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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 residues present at the 3' side of the UAG codon . The most important role appears to be played by the base that is immediately adjacent to the codon . When this base is a purine, the amber codon is suppressed more efficiently than when a pyrimidine is in the same position . Superimposed on this initial pattern, the influence of bases further downstream to the UAG triplet can be detected also . The possibility is discussed that context effects are produced by the whole codon following UAG in the message.

J Mol Biol, 1983 Feb 15, 164(1), 59 - 71
Effects of surrounding sequence on the suppression of nonsense codons; Miller JH et al.; Using a lacI-Z fusion system, we have determined the efficiency of suppression of nonsense codons in the I gene of Escherichia coli by assaying beta-galactosidase activity . We examined the efficiency of four amber suppressors acting on 42 different amber (UAG) codons at known positions in the I gene, and the efficiency of a UAG suppressor at 14 different UGA codons . The largest effects were found with the amber suppressor supE (Su2), which displayed efficiencies that varied over a 35-fold range, and with the UGA suppressor, which displayed a 170-fold variation in efficiency . Certain UGA sites were so poorly suppressed (less than 0.2%) by the UGA suppressor that they were not originally detected as nonsense mutations . Suppression efficiency can be correlated with the sequence on the 3' side of the codon being suppressed, and in many cases with the first base on the 3' side . In general, codons followed by A or G are well suppressed, and codons followed by U or C are poorly suppressed . There are exceptions, however, since codons followed by CUG or CUC are well suppressed . Models explaining the effect of the surrounding sequence on suppression efficiency are considered in the Discussion and in the accompanying paper.

Nucleic Acids Res, 1983 Feb 11, 11(3), 719 - 26
Molecular cloning and nucleotide sequencing of human immunoglobulin epsilon chain cDNA; Seno M et al.; DNA complementary to mRNA of human immunoglobulin E heavy chain (epsilon chain) isolated and purified from U266 cells has been synthesized and inserted into the PstI site of pBR322 by G-C tailing . This recombinant plasmid was used to transform E . coli chi 1776 to screen 1445 tetracycline resistant colonies . Nine clones (pGETI - 9) containing cDNA coding for the human epsilon chain were recognized by colony hybridization and Southern blotting analysis with a nick-translated human IgE genome fragment . The nucleotide sequence of the longest cDNA contained in pGET2 was determined . The results indicate that the sequence of 1657 nucleotides codes for 494 amino acids covering a part of the variable region and all of the constant region of the human epsilon chain . Most of the amino acid sequence deduced from the nucleotide sequence is in substantial agreement with that reported . Furthermore a termination codon after the -COOH terminal amino acid marks the beginning of a 3' untranslated region of 125 nucleotides with a poly A tail . Taking this into account, the structure of the human epsilon chain mRNA, except a part of the 5' end, is conserved fairly well in the cDNA insert in pGET2.

Nucleic Acids Res, 1983 Feb 11, 11(3), 671 - 86
Determination of the promoter strength in the mixed transcription system: promoters of lactose, tryptophan and ribosomal protein L10 operons from Escherichia coli; Kajitani M et al.; In vitro transcription was performed in a single reaction mixture, which contained three species of truncated E . coli DNA template, each carrying one specific promoter, lacP (UV5), trpP or rplJp, and the transcripts of distinct sizes were analyzed by electrophoresis on the same gel . Using this "mixed transcription" system, the order of the promoter strength, i.e., the capacity to form stable open complex, was determined in the single-round transcription under the standard conditions (50 mM NaCl and 37 degrees C) to be lacP greater than trpP greater than rplJp, the latter two promoters being 30--40% and 5--10% the strength of lacP, respectively . After the multiple-round transcription, however, the level of trp transcription was the lowest due to low cyclic-reaction rate but became the highest when another trp fragment containing the natural terminator was used as the template . The order of the transcription level also varied depending on the ionic strength and the reaction temperature and, as a result, lacP was no more the strongest under high salt concentration and at high temperature.

Science, 1983 Feb 11, 219(4585), 614 - 20
Rabies virus glycoprotein analogs: biosynthesis in Escherichia coli; Yelverton E et al.; The surface of rabies virus is composed of an approximately 60,000 dalton glycoprotein, in which most of the antigenic and immunogenic determinants of the virus reside . We have constructed plasmids for the direct expression in Escherichia coli of the mature full length rabies glycoprotein gene and also for the expression of a glycoprotein gene which has been truncated to exclude the coding region for a hydrophobic, possibly transmembrane, domain of the protein . Escherichia coli harboring the plasmids synthesize analog proteins which conform by several biochemical and antigenic criteria to rabies glycoprotein.

Nucleic Acids Res, 1983 Feb 11, 11(3), 687 - 706
Constitutive, long-term production of human interferons by hamster cells containing multiple copies of a cloned interferon gene; Haynes J et al.; Hybrid plasmids containing the mouse dihydrofolate reductase (dhfr) and a human interferon (either IFN-alpha 5 or IFN-gamma) coding sequence under the control of viral promoters were transfected into dhfr- Chinese hamster ovary (CHO) cells . dhfr+ colonies produced IFN at 10-1000 units X ml-1 X day-1 . Clones selected in methotrexate had a 20-50-fold increase in the IFN-alpha 5 and dhfr DNA and mRNA content and secreted IFN at 20,000-100,000 units X ml-1 X day-1 . SDS-polyacrylamide gel electrophoresis of partially purified 35S-HuIFN-gamma from CHO cells showed a multiple of labeled bands with a mobility corresponding to 22,400 to 23,400 daltons which was absent in the supernatants of non-transformed CHO cells . The higher apparent molecular weight of human IFN-gamma from CHO cells as compared to that of human IFN-gamma from E . coli (about 18,800) suggests that the former was glycosylated.

Nucleic Acids Res, 1983 Feb 11, 11(3), 659 - 69
A versatile method for the coupling of protein to DNA: synthesis of alpha 2-macroglobulin-DNA conjugates; Cheng S et al.; We describe a simple, general method to link proteins covalently to DNA . The method uses two reagents, N-acetyl-N'-(p-glyoxylylbenzoyl)cystamine and 2-iminothiolane . The former reacts specifically with nonpaired quanine residues and upon reduction generates a free sulfhydryl group . The latter reacts with a protein to provide another sulfhydryl group which is subsequently conjugated to DNA by an intermolecular disulfide interchange reaction . Using this method alpha 2-macroglobulin was conjugated to plasmid DNA encoding the Herpes simplex virus-1 thymidine kinase gene or a DNA fragment containing the E . coli chloramphenicol acetyltransferase gene . Up to 20% of the total DNA was conjugated to alpha 2-macroglobulin and the alpha 2-macroglobulin-DNA conjugate had a protein/DNA molar ratio of approximately two . The whole reaction takes place under very mild conditions in aqueous solution . The structure of DNA appears not to be significantly affected by the chemical modification . This method may prove useful in ligand directed gene transfer studies.

Nucleic Acids Res, 1983 Feb 11, 11(3), 555 - 73
Structure and expression of cloned murine IFN-alpha genes; Shaw GD et al.; The mouse has an interferon-alpha (MuIFN-alpha) gene family containing at least four, and likely more than ten members . A segment of mouse chromosomal DNA and cDNAs encoding murine alpha IFNs have been cloned, and the sequence of two MuIFN-alpha DNAs determined . No intron was found in the chromosomal gene . The two coding sequences produced biologically active IFN when expressed in monkey cells under the control of an SV40 promoter, and in E.coli under the control of the ampicillinase promoter . MuIFN-alpha 1 had no detectable activity on human cells, while MuIFN-alpha 2 was 20% as active on human as on mouse cells.

