes, affecting selectively the accumulation of enzymes by the cells and lysosomal membrane permeability . The action of PS is dose-dependent . Under experimental conditions in question, the most favourable effect on local cellular reaction was exerted by PS in a dose of 20 micrograms per mouse. Am J Physiol, 1983 Mar, 244(3), R368 - 73 Relationship of trace metals to fever during infection: are prostaglandins involved? Tocco RJ, Kahn LL, Kluger MJ, Vander AJ. Endogenous pyrogen/leukocyte endogenous mediator (EP/LEM) induces, in the host organism, a fever that is thought to be mediated to some extent via the production of prostaglandins . The role of prostaglandins in the EP/LEM-induced fall in plasma iron and zinc is less clear . To study this relationship, rabbits and rats were injected with an antipyretic dose of prostaglandin synthase inhibitors concurrent with heat-killed bacteria or endotoxin . These inhibitors, indomethacin and sodium salicylate, were successful in partially and totally blocking fever in rabbits and rats, respectively; however, they had no effect on the hypoferremia and hypozincemia of infection . We conclude that prostaglandins are probably not involved in the fall in plasma iron and zinc during acute bacterial infection . Further, since hypoferremia and hypozincemia occurred even though fever was blocked, the fall in trace metals due to infection is not dependent on the rise in both temperature. Am J Physiol, 1983 Mar, 244(3), H370 - 7 Canine left ventricular performance during LD50 endotoxemia; Goldfarb RD et al.; Previous reports of the effect of endotoxin shock on cardiac performance have not achieved uniform results . These discrepancies have possibly been caused by the use of indices of cardiac performance that may have been sensitive to altered heart rate or preconditions of cardiac contraction as well as altered cardiac performance . We tested the hypothesis that, following a median lethal dose (LD50) of E . coli endotoxin, cardiac performance would be diminished in nonsurviving animals and maintained in surviving animals . We elected to employ the analysis of the end-systolic pressure-diameter relationship (sigma ES) as well as other measurements of cardiac performance to test this hypothesis . We established that the sigma ES measurement was independent of increased and decreased afterload and relatively insensitive to altered heart rate . In the nonsurviving animals, sigma ES exhibited a marked depression following endotoxin a administration . In the surviving animals, sigma ES exhibited a nonsignificant decrease followed by a return toward preendotoxin values . All other cardiodynamic measurements were uninterpretable due to the marked changes in heart rate, peripheral vascular function, aortic pressure, and cardiac output . We conclude that, following endotoxin administration, those animals that exhibited a diminished myocardial contractility failed to survive more than 2.5 h postendotoxin, whereas the surviving animals were able to restore normal cardiac contractility . Thus survival of endotoxin administration is associated with the maintenance of normal cardiac contractility. J Bacteriol, 1983 Mar, 153(3), 1502 - 12 Inverted repeats in the DNA of plasmid pCU1; Konarska-Kozlowska M et al.; Renaturable regions in the DNA strands of the N group plasmid pCU1 have been visualized as stem-loop structures by electron microscopy . Four such distinct structures are described, the smallest of which is within the loop of a larger one . The region of pCU1 in which these structures occur has several restriction sites . This and the availability of plasmid deletions and recombinants has permitted the mapping of these structures relative to one another and to the restriction and functional map of the plasmid . The replication and maintenance region of the plasmid is located within one of these stem-loop structures. J Bacteriol, 1983 Mar, 153(3), 1379 - 87 Cell elongation and division probability during the Escherichia coli growth cycle; Kubitschek HE et al.; A new method is presented for determining the growth rate and the probability of cell division (separation) during the cell cycle, using size distributions of cell populations grown under steady-state conditions . The method utilizes the cell life-length distribution, i.e., the probability that a cell will have any specific size during its life history . This method was used to analyze cell length distributions of six cultures of Escherichia coli, for which doubling times varied from 19 to 125 min . The results for each culture are in good agreement with a single model of growth and division kinetics: exponential elongation of cells during growth phase of the cycle, and normal distributions of length at birth and at division . The average value of the coefficient of variation was 13.5% for all strains and growth rates . These results, based upon 5,955 observations, support and extend earlier proposals that growth and division patterns of E . coli are similar at all growth rates and, in addition, identify the general growth pattern of these cells to be exponential. J Bacteriol, 1983 Mar, 153(3), 1361 - 7 Isolation of conditional lethal mutator mutants of Escherichia coli by localized mutagenesis; Maki H et al.; By using localized mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine, we isolated 39 temperature-sensitive growth mutants that exhibited high mutability when the bacteria were grown at the permissive temperature . Two of the mutations, dnaQ186 and dnaQ231, were shown to be new alleles of the dnaQ gene by genetic mapping and complementation tests with the dnaQ49 mutation previously isolated . They shared common properties with the dnaQ49 strain, but their mutator activity was not temperature dependent . The dnaQ mutants exhibited increased sensitivity to inhibitors of DNA gyrase and to DNA intercalating and alkylating agents. J Bacteriol, 1983 Mar, 153(3), 1301 - 7 A new gene (alkB) of Escherichia coli that controls sensitivity to methyl methane sulfonate; Kataoka H et al.; Seven mutants of Escherichia coli were isolated that are sensitive to methyl methane sulfonate but not to UV light . They exhibited decreased host cell reactivation capacity for methyl methane sulfonate-treated phage lambda . Five of the mutations were mapped in the same region as alkA (previously called alk) and may indeed be identical to known mutations . Another mutation was found near nalA, and the gene responsible was named alkB . Its phenotype was different from that of ada, since the alkB mutant exhibited a normal adaptive response to N-methyl-N'-nitro-N-nitrosoguanidine . A third type of mutation was mapped near polA, but this mutant contained an almost normal level of DNA polymerase I activity. Obstet Gynecol, 1983 Mar, 61(3), 271 - 4 Impairment of tissue defenses by vasoconstrictors in vaginal hysterectomies; England GT et al.; This paper examines the role of vasoconstrictors, specifically epinephrine, as an etiologic factor in the impairment of the host's defense mechanism . One hundred patients were injected circumferentially around the cervix with a dilute solution of epinephrine before vaginal hysterectomy; these patients were compared to a control group of 100 patients injected in the same manner with a normal saline solution . This randomized prospective study shows that vasoconstrictors significantly predisposed the vaginal cuff surgical site to infection compared to the matched control group . This study shows also that the use of a vasoconstrictor did not significantly reduce blood loss . Based on these data, it is recommended that a locally injected vasoconstrictor not be used during vaginal hysterectomy. J Gen Microbiol, 1983 Mar, 129 (Pt 3), 801 - 7 Relationship of the replication and incompatibility genes of an IncFVI haemolysin plasmid with other F-like haemolysin plasmids; Zabala JC et al.; The replication and incompatibility region of the IncFVI plasmid pSU502 has been isolated by in vitro DNA manipulation as part of a 12.6 kb plasmid, denominated pSU503 . Plasmid pSU503 was strongly incompatible with its parental plasmid, pSU1, but was fully compatible with the haemolytic plasmids pSU316 (IncFIII/IV), pHly152 (IncI2) and pSU233 (Inc-pSU233) . Furthermore, the 6.9 kb EcoRI fragment of pSU503 which carries the replication and incompatibility determinants of pSU1 did not show any detectable homology (less than 70%) with any of the haemolysin-determining plasmids with which it is compatible . Thus, homologous haemolysin determinants have become linked to apparently unrelated replicons. J Gen Microbiol, 1983 Mar, 129 (Pt 3), 671 - 80 Hybrid plasmids containing the pyruvate dehydrogenase complex genes and gene-DNA relationships in the 2 to 3 minute region of the Escherichia coli chromosome; Guest JR et al.; A sample of colonies from the Clarke-Carbon ColE1-Escherichia coli DNA plasmid gene bank was screened by conjugation for complementation of the lipoamide dehydrogenase lesion of a deletion strain lacking all components of the pyruvate dehydrogenase complex, delta (aroP aceE aceF lpd) . Two ColE1-lpd+ hybrid plasmids were identified: pGS2 (ColE1-ace lpd+; 24 kb) and pGS5 (ColE1-lpd+; 14 kb) . Enzymological studies confirmed that pGS2 expressed all the activities of the pyruvate dehydrogenase complex, whereas pGS5 expressed the lipoamide dehydrogenase and acetyltransferase activities (the latter from a ColE1 promoter) . These and other plasmids were used to construct a 47-site (15 enzymes) restriction map for a 24.2 kb segment of bacterial DNA in the nadC-lpd region . A further 13 sites (six enzymes) were defined in a 5.4 kb sub-segment containing the lpd gene . lambda phage derivatives containing specific fragments were constructed and used in transduction studies which located the ace and lpd genes in a 7.78 kb sub-segment flanked by AccI and NruI sites. Plasmid, 1983 Mar, 9(2), 201 - 14 Gene rearrangements leading to the expression of an insertion-inactivated tetracycline resistance gene in pBR322; Brunel F et al.; Cloning into the HindIII site of plasmid pBR322 inactivates the tetR promoter and usually prevents the expression of the tetR gene . The