J Biol Chem, 1983 Feb 10, 258(3), 1985 - 90
The rfaD gene codes for ADP-L-glycero-D-mannoheptose-6-epimerase . An enzyme required for lipopolysaccharide core biosynthesis; Coleman WG Jr; The rfaD gene product, ADP-L-glycero-D-mannoheptose-6-epimerase, is necessary for the conversion of ADP-D-glycero-D-mannoheptose to ADP-L-glycero-D-mannoheptose . The nucleotide ADP-D-glycero-D-mannoheptose accumulates in rfaD mutant strains . Two chimeric colicin E1 plasmids carrying the coding sequence of the rfaD+ locus have been selected and shown to complement the rfaD phenotype . These clones also result in an amplification of ADP-L-glycero-D-mannoheptose-6-epimerase activity.

J Biol Chem, 1983 Feb 10, 258(3), 1954 - 9
Initiation factor and ribosome levels are coordinately controlled in Escherichia coli growing at different rates; Howe JG et al.; The levels of initiation factors and other translational components were compared in crude lysates of Escherichia coli grown at different rates . Cells were grown in media containing {35S}sulfate and different carbon sources, and harvested during mid-exponential phase after about 10 generations of balanced growth . Initiation factors (IF), elongation factors (EF), and a number of ribosomal proteins were identified unambiguously in gel patterns following two-dimensional polyacrylamide gel electrophoresis . The molar concentration of each protein was calculated from measurements of the radioactivity in excised gel spots and knowledge of the sulfur content of each protein . We found that the ribosomal proteins and elongation factors were present in equimolar amounts except for L7/L12 and EF-Tu which were 4-fold and 5-fold more abundant, respectively, and that the levels of each protein increased in proportion to growth rate . These results are similar to ones obtained previously by other methods, and serve to confirm the validity of our method . We found that the levels of IF2a and IF3 also were approximately proportional to growth rate . We also measured the levels of all three initiation factors using a radioimmune assay, showed that the factors are present in equimolar amounts, and confirmed that their abundance increased in parallel with ribosomes . We conclude that initiation factor levels are coordinately regulated with one another and with ribosome and elongation factor levels.

J Biol Chem, 1983 Feb 10, 258(3), 1747 - 51
Photoaffinity labeling of catechol O-methyltransferase with 8-azido-S-adenosylmethionine; Kaiser II et al.; An in vitro system using an enzyme extract containing ATP:L-methionine S-adenosyltransferase from Escherichia coli MRE 600 cells was used to synthesize 8-azido-S-adenosyl-L-methionine from methionine and 8-azidoadenosine 5'-triphosphate . In the absence of ultraviolet light and analog can serve as a methyl donor for porcine catechol O-methyltransferase . Photolysis of 8-azido-S-adenosyl{35S}methionine in the presence of catechol O-methyltransferase results in covalent incorporation . Addition of either authentic S-adenosylmethionine or S-adenosylhomocysteine, but not adenosine 5'-monophosphate, to the photolysis reaction mixture eliminates the photoincorporation . These results indicate that the incorporation is occurring at the S-adenosylmethionine binding site in the catechol O-methyltransferase.

J Biol Chem, 1983 Feb 10, 258(3), 1714 - 9
Release of transcript and template during transcription termination at the trp operon attenuator; Berlin V et al.; We studied release of trp leader RNA and trp template DNA from RNA polymerase during transcription termination at the attenuator of the trp operon of Escherichia coli . Preliminary evidence had suggested that a stable ternary complex was formed at the trp attentuator . We observed that the complexes between RNA polymerase and trp leader RNA and the DNA template produced during transcription were labile at high salt concentrations and were undetectable when transcription was performed in the presence of heparin . These characteristics are atypical of the stable transcription termination complexes described by others (Richardson, J . P., and Conaway, R . (1980) Biochemistry 19, 4293-4299; Shigesada, K., and Wu, C . (1980) Nucleic Acids Res . 8, 3355-3369) . We successfully reconstituted polymerase-trp leader RNA complexes in simple mixing experiments; these and other studies indicated that it is core polymerase that binds the leader transcript and the DNA template . In agreement with this conclusion, it was observed that sigma factor inhibited binding of RNA polymerase to the trp leader transcript and the DNA template and displaced leader RNA from RNA polymerase during transcription . It seems likely that small amounts of core polymerase present in the holoenzyme preparation, or generated during transcription, are responsible for the nonspecific binding of RNA transcript and DNA template . Our findings, therefore, suggest that the transcription termination event at the trp attenuator normally involves spontaneous dissociation of polymerase, template, and RNA transcript.

J Biol Chem, 1983 Feb 10, 258(3), 1625 - 31
Mechanism of ribonucleoside diphosphate reductase from Escherichia coli . Evidence for 3'-C--H bond cleavage; Stubbe J et al.; Incubation of the pyrimidine {3'-3H}UDP with ribonucleotide reductase resulted in an isotope effect on the conversion to dUDP which varied as a function of pH and allosteric effectors (pH, kH/kT, effector): 6.6, 4.7, ATP; 7.6, 3.3, ATP; 7.6, 2.6, dATP; 7.6, 2.0, TTP; 8.4, 2.8, ATP . During this reaction 3H2O was also released . The lower the pH of the reaction, the larger the isotope effect, and the smaller the amount of 3H2O produced . At 50% conversion of UDP to dUDP and at pH 7.6, approximately 0.5% of total 3H present in solution was volatilized, while at pH 8.4, approximately 0.9% was volatilized . Similar experiments in which the purine {3'-3H}ADP was incubated with ribonucleotide reductase also resulted in an isotope effect on its conversion to dATP which varied as a function of pH (pH, kH/kT with dGTP as an effector); 6.6, 1.9; 7.6, 1.7; 8.6, 1.4 . Furthermore, 3H2O was also released as a function of the extent of the reaction . At 50% turnover and pH 7.6, approximately 0.6% of 3H2O was volatilized, while at pH 8.6 approximately 1.25% was released . Two control experiments in which either the B1 subunit of ribonucleotide reductase was inactivated with 2'-chloro-2'-deoxyuridine 5'-diphosphate or the B2 subunit of ribonucleotide reductase was inactivated with 2'-azido-2'-deoxyuridine 5'-diphosphate and then the enzyme incubated with {3'-3H}ADP or {3'-3H}UDP indicated that in neither case was 3H released . Both B1 and B2 subunits are required for cleavage of the 3'-C--H bond . Incubation of {3'-3H}dADP or {3'-3H}dUDP with ribonucleotide reductase produced no measurable release of 3H . These data clearly indicate that conversion of a purine or pyrimidine diphosphate to a deoxynucleotide diphosphate by Escherichia coli ribonucleotide reductase requires cleavage of the 3'-C--H bond of the substrate . The fate of the 3'-H of the substrate was also determined . Incubation of {3'-2H}UDP with ribonucleotide reductase resulted in the production of {3'-2H}dUDP.

J Biol Chem, 1983 Feb 10, 258(3), 2045 - 51
Purification and properties of T4 phage thymidylate synthetase produced by the cloned gene in an amplification vector; Belfort M et al.; We have introduced the T4 thymidylate synthetase gene, resident in a 2.7-kilobase EcoRI restriction fragment, into an amplification plasmid, pKC30 . By regulating expression of this gene from the phage lambda pL promoter within pKC30 in a thyA host containing a temperature-sensitive lambda repressor, the T4 synthetase could be amplified about 200-fold over that after T4 infection . At this stage, a 20-fold purification was required to obtain homogeneous enzyme, mainly by an affinity column procedure . The purified plasmid-amplified T4 synthetase appeared to be identical with the T2 phage synthetase purified from phage-infected Escherichia coli in molecular weight, amino end group analysis, and immunochemical reactivity . The individual nature of the phage and host proteins was revealed by the fact that neither the T2 nor the T4 enzyme reacted with antibody to the E . coli synthetase, nor did antibody to the phage enzymes react with the E . coli synthetase . These differences were corroborated by DNA hybridization experiments, which revealed the absence of apparent homology between the T4 and E . coli synthetase genes . The techniques and genetic constructions described support the feasibility of employing similar amplification methods to prepare highly purified thymidylate synthetases from other sources.

J Biol Chem, 1983 Feb 10, 258(3), 1908 - 13
Identification and expression of a cloned yeast heat shock gene; Finkelstein DB et al.; We have isolated the yeast HSP90 gene which encodes the Mr = 90,000 heat shock-inducible protein of this organism . When this gene is introduced into yeast on a multicopy plasmid vector, a dramatic increase is observed in the level of synthesis of the Mr = 90,000 heat shock-inducible protein . This protein overproduction is due to expression of the plasmid-borne HSP90 gene, which is under the same heat shock regulation as its chromosomal counterpart . The presence of an increased dosage of the HSP90 gene has no effect on the synthesis of the other major heat shock-inducible proteins and does not alter the heat shock-associated phenotype of thermal tolerance.

J Biol Chem, 1983 Feb 10, 258(3), 1978 - 84
Terminal adenylation in the synthesis of RNA by Q beta replicase; Bausch JN et al.; We investigated the apparent requirement that Q beta replicase must add a nontemplated adenosine to the 3' end of newly synthesized RNA strands . We used abbreviated MDV-1 (+)-RNA templates that lacked either 62 or 63 nucleotides at their 5' end in Q beta replicase reactions . The MDV-1 (-)-RNA strands synthesized from these abbreviated (+)-strand templates were released from the replication complex, yet they did not possess a nontemplated 3'-terminal adenosine . These results imply that, despite observations that all naturally occurring RNAs synthesized by Q beta replicase possess a nontemplated 3'-adenosine, the addition of an extra adenosine is not an obligate step for the release of completed strands . Since the abbreviated templates lacked a normal 5' end, it is probable that a particular sequence at the 5' end of the template is required for terminal adenylation to occur.

J Biol Chem, 1983 Feb 10, 258(3), 1456 - 66
The interaction in vitro of the intermediate filament protein vimentin with naturally occurring RNAs and DNAs; Traub P et al.; The binding of the intermediate filament protein vimentin to a variety of naturally occurring RNAs and DNAs was studied . The relative capacities of the various nucleic acids to associate with pure {3H}vimentin were determined in competition experiments with 28 S rRNA from Ehrlich ascites tumor cells . The reaction products were analyzed by sucrose gradient centrifugation at low ionic strength and in the presence of EDTA . Under these ionic conditions, vimentin reacted preferentially with single-stranded nucleic acids, particularly with those of high (G + C) content . The vimentin binding potentials of single-stranded RNAs and DNAs were largely comparable . However, when the concentrations of mono- and divalent cations were raised to physiological and higher values, only single-stranded DNA retained its vimentin binding capacity . With increasing KCl concentrations at 0 to 1 mM Mg2+, increasing amounts of vimentin were detected in complexes which sedimented considerably faster than the bulk of the DNA, suggesting cooperative binding of vimentin . The salt optimum of this cooperativity was at 200 mM KCl . Thus, the capability of vimentin to discriminate between single-stranded RNA and DNA under physiological ionic conditions points to specificity of the interaction of vimentin with nucleic acids.

J Biol Chem, 1983 Feb 10, 258(3), 1678 - 83
Guanosine 5'-triphosphate, 3'-diphosphate 5'-phosphohydrolase . Purification and substrate specificity; Hara A et al.; The regulatory nucleotide guanosine 5'-diphosphate, 3'-diphosphate (ppGpp) and its precursor guanosine 5'-triphosphate, 3'-diphosphate (pppGpp) are accumulated during stringent response in bacterial cells . The enzyme pppGpp-5'-phosphohydrolase, which catalyzes the conversion of pppGpp to ppGpp, was partially purified from Escherichia coli . It has Mr = 140,000 and an apparent Km of 0.11 mM for pppGpp . It requires Mg2+ and a monovalent cation . NH4+ is preferred over K+, while Na+ is inactive . The enzyme does not hydrolyze GTP, ATP, pppApp, or ppGpp . It is also not effectively inhibited by these nucleotides . pppGpp-5'-phosphohydrolase hydrolyzes the 3'-monophosphate analog pppGp equally well (apparent Km of 0.13 mM), yielding the recently identified MS III nucleotide (ppGp) . pppGpp-5'-phosphohydrolase does not have RNA 5'-terminal gamma-phosphatase activity; however, 5'-terminal phosphates are released by pppGpp-5'-phosphohydrolase when the GTP-terminated RNA chains are first converted into oligonucleotides by RNase A treatment . pppGpp-5'-phosphohydrolase was found to actively hydrolyze the dinucleotide fragment pppGpNp but exhibited very low activity toward longer chain fragments . The 3'-unphosphorylated dinucleotide pppGpN was, however, not hydrolyzed . The ability of pppGpp-5'-phosphohydrolase to hydrolyze pppGpp, pppGp, and pppGpNp, but not pppG and pppGpN, indicates that pppGpp-5'-phosphohydrolase is rather nonspecific toward the 3'-OH substitutions of the substrates although a free, unsubstituted phosphate group at the 3'-OH position is essential.

FEBS Lett, 1983 Feb 7, 152(1), 125 - 30
Susceptibility of ribosomes of the tetracycline-producing strain of Streptomyces aureofaciens to tetracyclines; Mikulik K et al.; Ribosomes from cells of Streptomyces aureofaciens producing tetracycline antibiotics (Tc-ribosomes) differ in electrophoretic mobility of ribosomal proteins S2, S10 and L19 from those of the same strain, where the production of tetracyclines was suppressed by changed cultivation conditions (C-ribosomes) . Purified tight vacant couples C- and Tc-ribosomes are equally active in the translation of poly(U) . Both types of S . aureofaciens ribosomes are more sensitive to tetracycline and chlortetracycline than ribosomes of Escherichia coli in the Phe-tRNA binding and the translation of poly(U).

FEBS Lett, 1983 Feb 7, 152(1), 1 - 5
Amino acid sequence of a heat-stable enterotoxin isolated from enterotoxigenic Escherichia coli strain 18D; Takao T et al.; A heat-stable enterotoxin produced by a strain of enterotoxigenic Escherichia coli 18D was purified by ion-exchange and reversed-phase high-pressure liquid chromatography . The amino acid sequence of the purified toxin was determined by Edman-degradation and a combination of fast atom bombardment mass spectrometry and carboxypeptidase digestion to be Asn-Thr-Phe-Tyr-Cys-Cys-Glu-Leu-Cys-Cys-Asn-Pro-Ala-Cys-Ala-Gly-Cys-Tyr.