corresponding clones revert to tetracycline resistance at a low frequency . Such reversion is caused by gene rearrangement within the plasmids . DNA sequence analysis reveals three classes of revertants . The first class contains plasmids with partial duplications, which result in the fusion of the promoter of the RNA I species to the tetR gene . The event itself destroys the region encoding the RNA primer for replication and thus the plasmids would be replication defective if the duplication did not also include this region of the molecule . The plasmids from the second class are simple deletions which again fuse the tetR region to the RNA I promoter . In one case, the junction takes place at the end of the RNA I transcript, leaving RNA I and the RNA primer virtually intact . However, it removes the promoter of the RNA primer, the latter now being read from the cloned material . The second member of this class has fused the tetR gene well upstream of the RNA I region so that the RNA primer is still read from its own promoter . The low-level tetracycline resistance is probably due to partial read-through of the RNA I terminator . The third class of revertants differs from the previous two by the acquisition of foreign DNA in the form of an IS2-type insertion element which is known to promote transcription. Mol Biol (Mosk), 1983 Mar-Apr, 17(2), 418 - 29 {Dependence of the activity of phi X 174 B-promoter in the expression of Escherichia coli gal-operon on the number of its copies and their orientation}; Fedchenko VI et al.; Promoter activities of different restriction fragments of the R8 DNA region of phage phi X 174 were compared . The studied DNA fragments included HindII fragment R8 (B-promoter), its left portion 49 nucleotide long, and the central segment containing 113 nucleotides generated by AluI . The promoter activity of these fragments was quantitated by the appearance of uridyltransferase and galactokinase activities in Escherichia coli clones carrying plasmids pHD68-17 . The gal-promoters of these plasmids was substituted for the three aforementioned restriction fragments . The R8 region and its central part (BII-promoter) had comparable promoter activities while the left part containing the putative BI-promoter, did not induce clones with the expressed gal-operon . Clones containing 1, 2, 3 copies of the promoter fragment R8 were selected . No clones were revealed with more copies . All selected di- and tri-promoter clusters in plasmids had the same correct orientation of all inserted promoters with respect to the gal-operon . The expression of the gal-operon in E . coli was nearly directly proportional to the number of the phi X 174 B-promoters inserted before the operon. Mol Biol (Mosk), 1983 Mar-Apr, 17(2), 362 - 72 {Spectral division of conformational states of spin-labeled tRNA Phe from Escherichia coli}; Isaev-Ivanov VV et al.; A spectral division method of two conformational states of spin-labeled macromolecules is presented . The method is suitable in conditions of highly anisotropic motion of spin label and is based on titration of experimental spectra of spin-labeled macromolecule by theoretical ones . Theoretical spectra simulation uses the Freed theory and spin-Hamiltonian parameters, derived from independent experiments . Nomogrammes and formula for calculation of order parameter Sz and correlation time tau c in temperature-viscosity experiment are available . The method was applied to spectral division of two conformational states of spin-labeled tRNAPhe from E . coli and spectral parameters Sz and tau c were obtained for both states . ESR spectra of these conformational states at t degree = 20 degrees differ strongly from one another by order parameter Sz . The first conformer, that is characterised by a greater order parameter has no globular conformational transition (in terms of changes of the hydrodynamic macromolecule radius) between 2 degrees and 20 degrees, but local conformational changes take place in this temperature region. J Vet Pharmacol Ther, 1983 Mar, 6(1), 3 - 12 The recombinant DNA technology; Tiemeier DC; The recombinant DNA technology or DNA cloning permits the isolation, amplification, and precise manipulation of specific DNA fragments . This is generally accomplished by linking or recombining the desired DNA fragment with a DNA molecule, termed the vector, which is capable of directing the replication of itself in a suitable host cell and any DNA segment covalently attached to it . Using this and associated technologies, it is possible to produce large amounts of specific proteins and to modify cell types by introducing the genes for proteins that are otherwise absent . Moreover, it is now possible to construct variants of naturally-occurring proteins with improved biological or physical properties. Genetika, 1983 Mar, 19(3), 425 - 34 {Temperature-sensitive mutant Escherichia coli for the B-subunit of DNA-gyrase . The effect on replication and transcription}; Mirkin SM et al.; A temperature sensitive mutant affecting the B-subunit of DNA gyrase was isolated . The mutation leads to the thermolability of DNA gyrase in vitro and to the plasmid DNA relaxation in vitro . The immediate stop of the increase in mutant culture titre has been observed under nonpermissive conditions . However, DNA synthesis does not cease, though its efficiency is reduced by a factor of three . The transcription rate is also reduced two - three times, but more quickly than the rate of replication . This would mean that the transcription apparatus is the first to react to the change of DNA supercoiling in the cell . This suggestion is supported by the facts that small amounts of rifampicin increase the viability of ts mutant cells under nonpermissive conditions and also by the previously obtained results concerning the change in sensitivity of RNA polymerase mutants to DNA gyrase inhibitors. Genetika, 1983 Mar, 19(3), 388 - 96 {Independence of the processes of transposition and genome rearrangements induced by transposons}; Nechaeva EV et al.; The data on the influence of the tnm mutations affecting transposition process on the deletion formation promoted by Tn and IS elements are presented . It was shown that the tnm mutations did not affect the frequency of deletion formation . The results of genetic analysis of the tnm mutant deficient in both transposition and genomic rearrangements induced by Tn9 inserted into lambda prophage, indicated that the mutant phenotype was caused by two different but linked mutations . A mutation affecting the process of genomic rearrangements was designated gerA2 . The gerA2 mutation decreased sharply the frequency of rearrangements promoted by Tn9, Tn10 or Tn601 inserted into lambda prophage . However, this mutation had no influence upon transposition of the same Tn elements . The data obtained could be interpreted as indicating the independence of the processes of transposition and genomic rearrangements or as indication of the existence of specific steps of these processes. Proc Natl Acad Sci U S A, 1983 Mar, 80(6), 1492 - 5 Enzymic modification of a tyrosine residue to a stable free radical in ribonucleotide reductase; Barlow T et al.; Protein B2, a subunit of ribonucleotide reductase from Escherichia coli, contains in its active form a tyrosyl free radical as part of the polypeptide chain and a dimeric iron center that stabilizes the radical . The enzyme depends on this radical for its catalytic activity . Treatment with hydroxyurea scavenges the radical without disturbing the iron center and, thereby, results in an inactive form of the subunit, B2/HU . A second inactive form, apoB2, lacking both the radical and the iron center, is obtained by treatment of B2 with 8-hydroxyquinoline . Here we describe an enzyme activity in extracts from E . coli that transforms the catalytically inactive B2/HU form into the active B2 subunit by regeneration of the tyrosyl radical . This reaction requires the presence of oxygen, dithiothreitol, and Mg2+ and does not proceed through apoB2 . Under anaerobic conditions, we obtained evidence for a second activity in the bacterial extract that destroys the free radical and transforms B2 into B2/HU . We suggest that this novel type of protein modification is functionally related to the synthesis of deoxyribonucleotides and DNA. J Virol, 1983 Mar, 45(3), 1195 - 9 Cloning and analysis of reverse transcript P160 genomes of Abelson murine leukemia virus; Latt SA et al.; Circular duplex reverse transcripts of the genome of a strain of Abelson murine leukemia virus that encodes a 160,000-molecular-weight protein were isolated, cleaved with HindIII restriction endonuclease, and cloned into the unique HindIII site of lambda phage Charon 21A . Recombinant phage clones, some of which were infectious in transfection assays, were found to contain a 789-base-pair region specific for Abelson murine leukemia virus; this region is not found in other strains of this virus . The extra sequence was localized by restriction endonuclease and electron microscopic heteroduplex analysis . Sequence analysis showed no homology at the ends of the extra sequence, implying that it was deleted by an event that did not utilize sequence homology . The sequence of this unique region has an open reading frame through its entirety. Eur J Biochem, 1983 Mar 1, 131(1), 137 - 41 Evidence for a restriction/modification-like system in Anacystis nidulans infected by cyanophage AS-1; Szekeres M et al.; Anacystis nidulans infected by AS-1 cyanophage contains an endonuclease (AS-1 endonuclease) which splits host DNA but not AS-1 phage DNA {Szekeres, M . (1981) Virology, 111, 1-10} . AS-1 phage DNA proved to be resistant not only to AS-1 endonuclease but also to a number of restriction endonucleases the recognition sites of which contain a central dG-dC dinucleotide . Since an unmodified 5'dG-dC dinucleotide was shown to be present at the sites at which DNA is cleaved by AS-1 endonuclease, the results suggest that the sites attacked preferentially by the AS-1 endonuclease are specifically protected on the AS-1 DNA molecule . The modification of AS-1 DNA was shown to occur specifically in infected