Clin Chim Acta, 1983 Feb 7, 127(3), 353 - 63
Immunoassay of three enolase isozymes in human serum and in blood cells; Kato K et al.; Sandwich enzyme immunoassay procedures for measurement of three human enolase isozymes (alpha alpha, alpha gamma and gamma gamma forms) were developed by use of purified antibodies specific to the alpha or the gamma subunit of enolase . The assay systems consisted of polystyrene balls with immobilized antibody F(ab')2 fragments and antibody Fab' fragments labeled with beta-D-galactosidase from E . coli . The measurable range was from 3 pg to 10 ng of each enolase isozyme per assay tube, and the levels of the three enolases in human sera and isolated blood cells were determined . Serum samples from healthy adults contained about 60, 8, and 2 ng/ml of alpha alpha, alpha gamma, and gamma gamma enolases, respectively . Red blood cells and platelets had about 4, 1, and 0.1 ng/10(6) cells of alpha alpha, alpha gamma, and gamma gamma enolases, respectively . Lymphocytes (mononuclear cells) contained larger amounts of all three enolases (alpha alpha, 370 ng/10(6) cells; alpha gamma, 30 ng/10(6) cells; gamma gamma, 2.7 ng/10(6) cells) . The levels of three enolase isozymes in sera of patients with small-cell cancer of the lung were also determined.

J Mol Biol, 1983 Feb 5, 163(4), 513 - 32
Complete nucleotide sequence of phoE, the structural gene for the phosphate limitation inducible outer membrane pore protein of Escherichia coli K12; Overbeeke N et al.; The complete nucleotide sequence of the gene phoE, which codes for the phosphate limitation inducible outer membrane pore protein of Escherichia coli K12 was established . The results show that PhoE protein is synthesized in a precursor form with a 21 amino acid residue amino-terminal extension . This peptide has the general characteristics of a signal sequence . The promoter region of phoE has no homology with the consensus sequence of E . coli promoter regions, but homologous sequences with the promoter region of phoA, the structural gene for alkaline phosphatase, were observed . The deduced amino acid sequence showed that the mature PhoE protein is composed of 330 amino acid residues with a calculated molecular weight of 36,782 . A number of 81 charged amino acids was found scattered throughout the protein while no large stretches of hydrophobic amino acids were observed . Hydrophobicity and hydration profiles of PhoE protein showed five pronounced hydrophilic maxima which are all located in the region from the amino terminus to residue 212 . When the deduced amino acid sequence of PhoE protein was compared with the established sequence of the OmpF pore protein, a number of 210 identical residues was found . Some aspects of the structure-function relationship of PhoE protein are discussed in view of the hydrophobicity and hydration profiles, and the homology between PhoE protein and OmpF protein.

J Mol Biol, 1983 Feb 5, 163(4), 553 - 73
Ribosomal proteins L7/L12 of Escherichia coli . Localization and possible molecular mechanism in translation; Moller W et al.; Experiments were performed in order to determine the minimal requirement for the proteins L7/L12 in polyphenylalanine synthesis and elongation factor EF-G-dependent GTP hydrolysis . Via reconstitution, ribosomal particles were prepared containing variable amounts of L7/L12 . The L7/L12 content of these particles was carefully determined by the use of 3H-labelled L7/L12 and by radioimmunoassay . The activity of the particles was determined as a function of the L7/L12 content . Our results show that only one dimer of L7/L12 is required for full activity in EF-G-dependent GTP hydrolysis . On the other hand, two L7/L12 dimers are required for polyphenylalanine synthesis . In addition, we have determined the relation between the number of L7/L12 stalks, as observed by electron microscopy, and the L7/L12 content of the 50 S particles . Our interpretation of these results is that each ribosomal particle possesses two L7/L12 binding sites, each site being involved in binding one dimer . Binding of L7/L12 dimer in one site gives rise to formation of the L7/L12 stalk, whereas binding in the other site has no effect on the number of visible stalks.

Ann Trop Med Parasitol, 1983 Feb, 77(1), 45 - 9
Recovery of soil amoebae from the air during the harmattan in Zaria, Nigeria; Lawande RV; An attempt was made to recover soil amoebae from the air during the harmattan in Zaria, Nigeria . Non-nutrient agar plates seeded with Escherichia coli were used as settle plates and exposed to the air for 30 minutes to four hours, after which they were incubated at 27 degrees C or at 37 degrees C . A total of 38 strains of amoebae were cultured: 21 of the genus Hartmannella, eight Naegleria, four Schizopyrenus, three Didascalus and two Tetramitus . Three pathogenic species, H . culbertsoni, N . fowleri and H . rhysodes, were also recovered and all killed mice . These results suggest the possibility of airborne primary amoebic meningoencephalitis infections in Nigeria.

Exp Cell Res, 1983 Feb, 143(2), 335 - 41
The effect of light on morphogenesis of Dictyostelium mucoroides; Chang MT et al.; The effect of light on the production of macrocysts and sorocarps of Dictyostelium mucoroides, strain DM-7, has been studied with surface cultures grown on dilute lactose-peptone agar at 22 degrees C with Escherichia coli, strain B/r, as food bacteria . The production of sorocarps or macrocysts can be controlled by altering the light component of the environment . Far red light had no effect on macrocyst production, whereas visible light from 440 to 700 nm inhibited macrocyst production with production decreasing with increasing light intensity . Fluence response curves for macrocyst production were determined for twelve wavelengths of light between 400 and 700 nm . An action spectrum calculated from the fluence response curves shows a single major peak at about 425-430 nm.

Environ Res, 1983 Feb, 30(1), 142 - 51
Rabbit lungs after long-term exposure to low nickel dust concentration . II . Effects on morphology and function; Johansson A et al.; For 4 and 8 months (5 days/week, 6 hr/day) rabbits were exposed to 0.13 +/- 0.05 (mean +/- SD) mg/m3 of metallic nickel dust . Volume density of alveolar type II cells was estimated with electron microscopy . Lavaged alveolar macrophages were studied with light and electron microscopy and their abilities to reduce nitroblue tetrazolium (NBT) and to phagocytize particles were tested . The effects seemed to be similar after 4 and 8 months of exposure and when the exposed animals were combined, volume density of type II cells was increased and also significantly correlated with concentration of disaturated phosphatidylcholines in the lung . The macrophages had an active surface . Their NBT activity at rest was increased but a further increase during stimulation with E . coli was low, suggesting an impaired function . Phagocytic activity, however, was not significantly changed.

Bioorg Khim, 1983 Feb, 9(2), 276 - 9
{Cloning and primary structure of DNA copies of genome fragments of the tick-borne encephalitis virus}; Chumakov MP et al.; RNA of a flavivirus-tick-borne encephalitis virus (Far-East, type 1, strain Sofin) was subjected to reverse transcription and the DNA copy was transformed into double-stranded DNA by action of E . coli DNA-polymerase (Klenow's fragment) without primer . The hairpin structures were removed by S1 nuclease . Oligo-dC ends were attached to ds-cDNA thus obtained, and this DNA was annealed with pBR322 plasmid cut by PstI and equipped with oligo-dG termini . The recombinant plasmids were cloned in E . coli HB101 . Of the 360 TcrAps clones obtained, 187 clones efficiently hybridized with partially degraded 32P-RNA of TBE virus . The sequence of the insert of one of the clones was determined by the Maxam-Gilbert method . The 720 b.p . sequence is translatable into an amino acid sequence without interruption . Nearby the 3'-terminus of the insert, the sequence ACACAGG is present which is homologous with that found in RNA of the flavivirus West Nile.

Proc Natl Acad Sci U S A, 1983 Feb, 80(3), 802 - 6
Isolation of a cDNA clone for human X-linked 3-phosphoglycerate kinase by use of a mixture of synthetic oligodeoxyribonucleotides as a detection probe; Singer-Sam J et al.; We have obtained a cDNA clone encoding most of human X-linked 3-phosphoglycerate kinase (PGK; ATP:3-phospho-D-glycerate 1-phosphotransferase, EC 2.7.2.3) . Total mRNA was prepared from human adenocarcinoma-derived cell line LS174T and used for cDNA preparation . Double-stranded cDNA was inserted, after tailing with oligo(dC), into the plasmid vector pBR327 and cloned in Escherichia coli K-12 . Transformants were screened by colony hybridization with a mixture of 32P-labeled oligodeoxyribonucleotides . A pool of hexadecamers complementary to all 32 possible sequences encoding amino acids 291-296 of X-linked PGK was used for the initial screen . One clone among 2,500 gave a strong positive signal . Plasmid DNA from this clone was purified and characterized by hybridization first to the hexadecamer probe mixture and then to an undecamer probe consisting of a mixture of four sequences . The cloned fragment hybridizes preferentially to DNA from human cells with five X chromosomes . DNA sequence analysis has established that the 1.2-kilobase-pair fragment encodes PGK from amino acid 121 through the COOH terminus.

Eur J Cancer Clin Oncol, 1983 Feb, 19(2), 163 - 71
Bacteriocin and flow cytometry in laboratory diagnosis of leukemic peripheral blood lymphocytes and bone marrow cells; Musclow E et al.; The bacteriocin, colicin HSC10, produced by Escherichia coli HSC10, was studied as a laboratory tool for detection and differentiation of leukemic from normal lymphocytes in human peripheral blood . Flow cytometry studies detected DNA loss in bacteriocin-affected cells by computerized histograms . Differential analysis is given for the peripheral blood of 26 individuals using bacteriocin, cytochemistry and surface markers . Sensitivity to colicin was detected in 10 (83%) of the 12 patients with chronic lymphocytic leukemias and other leukemias with morphologically immature lymphocytes . Cells were lost from the G0/G1 phase and accumulated in the 'pre-G1' channels of the histogram, indicative of cells with reduced DNA content . The lymphocytes of 14 normals, however, were not or only slightly affected by the bacteriocin (P less than 0.001) . Similarly, normal bone marrow cells exposed to bacteriocin remained unaffected (P greater than 0.2) . Thus, immaturity per se was not recognized by bacteriocin . The bacteriocin effect was more discriminatory than other laboratory tests reported here and in most cases differentiated malignant from normal cells.

Mutat Res, 1983 Feb, 107(2), 219 - 27
Antimutagenic effects of cinnamaldehyde on chemical mutagenesis in Escherichia coli; Ohta T et al.; Antimutagenic effects of cinnamaldehyde on mutagenesis by chemical agents were investigated in Escherichia coli WP2 uvrA- trpE- . Cinnamaldehyde, when added to agar medium, greatly reduced the number of Trp+ revertants induced by 4-nitroquinoline 1-oxide (4-NQO) without any decrease of cell viability . This antimutagenic effect could not be explained by inactivation of 4-NQO caused by direct interaction with cinnamaldehyde . Mutagenesis by furylfuramide (AF-2) was also suppressed significantly . Mutations induced by methyl methanesulfonate (MMS) and ethyl methanesulfonate (EMS) were slightly inhibited . However, cinnamaldehyde was not at all effective on the mutagenesis of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) . Two derivatives of cinnamaldehyde, cinnamyl alcohol and trans-cinnamic acid, did not have as strong antimutagenic effects on 4-NQO mutagenesis as cinnamaldehyde had . Because cinnamaldehyde showed marked antimutagenic effects against mutations induced by UV-mimic mutagens but not those induced by MNNG or EMS, it seems that cinnamaldehyde might act by interfering with an inducible error-prone DNA repair pathway.

Vet Microbiol, 1983 Feb, 8(1), 35 - 43
Intestinal changes associated with rotavirus and enterotoxigenic Escherichia coli infection in calves; Tzipori S et al.; Newborn calves inoculated with rotavirus, enterotoxigenic Escherichia coli (ETEC) serotype 020:K' x 106':K99:HNM, either alone or in combination, became depressed, anorectic, diarrhoeic and dehydrated . ETEC did not adhere to the intestine although there was extensive proliferation in the lumen . Only slight mucosal changes were induced by ETEC and the activity of membrane bound lactase remained normal . More severe mucosal damage and a decrease in lactase activity were found in newborn calves inoculated with either rotavirus or rotavirus and ETEC in combination . The most severe clinical illness was found in calves inoculated with both rotavirus and ETEC . Calves inoculated at 1 week of age with either rotavirus or ETEC remained clinically normal . Rotavirus infection produced slight mucosal changes and a reduction of lactase activity . In contrast, colostrum-fed or suckling calves up to 2 weeks old inoculated with both rotavirus and ETEC became clinically affected, showed severe mucosal damage and decreased lactase activity . There was no bacterial adhesion to the intestinal mucosa as observed by immunofluorescent labelling and light microscopy.

Proc Natl Acad Sci U S A, 1983 Feb, 80(3), 825 - 9
Immunoglobulin gene expression in transformed lymphoid cells; Oi VT et al.; Myeloma, hybridoma, and thymoma cell lines have been successfully transfected for the Escherichia coli xanthine-guanine phosphoribosyltransferase gene (gpt) by using the plasmid vector pSV2-gpt . The transformed cells synthesize the bacterial enzyme 5-phospho-alpha-D-ribose-1-diphosphate:xanthine phosphoribosyltransferase (XGPRT; EC 2.4.2.22) and have been maintained in selective medium for over 4 months . Lymphoid cell lines expressing a K immunoglobulin light chain were obtained by transfecting cells with pSV2-gpt containing a rearranged K light chain genomic segment from the S107 myeloma cell line . The S107 light chain is synthesized in gpt-transformed J558L myeloma cells and is identical to the light chain synthesized by the S107 myeloma cell line, as judged by immunoprecipitation and two-dimensional gel electrophoresis . Furthermore, this light chain is synthesized and secreted as part of an intact antibody molecule by transformed hybridoma cells that normally secrete an IgGl (gamma, K) antibody molecule . No light chain synthesis was detected in a similarly transformed rat myeloma or a mouse thymoma line.

Arch Biochem Biophys, 1983 Feb 1, 220(2), 519 - 29
Molecular asymmetry in alkaline phosphatase of Escherichia coli; Malhotra OP et al.; Thermal inactivation of alkaline phosphatase of Escherichia coli has been studied at different temperatures (45 to 70 degrees C) and pHs (7.5, 9.0, and 10.0) for the commercial, buffer-dialyzed (pH 9.0) and EDTA-dialyzed (pH 9.0) enzymes . In each case, the inactivation exhibits biphasic kinetics consistent with the rate equation, (formula; see text) where A0 and A are activities at time zero and t, and k1 and k2 are first-order rate constants for the fast and slow phase, respectively . Values of k1 and k2 change independently with temperature, pH, and pretreatment (dialysis) of the enzyme . Time course of inactivation of the enzyme with excess EDTA and effect of Zn2+ ion concentration on the activity of EDTA-dialyzed enzyme have been investigated . The data suggest that the dimeric enzyme protein has two types of catalytic sites which have equal catalytic efficiency (or specific activity) but differ in several other properties . Structural implications of these results have been discussed.