Anacystis because AS-1 DNA fragments which are normally resistant to AS-1 endonuclease became susceptible to this enzyme if inserted into pBR322 plasmid and cloned in Escherichia coli . AS-1 DNA was shown to contain about 5% of a modified nucleotide which was not 5-methyldeoxycytidylic acid . Results presented and our earlier data suggest that in Anacystis infected by AS-1 phage, a restriction/modification-like system operates which is able to eliminate 'unwanted' (host) DNA selectively. Cell, 1983 Mar, 32(3), 783 - 8 A control element within a structural gene: the gal operon of Escherichia coli; Irani MH et al.; The gal operon of Escherichia coli is transcribed from two overlapping promoters, PG1 and PG2 . Cyclic AMP and its receptor protein (CRP) modulate the two promoters in opposite directions by binding to a single cat locus . Both the promoters are negatively regulated by a single repressor, the product of the galR gene . An operator site, defined by several mutations, has previously been located upstream from the cat locus . We have isolated and characterized a new set of cis-dominant constitutive mutations of the gal operon and determined their locations by DNA sequencing . From these studies, we propose the existence of a second functional gal operator element at an extraordinary site--within galE, the first structural gene . Both the operators, OE (exterior) and OI (interior), are involved in the repression of PG1 and PG2 . This would be the first example of the presence of a functional operator element within a structural protein-coding region. J Bacteriol, 1983 Mar, 153(3), 1513 - 20 chlC (nar) operon of Escherichia coli includes structural genes for alpha and beta subunits of nitrate reductase; Edwards ES et al.; The synthesis of the alpha and beta subunits of nitrate reductase by 20 chlC::Tn5 insertion mutants of Escherichia coli was determined by immune precipitation of the subunits from fractions of cell extracts . Only two of the mutants produced either subunit in detectable amounts; these two accumulated the alpha subunit, but no beta subunit . In both cases the alpha subunit was present in the cytosolic fraction, in contrast to wild-type cells, in which both subunits are present mainly in the membrane fraction . EcoRI restriction fragments containing the Tn5 inserts from five of the mutants were cloned into pBR322 . The insertions were localized on two contiguous EcoRI fragments spanning a 5.6-kilobase region that overlapped the contiguous ends of the two fragments . An insertion that permitted alpha subunit formation defined one end of the 5.6-kilobase region . The results indicated that the genes encoding the alpha and beta subunits of nitrate reductase were part of a chlC (nar) operon that is transcribed in the direction alpha leads to beta. J Bacteriol, 1983 Mar, 153(3), 1479 - 85 cea-kil operon of the ColE1 plasmid; Sabik JF et al.; We isolated a series of Tn5 transposon insertion mutants and chemically induced mutants with mutations in the region of the ColE1 plasmid that includes the cea (colicin) and imm (immunity) genes . Bacterial cells harboring each of the mutant plasmids were tested for their response to the colicin-inducing agent mitomycin C . All insertion mutations within the cea gene failed to bring about cell killing after mitomycin C treatment . A cea- amber mutation exerted a polar effect on killing by mitomycin C . Two insertions beyond the cea gene but within or near the imm gene also prevented the lethal response to mitomycin C . These findings suggest the presence in the ColE1 plasmid of an operon containing the cea and kil genes whose product is needed for mitomycin C-induced lethality . Bacteria carrying ColE1 plasmids with Tn5 inserted within the cea gene produced serologically cross-reacting fragments of the colicin E1 molecule, the lengths of which were proportional to the distance between the insertion and the promoter end of the cea gene. J Bacteriol, 1983 Mar, 153(3), 1368 - 78 Isolation and characterization of Tn5 insertion mutations in the lexA gene of Escherichia coli; Krueger JH et al.; A Mu d(Ap lac)-generated fusion of lacZ to dinD, a gene induced by DNA damage, was used to isolate Tn5 insertion mutations that affect the regulation of the SOS responses . Three mutants were obtained that contained Tn5 insertions genetically linked to the lexA gene and had properties that suggested the mutants were deficient in lexA expression . The lexA protein has been shown to function as the repressor for genes involved in the SOS responses . By Southern blotting experiments, the three Tn5 insertions were physically mapped to distinct locations within the coding region of the lexA gene . The introduction of these mutations in six strains carrying lacZ fusions to different damage-inducible genes resulted in high expression of beta-galactosidase in all but one of the strains . In the dinF fusion strain, lacZ expression was reduced below that seen in a lexA+ background . Physical mapping studies of the dinF locus gave results consistent with the notion that dinF is part of the lexA transcription unit and that a lexA::Tn5 mutation has a polar effect on dinF expression . With certain din-lac fusion strains, a correlation was seen between the amount of beta-galactosidase production and the location of the particular Tn5 insertion within the lexA gene. J Bacteriol, 1983 Mar, 153(3), 1352 - 60 Plasmids of enterotoxigenic Escherichia coli H10407: evidence for two heat-stable enterotoxin genes and a conjugal transfer system; Yamamoto T et al.; Three species of plasmids, associated with virulence and conjugal transfer, were identified in a clinically isolated enterotoxigenic Escherichia coli strain, H10407 (serotype O78:H11) . pCS1, a non-self-transmissible plasmid species with a molecular weight of 62 X 10(6) and a 47 mol% guanine-plus-cytosine content, specified colonization factor antigen I and heat-stable enterotoxin (ST) production, as reported by others previously . A second non-self-transmissible plasmid species, designated pJY11, with a molecular weight of 42 X 10(6) and a 51 mol% guanine-plus-cytosine content, specified ST and heat-labile enterotoxin production and manifested T5/T6 phage restriction . The third plasmid species, pTRA1, also had a molecular weight of 42 X 10(6) and had a guanine-plus-cytosine content of 51 mol%; this species was self-transmissible and promoted transfer of both pCS1 and pJY11 to other bacterial cells . pCS1 may have originated from species of bacteria with a lower guanine-plus-cytosine content than E . coli . Finally, although demonstrating some heterogeneity with each other, both STs encoded by pCS1 and pJY11 belonged to the STa group. J Bacteriol, 1983 Mar, 153(3), 1187 - 95 Adenylate cyclase is required for chemotaxis to phosphotransferase system sugars by Escherichia coli; Black RA et al.; We report that in Escherichia coli, chemotaxis to sugars transported by the phosphotransferase system is mediated by adenylate cyclase, the nucleotide cyclase linked to the phosphotransferase system . We conclude that adenylate cyclase is required in this chemotaxis pathway because mutations in the cyclase gene (cya) eliminate or impair the response to phosphotransferase system sugars, even though other components of the phosphotransferase system known to be required for the detection of these sugars are relatively unaffected by such mutations . Moreover, merely supplying the mutant bacteria with the products of this enzyme, cyclic AMP and cyclic GMP, does not restore the chemotactic response . Because a residual chemotactic response is observed in certain strains with residual cyclic GMP synthesis but no cyclic AMP synthesis, it appears that the guanylate cyclase activity rather than the adenylate cyclase activity of the enzyme may be required for chemotaxis to sugars transported by the phosphotransferase system . Mutations in the cyclic nucleotide phosphodiesterase gene, which increase the level of both cyclic AMP and cyclic GMP, also reduce chemotaxis to these sugars . Therefore, it appears that control of the level of a cyclic nucleotide is critical for the chemotactic response to phosphotransferase system sugars. Radiat Res, 1983 Mar, 93(3), 609 - 12 Inducible repair of thymine ring saturation damage in phi X174 DNA; Achey PM et al.; The susceptibility to inducible SOS repair of 5,6-dihydroxy-dihydrothymine (t') damage in single-stranded phi X174 DNA has been measured . Following exposure to osmium tetroxide, which introduces t' damage in DNA under the conditions used, biological survival of the DNA infected into spheroplasts of Escherichia coli which had received prior exposure to ultraviolet light was higher than in unexposed spheroplasts . From our results, we conclude that approximately 63% of the biological damage from t' products, which is one of the classes of damage present in DNA following ionizing radiation, is susceptible to repair by the inducible SOS repair system. Gene, 1983 Mar, 21(3), 211 - 6 Direct transfer of coliphage lambda DNA from Escherichia coli to cellular slime mold Dictyostelium discoideum; Ishikawa A et al.; Dictyostelium discoideum myxamoebae were cultured with Escherichia coli cells infected with lambda phage in the presence of chloramphenicol . After eliminating the uningested bacteria by repeated centrifugation in a Percoll gradient, we examined the myxamoeba cytoplasm (not the food vacuole) for the presence of phage DNA . A significant amount of DNA extracted from the myxamoebae was hybridizable with purified phage lambda DNA, and capable of forming phage particles when packaged in vitro with phage lambda proteins . The EcoRI restriction maps of the phages recovered from the plaques were identical to that of the infecting phage . These results strongly suggest that phage DNA molecules were taken up by the cellular slime mold cells and that at least some fraction existed in intact form. Genetika, 1983 Mar, 19(3), 375 - 80 {Escherichia coli K-12 mutants with an increased efficiency of plasmid transformation}; Molchanova ES et al.; Escherichia coli K-12 mutants with an enhanced efficiency of plasmid transformation were obtained . In all the mutants, the efficiency of transfection with lambda vir phage DNA was changed, in comparison to the parent strain . However, these changes did not always correlate strictly with plasmid transformation alterations . For instance, two mutants with an increased plasmid transformation efficiency demonstrated 50-fold decrease in the level of transfection with lambda phage DNA . Polyacrylamide gel electrophoresis points to both quantitative and qualitative differences in protein composition of the mutant cell envelopes, as compared with the parent strain. Virology, 1983 Mar, 125(2), 257 - 64 Proteolytic processing of phage lambda tail protein gpH: timing of the cleavage; Tsui LC et al.; We describe a method for the rapid partial purification of intermediate structures of phage lambda tail assembly, using formaldehyde-fixed Escherichia coli cells to precipitate tail-related structures . The purification depends on the specific interaction between the E . coli lambda receptor protein and lambda tail protein gpJ . Protein compositions of tail assembly intermediates were analyzed to determine when in the assembly sequence the minor tail protein gpH is cleaved . gpH joins the tail precursor structure early in the pathway, during assembly of the initiator (a structure that becomes the tail tip) . However, gpH is not cleaved until after initiator assembly is complete and after the tail shaft has polymerized onto the initiator . These results suggest that each gpH molecule is extended along the length of the tail . Our results also appear to eliminate an ambiguity in the tail assembly pathway determined by earlier experiments: we argue that gene G acts between genes H and M. Proc Natl Acad Sci U S A, 1983 Mar, 80(5), 1411 - 5 Regulation of differentiated cell-specific functions; Savageau MA; The demand theory of gene regulation predicts that regulated cell-specific functions in high demand (i.e., high level of gene expression frequently required) are under the influence of a positive regulatory element whereas those in low demand (i.e., high level of gene expression not frequently required) are under the influence of a negative regulatory element . Furthermore, during differentiation, when the demand regimen for cell-specific functions changes, a switch in the regulatory mechanism itself is predicted . For the case in which a function is regulated in both demand regimens, the mode of regulation will switch from positive (high demand) to negative (low demand) or vice versa . These predictions are compared with published experimental evidence and found to be in good agreement. Proc Natl Acad Sci U S A, 1983 Mar, 80(5), 1194 - 8 Efficient isolation of genes by using antibody probes; Young RA et al.; A sensitive and general technique has been devised for the dual purposes of cloning genes by using antibodies as probes and isolating unknown proteins encoded by cloned DNA . The method uses an expression vector, lambda gt11 (lac5 nin5 cI857 S100), that permits insertion of foreign DNA into the beta-galactosidase structural gene lacZ and promotes synthesis of hybrid proteins . Efficient screening of antigen-producing clones in lambda gt11 recombinant cDNA libraries is achieved through lysogeny of the phage library in hflA (high-frequency lysogeny) mutant cells of Escherichia coli; lysogens produce detectable quantities of antigen on induction, even when plated at high cell densities . The vector is also designed to facilitate the isolation of proteins specified by previously cloned gene sequences . Hybrid proteins encoded by recombinant phage accumulate in strains defective in protein degradation (lon mutants) in amounts amenable to large-scale purification . Antibodies produced against the portion of the hybrid encoded by foreign DNA could in turn be used to isolate the native polypeptide from eukaryotic cells. Biochemistry, 1983 Mar 1, 22(5), 1123 - 8 Guanosine 5'-diphosphate 3'-diphosphate levels, carbon source, and ribonucleic acid synthesis in a mutant strain of Escherichia coli; Reynolds SH et al.; We have previously described a mutant strain of Escherichia coli (2S142) which shows a specific inhibition of stable RNA synthesis at 42 degrees C . The temperature-sensitive lesion mimics a carbon source downshift (diauxie lag) . We therefore measured RNA synthesis and levels of ppGpp (guanosine 5'-diphosphate 3'-diphosphate) on a number of different carbon sources . There is a 6-fold variation in ppGpp levels at 42 degrees C, depending on the carbon source present . Much of the variation in ppGpp levels at 42 degrees C can be explained by variations in the decay rate of ppGpp at 42 degrees C . The rates of ribosomal RNA and total RNA synthesis also vary with the carbon source at 42 degrees C . Linear regression analysis shows only a moderately good correlation (correlation coefficient = 0.62, P = 0.0001) between the ppGpp level at 42 degrees C and the rate of rRNA synthesis at 42 degrees C . In fact, ppGpp levels are a slightly better predictor of the rate of total RNA synthesis (correlation coefficient = 0.69, P = 0.0001) at 42 degrees C . Other variables such as rate of carbon source uptake appear to have very little, if any, relationship to the rate of rRNA synthesis on the different carbon sources . Segmented linear regression analysis indicates that ppGpp levels and rates of RNA synthesis correlate best when the carbon sources are divided into two groups: 6- and 12-carbon sugars and other carbon sources . The rate of rRNA synthesis in 2S142 at 42 degrees C appears to be relatively insensitive to ppGpp levels with 6- and 12-carbon sugars as the carbon source . These data raise the possibility that carbon source may affect rRNA synthesis in a manner that is at least partially unrelated to ppGpp levels. Biochemistry, 1983 Mar 1, 22(5), 1113 - 22 Isolation and characterization of two 5-fluorouracil-substituted Escherichia coli initiator methionine transfer ribonucleic acids; Hills DC et al.; Escherichia coli initiator methionine tRNA labeled in vivo with 5-fluorouracil (FUra) has been isolated and characterized . The tRNA, with essentially all its uracil and uracil-derived minor bases replaced by FUra, was purified by sequential chromatography, first on diethylaminoethylcellulose (DEAE-cellulose), at pH 8.9, followed by chromatography on Sepharose 4B, using a reverse salt gradient, then on DEAE-Sephadex A-50, and finally on benzoylated DEAE-cellulose . The last step resolved two FUra-substituted tRNAfMet-iso-accepting species, each with a specific activity over 1500 pmol/A260 . Kinetic analysis shows both are aminoacylated at the same rate; apparent KmS for the two are 0.92 and 0.94 microM, compared with 1.7 microM for normal tRNAfMet . Chromatographic differences between the two forms of fluorinated tRNAfMet persist after aminoacylation, and the two tRNAs are not interconverted by denaturation and renaturation . The isoacceptors have nearly identical nucleoside composition, and both contain 7-methylguanosine and 2'-O-methylcytidine as the only modified nucleosides . Analysis of complete RNase T1 digests of the two methionine tRNAs shows that they differ in only one oligonucleotide . The sequence 20FpApGp, derived from the dihydrouridine loop and stem region, which is found in one of the isoaccepting forms of the tRNA, is replaced by an oligonucleotide containing adenine and guanine, but no FUra in the other . A modified FUra, with the properties of a 5-fluoro-5,6-dihydrouracil derivative, is detected in this tRNA . 19F NMR spectra of the two species of FUra-substituted initiator tRNA show 9-10 resolved resonances for the 12 FUra residues incorporated . The spectra differ primarily in the shift of one peak in the form lacking the sequence 20FpApGp, from 4.8 ppm downfield from free FUra (= 0 ppm) to 14.9 ppm upfield from the standard. Arch Biochem Biophys, 1983 Mar, 221(2), 548 - 56 Carbon source transport, nucleotide levels, and stable RNA synthesis in a mutant strain of Escherichia coli; Reynolds SH et al.; We have previously described a temperature-sensitive mutant of Escherichia coli, 2S142 (rel-, met-, rns-, ilv-, ts-) which shows specific inhibition of stable RNA synthesis at 42 degrees C . This mutation mimics a carbon source downshift in that the decay of guanosine 5'-diphosphate, 3'-diphosphate (ppGpp) is inhibited at the restrictive temperature . In this paper we show that the temperature-sensitive lesion in 2S142 does affect the uptake of glucose or alpha-D-methylglucopyranoside (alpha DMG) at 42 degrees C . However, restoration of glucose or alpha DMG uptake by the insertion of a constitutive galactose permease gene or further restriction of glucose uptake by insertion of a ptsG mutation into 2S142 have no effect on rRNA synthesis at 42 degrees C (although ppGpp levels are lowered in both cases) . Furthermore, while restriction of uptake at 42 degrees C varies widely from carbon source to carbon source, severe restriction of rRNA synthesis is observed on all carbon sources tested at 42 degrees C . Levels of glycolytic intermediates, adenylate energy charge, ATP levels, and cAMP levels are all unaffected at the restrictive temperature . GTP levels decrease at 42 degrees C in glucose grown cells but that also does not appear to be related to the decrease in rRNA synthesis . These data were interpreted to suggest that the restriction of stable RNA synthesis in 2S142 at 42 degrees C can not be explained on the basis of decreased uptake and/or metabolism of carbon source . "Phantom spot" levels do decrease in 2S142 at 42 degrees C . In fact, "phantom spot" is the only putative regulatory molecule which correlates with restriction of rRNA synthesis on all carbon sources tested. J Virol, 1983 Mar, 45(3), 1190 - 4 Isolation and partial characterization of a monoclonal antibody to the Rous sarcoma virus transforming protein pp60src; Parsons SJ et al.; Transformation of cells by Rous sarcoma virus is mediated by the product of the viral src gene, pp60src . A hybridoma cell line producing an immunoglobulin G3 antibody to pp60src was isolated after lymph node cells from immune mice were fused with mouse myeloma cells (P3-NS1-1) . Mice were immunized