J Bacteriol, 1983 Feb, 153(2), 685 - 92
Use of cloned mtl genes of Escherichia coli to introduce mtl deletion mutations into the chromosome; Lee CA et al.; The Escherichia coli mtl operon, which contains the cis-dominant regulatory region mtlC, the mannitol-specific enzyme II structural gene mtlA, and the D-mannitol-1-phosphate dehydrogenase structural gene mtlD, was cloned into plasmid pBR322 . A 2-kilobase pair fragment of the mtl operon DNA containing the mtlA gene was subcloned into plasmid pACYC184 . The direction of transcription of the cloned mtlA gene was determined . Localization of the mtlA gene on the cloned mtl operon DNA allowed in vitro construction of plasmid derivatives containing specific deletions in the mtl region . These plasmid derivatives were used to generate the corresponding mtl chromosomal deletions by homologous recombination at frequencies greater than 10(-4).

Jpn J Pharmacol, 1983 Feb, 33(1), 189 - 200
Traxanox augments immune response to lipopolysaccharide in inbred mice: role of macrophages; Goto K et al.; Both C57BL/6 and BALB/c mice were immunized intravenously with lipopolysaccharide (LPS, 10 micrograms/mouse) on day 0, and hemolytic plaque forming cells (HPFC) in the spleen were assayed on day 4 . Traxanox given orally at a dose of 30 mg/kg augmented the HPFC production to LPS in both mice . This agent (3-30 mg/kg) restored significantly the suppressed HPFC production to LPS in the BALB/c mice pretreated with carrageenan (0.03 mg/mouse), but did not restore it in the BALB/c mice pretreated with cyclophosphamide (25 mg/kg) . The transfer of spleen adherent cells of the mice immunized with LPS and treated with traxanox to the syngeneic mice resulted in a significant increase in the HPFC production to LPS . The HPFC production to trinitrophenylated polyvinylpyrrolidone, a T cell-independent antigen was not affected by the treatment with traxanox or carrageenan . These results suggest that traxanox has a capacity to augment the immune response by affecting macrophage function.

Mutat Res, 1983 Feb, 107(2), 203 - 18
Misincorporation in DNA synthesis after modification of template or polymerase by MNNG, MMS and UV radiation; Miyaki M et al.; The synthetic DNA polymers, poly(dG-dC), poly(dC), poly(dA-dT), poly(dA) and poly(dT), were treated with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), methyl methanesulfonate (MMS) and UV irradiation . The modified polymers were used as templates to examine the incorporation of non-complementary nucleotides by E . coli DNA polymerase I . Methylation of poly(dG-dC) by MNNG predominantly induced the misincorporation of dTMP, whereas methylation by MMS induced that of dAMP . Treatment of poly(dT) with MNNG caused the misincorporation of dGMP to a considerable extent, but MMS did not enhance the error on poly(dT) . The misincorporation of dAMP on poly(dC) and that of dGMP on poly(dA) were also increased by these chemicals . UV irradiation of poly(dT) and poly(dC) induced the error of dGMP and dAMP, respectively . These data on MNNG and MMS in vitro were in fair agreement with the directions of mutation in vivo . But the predominant induction of transitions by UV in vitro did not agree with the UV-induced transversions in E . coli . This inconsistency suggested the participation of other factors than direct mispairing in UV-induced transversion . Modification of DNA polymerase I by MNNG changed the ratio of polymerase to 3' leads to 5' exonuclease activity altering the fidelity of this enzyme, whereas MMS and UV-irradiation did not alter the fidelity of the enzyme.

J Virol Methods, 1983 Feb, 6(2), 61 - 70
Comparison of hepatitis B core antigens derived from human liver or synthesized in Escherichia coli and evaluation of their use in diagnostic assays for anti-HBc IgM; Roggendorf M et al.; Hepatitis B core antigen (HBcAg) synthesized in Escherichia coli (clone PR1-11) was compared with HBcAg purified from a liver obtained at autopsy of a patient with chronic active hepatitis B . The molecular weight determined by SDS-PAGE with subsequent transfer of the proteins to nitrocellulose paper, incubation with 125I anti-HBc and exposure to X-ray film, was about 21,000 for HBcAg synthesized in E . coli and was slightly lower for the liver-derived HBcAg . In CsCl density gradients the liver-derived HBcAg had one peak at 1.32 g/ml, and the HBcAg synthesized in E . coli had two peaks at 1.34 and 1.30 g/ml . In a comparison of the immunological activity of both antigen preparations in an enzyme immunoassay, the liver-derived HBcAg was detected only up to a dilution of 1:320, whereas the HBcAg synthesized in E . coli was detected in dilutions up to 1:100,000 . With both antigens, the same percentage of sera were positive for anti-HBc IgM from patients with acute (100%), convalescent (61%), past (5%) and chronic active (29-37%) hepatitis . These results indicate that tests for anti-HBc IgM performed with HBcAg synthesized in E . coli are at least as sensitive and specific as those using liver-derived HBcAg.

Anal Biochem, 1983 Feb 1, 128(2), 481 - 9
A rapid mixing-photocrosslinking technique to study the dynamics of nucleic acid-protein interactions; Wu CW et al.; A rapid mixing-photocrosslinking technique has been developed to investigate the kinetics of protein-nucleic acid interactions . With this technique, binding of nucleic acid to protein is first synchronized by rapid mixing in a stopped-flow apparatus . The intermediates formed at different stages of the binding process are then "frozen" by photocrosslinking with a 10-microseconds uv light pulse at various times after mixing . By analyzing structural changes of these intermediates as a function of time, one can obtain the information concerning the dynamic aspects of the interaction . This technique may also be applied to other macromolecular interactions in biological systems.

Proc Natl Acad Sci U S A, 1983 Feb, 80(4), 1068 - 72
In vivo expression of rat insulin after intravenous administration of the liposome-entrapped gene for rat insulin I; Nicolau C et al.; A recombinant plasmid encoding rat preproinsulin I was encapsulated in large liposomes and intravenously injected in rats . Glycemia and blood, splenic, and hepatic insulin were assayed at various times beginning 6 hr after inoculation . The results were compared with controls that had received (i) empty liposomes, (ii) liposomes carrying the Escherichia coli pBR322 plasmid, (iii) the free rat insulin I gene, and (iv) no injection at all . Whereas all controls showed unchanged glucose and insulin levels, the treated animals had, 6 hr after inoculation, a blood glucose level of 72 +/- 5 mg of glucose/100 ml of blood as compared with 107 +/- 2 mg/ml for controls . Radioimmunoassay of blood insulin gave 61 +/- 8 microunits/ml as compared with 43 +/- 5 microunits/ml for controls and the spleen and liver values were 242 +/- 22 and 204 +/- 20 microunits/g of tissue, respectively, as compared with 112 +/- 20 and 87 +/- 15 microunits/g in controls . External gamma-camera imaging of the organ uptake of 111In-labeled liposomes permitted study of the kinetics and extent of uptake of the liposomes by spleen and liver, results that support the findings concerning insulin synthesis in the two organs.

Hoppe Seylers Z Physiol Chem, 1983 Feb, 364(2), 141 - 55
Direct micro-sequence analysis of peptides from Escherichia coli ribosomal proteins S11, L9 and L29 after separation by reversed phase chromatography; Kamp RM et al.; Tryptic peptides of the ribosomal proteins S11, L9 and L29 were separated by reversed phase chromatography under conditions which enabled direct micro-sequencing with the 4-(dimethylamino)azobenzene-4'-isothiocyanate/phenylisothiocyanate double coupling method {Chang, Brauer, Wittmann-Liebold (1978) FEBS Lett . 93, 205-214} . The peptides were separated on a RP-18 column employing volatile buffers at pH 2.0, 4.1 and 7.8 . Depending on the different chromatographic behaviour of the peptide mixture, the elution gradient was optimised for each hydrolysate using 20 micrograms of the hydrolysed protein . Preparative separations were made with 150-250 micrograms . At least 80% of the peptides could be isolated by these techniques and used for direct micro-sequencing without further purification or desalting . The results show that the high-performance liquid chromatographic method employed allows easy isolation and sequencing with minute amounts of peptides.