with p60src purified from Escherichia coli cells expressing the src gene product . The monoclonal antibody immunoprecipitated pp60src from Rous sarcoma virus-transformed cells and recognized an antigenic determinant located in the amino-terminal third of the pp60src protein. J Infect Dis, 1983 Mar, 147(3), 450 - 9 Effect of heat-stable enterotoxin of Escherichia coli on cultured mammalian cells; Thomas DD et al.; The binding of biologically active 125I-labeled heat-stable enterotoxin (ST) of Escherichia coli with cultured mammalian cells was dose dependent and could be inhibited with low concentrations of unlabeled toxin or by neutralization with specific antiserum . There was positive cooperativity among cell binding sites . A single cultured cell bound approximately 4 X 10(4) molecules of ST; the dissociation constant was 1.33 X 10(-10) M . The specific binding of ST was partially inhibited by Pronase (Sigma Chemical Company, St . Louis, Missouri) and trypsin, but not by lipid- or carbohydrate-specific enzymes, simple sugars, or saccharides . Addition of ST to cultures of rat basophilic leukemia cells resulted in a dose-dependent secretion of histamine . Pharmacologic agents that inhibited calcium uptake or prostaglandin synthesis decreased the amount of histamine released . These data demonstrate the specific binding of ST by cultured cells and support the contention that calcium and prostaglandins may be important in the molecular mechanism(s) whereby ST activates guanylate cyclase. Eur J Immunol, 1983 Mar, 13(3), 214 - 20 Clonal anergy: inhibition of antigen-driven proliferation among single B lymphocytes from tolerant animals, and partial breakage of anergy by mitogens; Pike BL et al.; Mice were injected with fluoresceinated human gamma globulin (FLU-HGG) either at 2-3 days of age or as pregnant females . At 2 weeks of age, the spleen cells of the injected suckling mice or offspring were fractionated on FLU-gelatin dishes to yield FLU-binding B cells . These B cells were then cloned in microcultures using one of two recently described systems in which single B cells grow in the absence of feeder or filler cells, namely following stimulation with FLU-polymerized flagellin (FLU-POL) and conditioned media containing B cell growth and differentiation factor(s); or mitogenic activation by a mixture of E . coli lipopolysaccharide (LPS) and dextran sulfate (DxS) . As such cultures permit visualization of clonal proliferation as well as ultimate harvesting of cultures for assay of hemolytic plaque-forming cells, it was possible to ask whether the lesion in the tolerant state affected the B cell's capacity to divide, to differentiate to antibody secretion, or both . The results indicated that, when stimulated with antigen, the anergic cells could neither divide nor differentiate . However, when the strong mitogen mixture was used, clonal anergy was partially broken . The cells proliferated, and a small proportion of them differentiated into anti-FLU antibody-forming cells . A marked variation in antigen-binding avidity of the FLU-binding cells made it difficult to quantitate the degree of uncoupling of proliferation and differentiation among tolerant, LPS plus DxS-stimulated cells . Nevertheless, a partial reversibility of clonal anergy must affect views on mechanisms of self-tolerance. Eur J Biochem, 1983 Mar 1, 131(1), 113 - 8 RNA polymerase: interaction of RNA and rifampicin with the subassembly alpha 2 beta; Huaifeng M et al.; We studied the inhibition of tryptic digestion of the subassembly alpha 2 beta of Escherichia coli DNA-dependent RNA polymerase to investigate its interaction with RNA and rifampicin . Both agents decreased distinctly the cleavage of subunit beta in the subassembly as well as the degradation of the transiently formed polypeptides (Mr greater than 80000) . Short RNAs with a chain length of approximately 35 nucleotides were most protective at a concentration of 1 mg/ml while long RNAs were less effective at the same concentration . DNA did not exert any observable protective effects . The association of RNA with alpha 2 beta was shown by chromatography on phosphocellulose, which separates alpha 2 beta bound to RNA from free alpha 2 beta . The association of alpha 2 beta with RNA was inhibited by rifampicin. J Bacteriol, 1983 Mar, 153(3), 1221 - 7 Expression of the Escherichia coli malPQ operon remains unaffected after drastic alteration of its promoter; Debarbouille M et al.; The malPQ operon, one of the three operons of the maltose regulon, is positively controlled by the product of gene malT . The starting point for malPQ transcription was deduced from experiments which involved a hybridization of in vivo-synthesized malPQ mRNA with adequate DNA probes, followed either by a digestion of nonhybridized DNA (S1 nuclease mapping) or by an extension of the hybridized probe (reverse transcriptase mapping) . In the wild-type strain, this starting point was 37 nucleotides upstream from the initiation codon for malP . This analysis was also performed on a double mutant which contained both a 13-base pair deletion and a 3-base pair insertion in the promoter region . This double mutant expressed the malPQ operon exactly as the wild-type strain did, in a maltose-inducible manner . In this strain, the starting point for malPQ transcription was shifted 11 nucleotides downstream from the wild-type location . An analysis of these results suggests that (i) the binding site for the malT product is located upstream from the region which is severely altered in the double mutant, i.e., upstream from position -31; and (ii) the 30-base pair sequence which precedes the transcription starting point contains very few positions which are essential for promoter activity. Rev Esp Fisiol, 1983 Mar, 39(1), 45 - 50 Purification and partial characterization of a K99-antigen associated adhesin in Escherichia coli (637 strain); Ferreiros CM et al.; The K99-antigen associated adhesin in Escherichia coli (637 Strain) has been purified to homogeneity by using conventional chromatographic procedures . Sodium deoxycholate was used in the precipitation steps to avoid hydrophobic interactions between the fimbriae and other membrane-associated components . Homogeneity of the purified adhesin was assessed by electrophoresis, isoelectrofocusing, analytical gel filtration and immunoprecipitation against K99 specific antiserum, being homogeneous in all cases . The purified adhesin is composed of protein sub-units with a molecular weight of 18,900 +/- 950 daltons . No sugars were detected in the molecule . The molecular weight of the adhesin was higher than 2 X 10(6) daltons, and its isoelectric point was estimated to be about 9.45. Appl Environ Microbiol, 1983 Mar, 45(3), 1060 - 5 Chlorine-induced damage to surface adhesions during sublethal injury of enterotoxigenic Escherichia coli; Walsh SM et al.; A comparison of the adhesive ability of noninjured and chlorine-injured enterotoxigenic Escherichia coli was made by in vitro attachment to human peripheral leukocytes . Chlorination selected for noninjured cells with greater capabilities for colonizing the small intestine . Injured populations exhibited reduced association with leukocytes . Maximum reduction was seen in populations with greater than 80% injury . These cells demonstrated less adhesive ability than nonpiliated populations . Electron micrographs suggested that reduced adhesive ability was due to the loss of surface structures as a consequence of sublethal chlorination . The data imply a reduced ability among chlorine-injured pathogens to colonize the small intestine and initiate disease. Infect Immun, 1983 Mar, 39(3), 1334 - 45 Enhancement of mannose-mediated stimulation of human granulocytes by type 1 fimbriae aggregated with antibodies on Escherichia coli surfaces; Perry A et al.; In the present study, we assayed protein iodination in human granulocytes after interaction of the cells with mannose-specific (MS) type 1 fimbriated (MS+) and nonfimbriated (MS-) phenotypes of Escherichia coli pretreated with various amounts of anti-E . coli and antifimbrial antibodies . The MS+ phenotype stimulated protein iodination in granulocytes and possessed potent MS activity as measured by yeast aggregometry . In contrast, the MS- phenotype lacked all these activities . MS+ pretreated with moderate concentrations of antibodies, however, showed up to a 15-fold increase in granulocyte stimulation as compared to granulocyte stimulation induced by the non-antibody-treated MS+ phenotype or by the antibody-treated MS- phenotype . This marked antibody-mediated increase in stimulation of granulocytes was (i) dependent on the antibody concentrations, (ii) markedly reduced by methyl-alpha-D-mannoside, (iii) caused by immunoglobulin G as well as by F(ab')2, (iv) caused only by antifimbrial antibodies, (v) associated with cross-linked fimbriae seen as "bundles" under an electron microscope, and (vi) mimicked by treating MS+ bacteria with a certain range of glutaraldehyde . The data taken together support the hypothesis that, although cross-linking of fimbriae reduced the density of functional MS fimbriae over the surface of antibody-treated bacteria and consequently reduced the ability of these organisms to agglutinate yeast cells, the resulting bundles of MS fimbriae were far more effective at stimulating granulocytes because, bound together, they were better equipped to aggregate the mannose-containing receptors on the granulocyte surface. Infect Immun, 1983 Mar, 39(3), 1280 - 4 Adherence of an enterotoxigenic Escherichia coli strain, serotype O78:H11, to purified human intestinal brush borders; Cheney CP et al.; The human enterotoxigenic Escherichia coli pathogen designated H10407 expresses two different types of surface pili, one designated type 1 pili and the other designated colonization factor antigen I (CFA/I), CFA/I pili are thought to promote the adherence of H10407 to the mucosa of the human small bowel . H10407 was grown under conditions which promoted the expression of either type 1 pili or CFA/I pili, and in each case, the adherence of H10407 to purified human intestinal brush borders was quantitated . The adherence assays revealed that H10407 adhered to human brush borders only when it expressed CFA/I pili . It appears that in vitro adherence of H10407 to human intestinal epithelial cells is dependent on the expression of CFA/I. Biochem Biophys Res Commun, 1983 Feb 28, 111(1), 188 - 93 Higher plants contain L-asparate oxidase, the first enzyme of the Escherichia coli quinolinate synthetase system; Hosokawa Y et al.; Cotton callus cells contain an L-aspartate oxidase which does not appear to be active with D-aspartate, L-glutamate or D- or L-alanine . The enzyme requires for activity a dialyzable cofactor with an apparent molecular weight of 1,050 . Since L-aspartate oxidase is the first enzyme of the pathway for de novo synthesis of the pyridine ring in Escherichia coli, this finding suggests that higher plants may use the L-aspartate-dihydroxyacetone phosphate pathway for de novo pyridine nucleotide biosynthesis. Biochem Biophys Res Commun, 1983 Feb 28, 111(1), 143 - 9 Mutants of Escherichia coli H+-ATPase defective in the delta subunit of F1 and the b subunit of F0; Noumi T et al.; Complete nucleotide sequence of the genes for subunits of the H+ ATPase of E.coli has been determined and several hybrid plasmids carrying various portions of these genes have been constructed . Genetic complementation and recombination tests of about forty mutants of E.coli defective in the ATPase were performed using these plasmids for identifying the locations of the mutations . Two mutants defective in the delta subunit and a novel type of mutant defective in the b subunit of F0 were identified . The delta subunit mutants showed no proton conduction, suggesting that this subunit has an important role for the proton conduction . The ATPase of the b subunit mutant has a normal activity of proton channel portion, which phenotype is clearly different from that of mutants of the b subunit reported previously. J Mol Biol, 1983 Feb 25, 164(2), 193 - 211 Amplification of ssb-1 mutant single-stranded DNA-binding protein in Escherichia coli; Chase JW et al.; The ssb-1 gene encoding a mutant Escherichia coli single-stranded DNA-binding protein has been cloned into plasmid pACYC184 . The amount of overproduction of the cloned ssb-1 gene is dependent upon its orientation in the plasmid . In the less efficient orientation, 25-fold more mutant protein is produced than in strains carrying only one (chromosomal) copy of the gene; the other orientation results in more than 60-fold overproduction of this protein . Analysis of the effects of overproduction of the ssb-1 encoded protein has shown that most of the deficiencies associated with the ssb-1 mutation when present in single gene copy, including temperature-sensitive conditional lethality and deficiencies in amplified synthesis of RecA protein and ultraviolet light-promoted induction of prophage lambda +, are reversed by increased production of ssb-1 mutant protein . These results provide evidence in vivo that SSB protein plays an active role in recA-dependent processes . Homogenotization of a nearby genetic locus (uvrA) was identified in the cloning of the ssb-1 mutant gene . This observation has implications in the analysis of uvrA- mutant strains and will provide a means of transferring ssb- mutations from plasmids to the chromosome . On a broader scale, the observation may provide the basis of a general strategy to transfer mutations between plasmids and chromosomes. J Biol Chem, 1983 Feb 25, 258(4), 2586 - 92 ADP-mediated dissociation of stable complexes of recA protein and single-stranded DNA; Cox MM et al.; The complete exchange of strands between circular single-stranded and full length linear duplex DNAs promoted by the recA protein of Escherichia coli is dependent upon the hydrolysis of ATP and is strongly stimulated by the single-stranded DNA binding protein (SSB) . In the presence of SSB, stable complexes of recA protein and single-stranded DNA are formed as an early step in the reaction . These complexes dissociate when the ADP/ATP ratio approaches a value of 0.6-1.5, depending upon reaction conditions . Thus, ATP hydrolysis never proceeds to completion but stops when 40-60% of the input ATP has undergone hydrolysis . recA protein can participate in a second round of strand exchange upon regeneration of the ATP . While 100-200 mol of ATP are hydrolyzed/mol of heteroduplex base pair formed under standard reaction conditions in the presence of SSB, this value is reduced to 16 at levels of ADP lower than that required to dissociate the complexes . ATP hydrolysis appears to be completely irreversible since efforts to detect exchange reactions using 18O probes have been unsuccessful. J Biol Chem, 1983 Feb 25, 258(4), 2577 - 85 On the role of single-stranded DNA binding protein in recA protein-promoted DNA strand exchange; Cox MM et al.; RecA protein-promoted DNA strand exchange is greatly stimulated by the single-stranded DNA binding protein (SSB) of Escherichia coli . Stimulation is not a consequence of the binding of SSB to excess single-stranded DNA . It results instead from stabilization of recA protein-single-stranded DNA complexes formed in the presence of ATP and SSB . In the presence of SSB, recA protein does not measurably dissociate from these complexes for up to 90 min . However, in its absence, recA protein moves rapidly between two populations of single-stranded DNA, and complete equilibration occurs with t 1/2 of 17 s . Rapid transfer of recA protein to single-stranded DNA occurs during all stages of DNA strand exchange and does not require ATP . The transfer involves an equilibrium between free and bound recA protein rather than a direct redistribution between single-stranded DNA molecules . Thus, SSB prevents dissociation of recA protein from single-stranded DNA, rendering the binding of the recA protein to single-stranded DNA irreversible . Under these conditions, the pairing phase of the strand exchange reaction is accelerated to the point that it is no longer rate-limiting . These results can explain the relative inefficiency of DNA strand exchange in the absence of SSB. J Mol Biol, 1983 Feb 25, 164(2), 313 - 27 X-ray diffraction analysis of matrix porin, an integral membrane protein from Escherichia coli outer membranes; Garavito RM et al.; An integral membrane protein forming channels across Escherichia coli outer membranes, porin, has been crystallized using a polyethylene glycol or salt-generated two-phase system . Monodispersity and homogeneity of protein-detergent complexes were found to be prerequisites for reproducible formation of crystals amenable to X-ray structural analysis . By varying pH, detergent and buffer type, large crystals of three different habits can be obtained, two of which are discussed in this paper . The tetragonal form (space group P4(2); unit cell dimensions, a = b = 155 A, c = 172 A) is suitable for X-ray analysis . Low temperature induces a change of the space group to P4(2)22, with a single trimer in the asymmetric unit . This crystal form diffracts to a resolution beyond 2.9 A . The hexagonal crystal form (space group P6(3)22; unit cell dimensions, a = b = 93 A, c = 220 A) is limited in resolution to 4.5 A, but reveals a packing arrangement very similar to that in two-dimensional membrane-like crystalline arrays. J Mol Biol, 1983 Feb 25, 164(2), 175 - 92 Proteins required for ultraviolet light and chemical mutagenesis . Identification of the products of the umuC locus of Escherichia coli; Elledge SJ et al.; umuC is the genetic locus showing the greatest specificity for the "error-prone repair" process that is responsible for u.v . and chemical mutagenesis in Escherichia coli . By generating a probe specific to umuC DNA, we have been able to clone the umuC locus . Through a combination of subcloning and Tn1000 mutagenesis, we have identified a region of 2.2 X 10(3) bases which contains the information necessary to complement umuC mutations . This region of DNA codes for two polypeptides with molecular weights of 16,000 and 45,000 . The genes for these proteins are organized in an operon that is repressed by the lexA protein . Complementation of previously isolated umuC mutations revealed that these proteins correspond to two complementation groups, umuC, which codes for the 45,000 Mr protein, and umuD, which codes for the 16,000 Mr protein, and that therefore both proteins are essential for "error-prone repair" in E . coli. Nucleic Acids Res, 1983 Feb 25, 11(4), 987 - 97 The dnaC protein of Escherichia coli . Purification, physical properties and interaction with dnaB protein; Lanka E et al.; E.coli dnaC protein was purified to near-homogeneity in using a dnaC complementation assay {S.Wickner, I.Berkower, M.Wright, and J.Hurwitz (1973) Proc . Natl . Acad . Sci . USA 70, 2369-2373} . Purification was achieved by taking advantage of the hydrophobic interaction of dnaC protein with aliphatic and aromatic matrixes and with Brij58 as stabilizing agent . A sedimentation coefficient for the dnaC protein of 2.6 S corresponding to a molecular weight of approximately 26,000 was estimated from glycerol gradient centrifugation . A polypeptide molecular weight of 28,000 was determined by densitometry on a denaturing gel . In the presence of ATP the dnaC protein forms a complex with dnaB protein {S.Wickner and J.Hurwitz (1975) Proc.Natl.Acad.Sci . USA 72, 921-925} . For the dnaB . dnaC complex a sedimentation coefficient of 14.5 S was measured by glycerol gradient centrifugation, indicating a molecular weight of about 400,000 . The ratio of the dnaC and dnaB polypeptides in the complex is approximately 1, as determined on a denaturing gel . It is suggested that the complex consists of the dnaB protein hexamer and six dnaC polypeptides amounting to a calculated molecular weight of about 450,000. Nucleic Acids Res, 1983 Feb 25, 11(4), 1117 - 22 Isolation and nucleotide sequence of a plant tRNA gene: petunia asparagine tRNA; Bawnik N et al.; A 14.3 kb petunia genomic DNA fragment was isolated and found to contain a single tRNA gene coding for asparagine tRNA . The nucleotide sequence of the asparagine tRNA gene and its flanking regions has been determined . This gene