Biochemistry, 1983 Feb 1, 22(3), 690 - 4
Effect of Escherichia coli initiation factors on the kinetics of N-Acphe-tRNAPhe binding to 30S ribosomal subunits . A fluorescence stopped-flow study; Wintermeyer W et al.; The mechanism of binding of N-AcPhe-tRNAPhe (yeast) to poly(U)-programmed Escherichia coli 30S ribosomal subunits and the effect of individual initiation factors (IF-1, IF-2, and IF-3) and GTP on this process have been studied by fluorescence stopped-flow kinetic measurements . The formation of the ternary complex was followed by an increase of both intensity and polarization of the fluorescence of a proflavin label located in the anticodon loop of the tRNA . The effect of the initiation factors and GTP is to increase the velocity of ternary complex formation (about 400-fold at 7 mM Mg2+) . In the presence of the three initiation factors and GTP the formation of the ternary complex could be resolved into two partial reactions: a fast apparently second-order step (k12 = 5 x 10(6) M-1 s-1, k21 = 1.4 s-1) followed by a slow rearrangement step (k23 less than or equal to 0.1 s-1) . The data suggest a mechanism in which the ternary complex is formed by at least two rearrangements of an initially formed preternary complex . The accelerating effects of both IF-2 and IF-3 can be understood by assuming a synergistic allosteric action of the factors on the 30S ribosomal subunit, whereas IF-1 appears to act indirectly by influencing the other two factors.

J Clin Microbiol, 1983 Feb, 17(2), 377 - 9
Multidose vials versus single-dose vials: a study in sterility and cost-effectiveness; Sheth NK et al.; A total of 197 multidose injectable vials were collected from 10 different nursing stations and evaluated for sterility . Experimental contamination studies were undertaken, and the cost-effectiveness of multidose vials was compared with that of single-dose vials . Our results showed that bacterial contamination of multidose injectable vials was not a significant hazard; in addition, contrary to common belief, the use of multidose vials was not always successful as a cost-containment measure.

Infect Immun, 1983 Feb, 39(2), 990 - 2
Immunization of swine with heat-stable Escherichia coli enterotoxin coupled to a carrier protein does not protect suckling pigs against an Escherichia coli strain that produces heat-stable enterotoxin; Moon HW et al.; Pregnant swine were immunized parenterally with purified heat-stable Escherichia coli enterotoxin that was made antigenic by coupling it to bovine immunoglobulin G . Immunized swine had high titers of antitoxin in serum and colostrum as measured by radioimmunoassay . However, the heat-stable enterotoxin neutralizing titers of the serum and colostrum from immunized swine were comparatively low . Newborn pigs suckling their immunized dams were not protected against challenge with porcine enterotoxigenic E . coli that produce heat-stable toxin but do not produce heat-labile toxin.

Proc Soc Exp Biol Med, 1983 Feb, 172(2), 255 - 9
D-Galactosamine liver injury: absorption of endotoxin and protective effect of small bowel and colon resection in rabbits; Camara DS et al.; D-Galactosamine is an amino sugar with unique hepatotoxic properties in animals . Although the mechanism of liver injury by galactosamine remains controversial, a role for bacterial endotoxin has been suggested . In the present study, using New Zealand rabbits, we show that the significant increase in serum glutamic oxaloacetic transaminase which followed the injection of 4.25 mmole/kg of D-galactosamine was completely prevented in animals subjected to resection of small bowel and colon . Using an immunoradiometric assay specific for E . coli 026 endotoxin we showed that after instillation of 50 mg of E . coli into the colon, serum levels of this endotoxin were higher in the animals injected with galactosamine than the controls injected with saline . However, the differences in endotoxin concentration between the two groups of animals was statistically significant only at 30 and 60 min . The role of endotoxin in the pathogenesis of galactosamine liver injury is reviewed and discussed.

Proc Natl Acad Sci U S A, 1983 Feb, 80(3), 687 - 91
Evidence for use of rare codons in the dnaG gene and other regulatory genes of Escherichia coli; Konigsberg W et al.; Amino acid sequence and composition data of Escherichia coli dnaG primase protein and its tryptic peptides have confirmed that the dnaG gene contains an unusually high number of codons that are not frequently used in most E . coli genes . In 25 E . coli proteins analyzed the codons AUA, UCG, CCU, CCC, ACG, CAA, AAT, and AGG are infrequently used, occurring as 4% of the total codons in the reading frame and 11% and 10% in the nonreading frames . In dnaG they occur as 11% in the reading frame and 12% in the nonreading frames . The rpsU and rpoD genes, which flank the dnaG gene {Smiley, B . L., Lupski, J . R., Svec, P . S., McMacken, R . & Godson, G . N . (1982) Proc . Natl . Acad . Sci . USA 79, 4550-4554}, however, have normal codon usage . Translational modulation using isoaccepting tRNA availability may therefore be part of the mechanism of keeping the dnaG gene expression low, while expression of the adjacent rpsU and rpoD genes on the same mRNA transcript is high.

Proc Natl Acad Sci U S A, 1983 Feb, 80(3), 668 - 72
Purification and characterization of trp aporepressor; Joachimiak A et al.; We have isolated homogeneous trp aporepressor from an overproducing strain of Escherichia coli carrying a plasmid containing trpR preceded by tandem trp operon promoters . Dye-affinity and ion-exchange chromatography were used in conjunction with a gel electrophoresis assay in which the repressor, when bound to the trp operator, protects an Rsa I restriction site from endonuclease cleavage . Crystals suitable for x-ray diffraction studies were grown from a variety of concentrated salt solutions . Hydrodynamic properties and electrophoretic analysis of unmodified and covalently crosslinked aporepressor show that the free aporepressor has an isoelectric point of 5.9 and is a dimer containing two identical 12.5-kilodalton subunits in the presence or absence of L-tryptophan . The repressor . operator complex binds poorly to nitrocellulose filters, but restriction-site protection studies indicate that, in the presence of tryptophan, one dimer is bound to the operator site with an apparent dissociation constant less than 2 X 10(-9) M . Preliminary equilibrium dialysis experiments suggest that tryptophan binds to the aporepressor with a dissociation constant of 1.6 X 10(-5) M.

Mutat Res, 1983 Feb, 112(1), 17 - 22
DNA replication past pyrimidine dimers in the absence of repair; Trgovcevic Z et al.; Post-UV DNA synthesis in Escherichia coli uvrA recA cells was studied . A low dose of UV radiation (0.07 J/m2), which caused no degradation of the dimer-containing DNA, was used . This enabled us to make a direct comparison between DNA synthesis on the normal template and DNA synthesis on the UV-damaged template . There was no change in the post-UV DNA synthesis kinetics during the first 60 min of post-irradiation incubation . A reduced rate of DNA synthesis was observed at later post-UV times when the dimers are expected to have passed through the normal replication complex . This reduced rate of DNA synthesis was associated with loss of the biological activity of the DNA . We suggest that the gaps opposite dimers rather than dimers per se interfere with normal replication, thus leading to cell death of uvrA recA bacteria.