does not contain intervening sequences nor the 3'-end CCA sequence of the mature tRNA and presents a similar overall sequence homology (70%) to both E . coli and mammalian asparagine tRNA . As in other eukaryotic tRNA genes the 5'-flanking region does not seem to contain any special sequence that could function as a regulatory element and the 3'-end is followed by a short cluster of T that may function as the transcription termination site. Nucleic Acids Res, 1983 Feb 25, 11(4), 1099 - 116 The coupled use of 'footprinting' and exonuclease III methodology for RNA polymerase binding and initiation . Application for the analysis of three tandem promoters at the control region of colicin El; Chan PT et al.; In order to determine the initiation site for three promoters P1, P2 and P3 (5' to 3') in close proximity in the colicin E1 control region we developed a new methodology that couples ternary complex formation and the analysis of the 3' border protected from exonuclease III digestion . The initiation of transcription could be detected by measuring the shift in the position of the 3' protected border when RNA polymerase moved from its binary complex position to its ternary complex position . The latter stops at a specific nucleotide because transcription is initiated with one or more NTPs missing . This approach, coupled with "footprinting", can also be used to decide whether the formation of an RNA polymerase binary or ternary complex at one site excludes or weakens binding at neighboring sites . The location of 3' protected borders reveals the formation of respective binary and ternary complexes at non-saturating RNA polymerase conditions, whereas at saturating conditions only the distal 3' boundary is seen and exonuclease cannot penetrate further . However, if "footprinting" reveals proximal 5' patterns this establishes that simultaneous binding has occurred on the same DNA fragment . The data showed that this was true for P1 and P3 which are only 8 nucleotides apart . P2 could only be detected at non-saturating conditions since it overlaps both P1 and P3 . The evidence from the literature and this study establishes P1 as the true colicin E1 promoter with the possibility that supercoiling may eliminate any role for P2 and P3. J Biol Chem, 1983 Feb 25, 258(4), 2720 - 8 Mechanism of the rifampicin induction of RNA polymerase beta and beta' subunit synthesis in Escherichia coli; Fukuda R et al.; The mechanism of the rifampicin induction of RNA polymerase beta and beta' subunit synthesis was investigated, employing an in vitro coupled system of transcription and translation as well as an in vitro transcription system with purified RNA polymerase holoenzyme . Two independent effects leading to the induction of beta and beta' polypeptide synthesis have been identified for the drug: 1) rifampicin binds to RNA polymerase holoenzyme and inhibits its autorepressor activity, thus relieving the autorepression exerted possibly on the translation of rpoBC mRNA; and 2) rifampicin-RNA polymerase complex functions as a positive effector stimulating the transcription of rpoBC genes, but not of rplL gene . Using a terminally labeled DNA fragment covering the intercistronic region between rplL and rpoB as a probe, the structure of both in vivo and in vitro RNA were analyzed by S1 nuclease-mapping assays . The experimental results indicated that the observed stimulation of the transcription by rifampicin was attributed, at least in part, to relaxation of the transcription attenuation immediately preceding the rpoB gene, resulting in the increase of the read-through transcript to rpoBC genes . Besides the read-through transcript and the processed mature rpoBC mRNA, another RNA species was found in in vivo RNA, which might be transcribed from a weak promoter located in the intercistronic region . However, no clear indication has been obtained of possible enhancement of the transcription initiation by rifampicin from the putative promoter. J Biol Chem, 1983 Feb 25, 258(4), 2394 - 8 The biosynthesis of membrane-derived oligosaccharides . A membrane-bound phosphoglycerol transferase; Jackson BJ et al.; Membrane-derived oligosaccharides, found in the Escherichia coli periplasmic space (Schulman, H., and Kennedy, E . P . (1979) J . Bacteriol . 137, 686-688), are composed of 8-10 units of glucose, the sole sugar, in beta 1 leads to 2 and beta 1 leads to 6 linkages (Schneider, J . E., Reinhold, V., Rumley, M . K., and Kennedy, E . P . (1979) J . Biol . Chem . 254, 10135-10138) . Oligosaccharides in this family are variously substituted with succinyl ester residues, as well as with sn-1-phosphoglycerol and phosphoethanolamine, both derived from membrane phospholipids . These negatively charged oligosaccharides may function in cellular osmoregulation since their synthesis is under osmotic control (Kennedy, E . P . (1982) Proc . Natl . Acad . Sci . U.S.A . 79, 1092-1095) . We now report initial characterization of an enzyme catalyzing transfer of phosphoglycerol residues from phosphatidylglycerol to membrane-derived oligosaccharides or to synthetic beta-glucoside acceptors . The products are sn-1,2-diglyceride and beta-glucoside-6-phosphoglycerol . Localized in the inner membrane, the transferase has a requirement for divalent cations, of which manganese is most effective, and a pH optimum of 8.9 in vitro. J Biol Chem, 1983 Feb 25, 258(4), 2410 - 4 Genetic mapping of antigenic determinants on a membrane protein; Gabay J et al.; The antigenic determinants recognized by two monoclonal antibodies were mapped on LamB, an outer membrane protein of Escherichia coli . The procedure consisted of performing immunoprecipitation experiments with extracts of strains which produced truncated fragments of LamB, either in a free form (deletion and nonsense mutants) or fused to another polypeptide (malK-lamB and lamB-lacZ fusion strains) . The conclusion is that the two antigenic determinants are located within 70 residues from the COOH-terminal end of LamB, which contains a total of 421 amino acids . Since these two antigenic sites were previously demonstrated to be exposed at the cell surface, it follows that a COOH-terminal portion of LamB must be located on the outer surface of the outer membrane. J Biol Chem, 1983 Feb 25, 258(4), 2118 - 21 Increase in S-adenosylhomocysteine concentration in interferon-treated HeLa cells and inhibition of methylation of vesicular stomatitis virus mRNA; de Ferra F et al.; A fraction of the viral mRNA synthesized in interferon-treated HeLa cells infected with vesicular stomatitis virus (VSV) lacks the 7-methyl group in the 5'-terminal guanosine of the cap; this mRNA is not associated with polyribosomes and does not bind to ribosomes in an assay for initiation of protein synthesis (de Ferra, F., and Baglioni, C . (1981) Virology 112, 426-435) . To establish whether this defect in methylation is due to changes in the level of the methyl donor S-adenosylmethionine (AdoMet) and of its competitive inhibitor S-adenosylhomocysteine (AdoHcy), we measured the concentration of these compounds in HeLa cells treated with interferon . An increase in both AdoMet and AdoHcy was detected 3 to 6 h after addition of interferon . The level of these compounds increased gradually and in proportion to the interferon concentration used . With 125 reference units/ml of beta interferon, for example, the AdoHcy concentration increased more than 3-fold and that of AdoMet about 1.5-fold with a consequent change in the AdoHcy/AdoMet ratio . An increased AdoHcy/AdoMet ratio was also found in HeLa cells treated with pure alpha 2 interferon produced in Escherichia coli by recombinant DNA techniques . When the methylation of VSV mRNA was measured in assays carried out with permeabilized virions at the AdoHcy and AdoMet concentrations found in interferon-treated cells, a preferential inhibition of the viral (guanine-7-)methyltransferase activity was observed . Such an inhibition may account for the synthesis of VSV mRNA lacking the 7-methyl group of guanosine in the cap. J Biol Chem, 1983 Feb 25, 258(4), 2246 - 53 Cascade control of Escherichia coli glutamine synthetase . Purification and properties of PII uridylyltransferase and uridylyl-removing enzyme; Garcia E et al.; Uridylyltransferase, a component of the covalent modification cascade system that controls glutamine synthetase activity in Escherichia coli, has been purified to apparent homogeneity . The purification was facilitated by the use of an E . coli strain which carries multiple copies of a ColE1-hybrid plasmid containing the glnD gene that encodes uridylyltransferase and which overproduces its synthesis by 25-fold . Gel electrophoresis and high pressure liquid chromatography studies show that the native enzyme is a single polypeptide chain of Mr = 95,000 +/- 5,000 . The purified enzyme catalyzes the uridylylation as well as the deuridylylation of the regulatory protein PII, demonstrating that a single bifunctional enzyme is involved in the covalent interconversion of PII . Gel filtration studies indicate that the enzyme undergoes slow irreversible aggregation during most steps of purification with a concomitant loss of activity. Biochim Biophys Acta, 1983 Feb 22, 755(3), 467 - 73 Accumulation of guanosine tetraphosphate induced by polymixin and gramicidin in Escherichia coli; Cortay JC et al.; The effects of two polypeptide antibiotics, polymixin B and gramicidin S, on the intracellular pool size and turnover of guanosine tetraphosphate (ppGpp) were analyzed in stringent (relA+) and relaxed (relA) strains of Escherichia coli . When either one of these two drugs was added to stringent bacteria cultures at a final concentration that blocked protein and RNA synthesis, ppGpp was found to accumulate . Under similar conditions of inhibition of macromolecular synthesis, ppGpp also appeared to accumulate in relaxed bacteria . Moreover, in either type of strain, no significant accumulation of guanosine pentaphosphate (pppGpp) could be detected upon drug treatment . It was, therefore, concluded that polymixin and gramicidin elicit ppGpp accumulation through a mechanism independent of the relA gene product and, consequently, quite distinct from the stringent control system triggered by amino acid starvation . Further experiments performed by using tetracycline as an inhibitor of ppGpp synthesis, showed that the increase in the level of this nucleotide induced by drug action was due, in fact, to a strong restriction of its degradation rate. Biochim Biophys Acta, 1983 Feb 22, 755(3), 326 - 31 Effect of polyamines on synthesis and degradation of guanosine 5'-diphosphate 3'-diphosphate; Igarashi K et al.; The effect of polyamines on the in vitro and in vivo synthesis and degradation on guanosine 5'-diphosphate 3'-diphosphate (ppGpp) has been studied in Escherichia coli . The presence of 2 mM spermidine lowered the optimal Mg2+ concentration for ppGpp formation from 17 mM to 11 mM . The formation of ppGpp in the presence of 2 mM spermidine and 11 mM Mg2+ was about 15% greater than that in the presence of 17 mM Mg2+ . At a concentration of less than 11 mM Mg2+, spermidine was found to stimulate ppGpp formation greatly . Putrescine did not cause any effect . When a polyamine-requiring mutant of E . coli (EWH319) was starved for an amino acid by the addition of valine, spermidine stimulated ppGpp formation . The degradation of ppGpp was not influenced significantly by polyamines. FEBS Lett, 1983 Feb 21, 152(2), 157 - 62 Interaction of polymerases with 2'-deoxyuridine-5'-triphosphate spin-labeled at the 5-position; Warwick-Koochaki PE et al.; 2'-Deoxyuridine-5'-triphosphate spin-labeled at the 5-position with N-{1-oxyl-2,2,6,6-tetramethyl-4-piperidinyl}-O- was found to be an inhibitor of some DNA and RNA polymerases including avian myeloblastosis virus reverse transcriptase . Furthermore, the spin-labeled nucleotide was found to be incorporated internally into polydeoxythymidylic acid via reverse transcriptase to an extent of 1.0 spin-labeled base per 10(3) bases . The incorporation, monitored by electron spin resonance, is analogous to some other nucleotide inhibitors of polymerases, and the results indicate that it may be feasible to obtain sequence specific, spin-labeled DNA, enzymatically. Nature, 1983 Feb 17-23, 301(5901), 623 - 6 Structure of the serine chemoreceptor in Escherichia coli; Boyd A et al.; Many biological processes depend on the function of proteins that detect changes in a cell's environment and transmit the information to the cytoplasm, for example, peptide hormone receptors . In Escherichia coli this class of proteins is exemplified by the sensory transducers (also called signalling proteins or methyl-accepting chemotaxis proteins) which have a central role in mediating chemotactic behaviour . The sensory transducers are the products of four genes: tsr, tar, tap and trg . Each transducer detects changes in the environmental concentration of one or a very few attractants: Tsr, serine; Tar, aspartate and maltose; Tap, unknown; and Trg, ribose and galactose . Tsr and Tar act directly as chemoreceptors for the amino acid attractants and signal changes in their degree of occupancy to the flagellar apparatus . Detection of these changes in occupancy is made possible as the transducers are methylated at multiple glutamate residues such that their level of methylation reflects the most recent chemoeffector concentration . Biochemical and genetic information concerning the serine transducer protein has been accumulating rapidly but little is known about the structure of the molecule . We present here the nucleotide sequence of the tsr gene of E . coli; the amino acid sequence derived from it suggests that the Tsr transducer protein has a relatively simple transmembrane structure that may place limits on the mechanisms available for the transmission of sensory information into the cell. Biochemistry, 1983 Feb 15, 22(4), 851 - 4 Involvement of histidine and tryptophan residues of glutamine binding protein in the interaction with membrane-bound components of the glutamine transport system of Escherichia coli; Hunt AG et al.; We treated the glutamine binding protein with diethyl pyrocarbonate (DEPC) and N-bromosuccinimide (NBS) to modify respectively the sole histidine and tryptophan residues and examined the effect of these modifications on the ability of the binding protein to bind glutamine as well as the ability to restore glutamine transport in membrane vesicles of Escherichia coli . Under the conditions used, both DEPC and NBS markedly inhibited the ability to restore glutamine transport in vesicles without any significant effect on glutamine binding . Moreover, saturating quantities of glutamine had no protective effect on the inactivation of the binding protein by DEPC or NBS . Fluorometric measurement and amino acid analysis indicate that the inactivation of the binding protein in restoring vesicle transport by NBS can be attributed to the oxidation of a single tryptophan residue . Similar analysis and the inability of hydroxylamine to reverse the effect of DEPC indicate that the effects of DEPC can probably be attributed to alterations of the sole histidine and/or one or more lysine residues of the binding protein . We conclude that the glutamine binding protein possesses at least two largely nonoverlapping functional domains, one responsible for glutamine binding and the other for the interaction with the other components of the glutamine transport system. Experientia, 1983 Feb 15, 39(2), 166 - 7 Stimulation of intestinal chromatin template activity by dietary carbohydrates in adult rats; Raul F et al.; Oral administration of a 70% solution of sucrose to starved adult rats resulted 1 h after feeding in a 3.5-fold stimulation of intestinal chromatin template activity assayed in vitro using E . coli RNA polymerase . A similar stimulatory effect was observed with fructose, whereas glucose exhibited a weaker effect, indicating that the nature of the ingested carbohydrate may have a direct effect on the extent of intestinal chromatin template activation. Eur J Biochem, 1983 Feb 15, 130(3), 427 - 35 Hyperproduction of phosphate-binding protein, phoS, and pre-phoS proteins in Escherichia coli carrying a cloned phoS gene; Morita T et al.; A large amount of phosphate-binding protein, the phoS gene product, accumulated in the periplasmic space of the cells when an Escherichia coli strain carrying a multicopy plasmid containing a chromosomal fragment of the phoS-phoT region (pSN507) was grown in a low-phosphate medium . When the same strain carrying a plasmid containing only the phoS gene (pSN518 or pSN5182) was grown in low-phosphate medium, phosphate-binding protein accumulated in the periplasm, and in addition a larger protein accumulated in the non-periplasmic fraction . The apparent Mr of this protein and the phosphate-binding protein were 39000 and 35000 respectively, as judged by sodium dodecyl sulfate/polyacrylamide gel electrophoresis . This larger protein showed immunological cross-reaction with the phosphate-binding protein . The 39000-Mr protein was also detected in cells carrying pSN507 when the proteins were pulse-labeled with radioactive amino acids . From these findings, together with the fact that this protein is recovered from the membrane fraction, we conclude that this protein is an unsecreted precursor protein of the phosphate-binding protein . Kinetics and regulation of accumulation of these proteins were studied . This system will be useful for preparation and purification of the precursor protein for biochemical studies in relation to the mechanism of protein secretion. Biochem J, 1983 Feb 15, 210(2), 395 - 403 Oxidative phosphorylation in Escherichia coli . Characterization of mutant strains in which F1-ATPase contains abnormal beta-subunits; Senior AE et al.; To facilitate study of the role of the beta-subunit in the membrane-bound proton-translocating ATPase of Escherichia coli, we identified mutant strains from which an F1-ATPase containing abnormal beta-subunits can be purified . Seventeen strains of E . coli, characterized by genetic complementation tests as carrying mutations in the uncD gene (which codes for the beta-subunit), were studied . The majority of these strains (11) were judged to be not useful, as their membranes lacked ATPase activity, and were either proton-permeable as prepared or remained proton-impermeable after washing with buffer of low ionic strength . A further two strains were of a type not hitherto reported, in that their membranes had ATPase activity, were proton-impermeable as prepared, and were not rendered proton-permeable by washing in buffer of low ionic strength . Presumably in these two strains F1-ATPase is not released in soluble form by this procedure . F1-ATPase of normal molecular size were purified from strains AN1340 (uncD478), AN937 (uncD430), AN938 (uncD431) and AN1543 (uncD484) . F1-ATPase from strain AN1340 (uncD478) had 15% of normal specific Mg-dependent ATPase activity and 22% of normal ATP-synthesis activity . The F1-ATPase preparations from strains AN937, AN938 and AN1543 had respectively 1.7%, 1.8% and 0.2% of normal specific Mg-dependent ATPase activity, and each of these preparations had very low ATP-synthesis activity . The yield of F1-ATPase from the four strains described was almost twice that obtained from a normal haploid strain . The kinetics of Ca-dependent ATPase activity were unusual in each of the four F1-ATPase preparations . It is likely that these four mutant uncD F1-ATPase preparations will prove valuable for further experimental study of the F1-ATPase catalytic mechanism. J Mol Biol, 1983 Feb 15, 164(1), 73 - 87 Context effects: translation of UAG codon by suppressor tRNA is affected by the sequence following UAG in the message; Bossi L; The efficiency of various suppressor tRNAs in reading the UAG amber codon has been measured at 42 sites in the lacI gene . Results indicate that: (1) for all suppressors, efficiency is not an a priori value; rather, it is determined at each site by the specific reading context of the suppressed codon; (2) the degree of sensitivity to context effects differs among suppressors . Most affected is amber suppressor supE (su2), whose activity varies over a 20-fold range depending on context; (3) context effects are produced by resid