J Med Chem, 1983 Feb, 26(2), 167 - 74
Analogues of 2'(3')-O-L-phenylalanyladenosine as substrates and inhibitors of ribosomal peptidyltransferase; Zemlicka J et al.; The chemical syntheses of 2'(3')-O-(L-3-amino-3-phenylpropionyl)adenosine (2e), the corresponding D stereoisomer 2f, 2'(3')-O-(DL-phenylglycyl)adenosine (2g), 2'(3')-O-(N-benzylglycyl)adenosine (2h), and 9(2-O-L-phenylalanyl-beta-D-xylofuranosyl)adenine (3b) are described . Compounds 2e-h were obtained by acylation of 5'-O-(4-methoxytrityl)adenosine with the appropriate N-benzyloxycarbonyl or N-tert-butoxycarbonyl amino acids with dicyclohexylcarbodiimide in pyridine . The corresponding reaction of N-(benzyloxycarbonyl)-D-phenylglycine led to an almost complete racemization of the aminoacyl residue (compounds 2c and 2g) . Subsequent chromatographic separation and deprotection of intermediates 2a-d afforded the desired target derivatives 2e-h . Product 3b was obtained by a similar acylation of 9-(3,5-O-isopropylidene-beta-D-xylofuranosyl)adenine with N-(benzyloxycarbonyl)-L-phenylalanine, followed by deblocking . The NMR spectra of 2' and 3' isomers of stereoisomers 2a and 2b are discussed . Compounds 2g and 3b are both substrates and inhibitors of Escherichia coli ribosomal peptidyltransferase, although the activity of 3b is low . Derivatives 2e,f,h do not accept AcPhe from N-AcPhe-tRNA in a peptidyltransferase-catalyzed reaction, but they inhibit the puromycin reaction in the same system . The order of inhibitory activity is 2e greater than 2f greater than 2h . The implications of these findings for the mechanism of peptidyltransferase and comparison of the latter with the action of chymotrypsin are discussed.

J Infect Dis, 1983 Feb, 147(2), 318 - 26
Vaccine for enterotoxigenic Escherichia coli based on synthetic heat-stable toxin crossed-linked to the B subunit of heat-labile toxin; Klipstein FA et al.; Synthetically produced Escherichia coli heat-stable toxin (ST) was conjugated to the nontoxic B subunit of the heat-labile toxin (LT) by the carbodiimide reaction . Modifying the molar ratio of toxins mixed and the ratio of carbodiimide added to the toxins permitted synthesis of conjugates with any desired degree of proportional antigenicity for each toxin component . Immunization of rats by the parenteral/peroral routes with cross-linked vaccine containing 39% ST and 61% B subunit antigenicity, with 0.06% residual ST toxicity, evoked fourfold to sevenfold increases over control values of serum IgG and mucosal secretory IgA antitoxin titers to each of the component toxins, thus providing significant (P less than 0.001) protection against challenge with either LT or ST or with viable heterologous strains which produce these toxins . These observations show that cross-linking synthetic ST to the B subunit results in a nontoxic vaccine that provides protection against all types of enterotoxigenic E . coli.

J Clin Pathol, 1983 Feb, 36(2), 224 - 7
Do urethral Escherichia coli cause abacterial cystitis?
Walpita P, Marsh FP.
To determine whether Escherichia coli in or near the urethra caused symptoms of abacterial cystitis, the results of serial cultures from the vaginal introitus of 92 patients with recurrent cystitis were compared with symptoms at clinic visits when they were abacteriuric . Similar comparisons were made in 15 of these patients using cultures from the external urethral meatus and proximal urethra . E coli were grown from the vaginal introitus, urethral meatus and proximal urethra at 41, 66 and 26% of visits respectively . Overall they were not significantly more often isolated when patients had cystitis, and the serotype was unrelated to symptoms . E coli was cultured significantly more often from the introital swabs of symptomatic intermittently bacteriuric (IB) women than from symptomatic persistently non-bacteriuric (NB) patients; and more often from symptomatic than from asymptomatic IB patients, although this difference was not significant . These findings were consistent with previous suggestions that symptoms of apparently "abacterial" cystitis in IB patients are due to occult coliform infection . We found no direct evidence that E coli were the cause of symptoms in persistently non-bacteriuric women, or that urethral colonisation caused them . However E coli were isolated from the introitus of control women only half as often as from both intermittently bacteriuric and persistently non-bacteriuric patients.

Eur J Biochem, 1983 Feb 1, 130(2), 409 - 17
tuf gene dosage effects on the intracellular concentration of EF-TuB; van der Meide PH et al.; In this paper we have studied the effect of raising the intracellular EF-Tu concentration on the expression of tufB . To this aim cells were transformed with multicopy plasmids carrying either tufA or tufB . The intracellular EF-Tu concentrations were determined by the specific immunoelectrophoresis assay described in the preceding paper in this journal . We have cloned the tufA gene in a plasmid, containing the powerful major leftward promoter (PL) of phage lambda . Transcription from PL can be repressed at low temperature by a temperature-sensitive repressor and activated by heat induction . Cloning occurred in two orientations in a single EcoRI site about 150 base pairs downstream of PL . Cells carrying either plasmid were shown to contain an almost doubled amount of EF-Tu at temperatures from 28 degrees C to 37 degrees C . This indicates that transcription of tufA can proceed from a possible binding site for RNA polymerase on these cloned fragments . The EF-Tu level was further increased to about 30% of total cellular protein after a temperature shift from 37 degrees C to 43 degrees C . The multicopy plasmid pTuB1 described by Miyajima et al . {FEBS Lett . 102, 207-210 (1979)} and a derivative (pTuBo, compare preceding paper in this journal) were used to study the expression of both chromosomal and plasmid-borne tufB . Transformation with either plasmid raised the intracellular EF-Tu concentration by 30-60% depending on the nutritional conditions . Suppression of tufB expression was observed when the intracellular level of EF-Tu increased after transformation with all plasmids mentioned above . The results are in accord with the concept that EF-Tu acts as an autogenous feedback inhibitor involved in the regulation of tufB.

Eur J Biochem, 1983 Feb 1, 130(2), 341 - 5
A stereochemical and positional isotope-exchange study of the mechanism of activation of methionine by methionyl-tRNA synthetase from Escherichia coli; Lowe G et al.; Methionyl-tRNA synthetase from Escherichia coli catalyses the activation of {18O2}methionine by adenosine 5'-{(R)-alpha 17O}triphosphate with inversion of configuration at P alpha . Furthermore methionyl-tRNA synthetase does not catalyse positional isotope exchange in adenosine 5'-{beta-18O2}triphosphate in the absence of methionine or in the presence of the competitive inhibitor, methioninol, which eliminates the possibility of either adenylyl-enzyme or adenosine metaphosphate intermediates being involved . These observations require that methionyl-tRNA synthetase catalyses the activation of methionine by an associative 'in-line' nucleotidyl transfer mechanism . A kinetic study of positional isotope exchange in adenosine 5'-{beta-18O2}triphosphate in the presence of methionine, Mg2+ and methionyl-tRNA synthetase showed that torsional equilibration (18O exchange into the P alpha--O--P beta bridge) occurs faster than tumbling (18O exchange into P gamma by rotation about the C2 axis of Mg{18O2}PPi), demonstratings that the positional isotope exchange occurs at least in part in the E X Met-AMP X Mg{18O2}PPi complex.

Arch Biochem Biophys, 1983 Feb 1, 220(2), 495 - 501
Monoclonal antibody to an integral membrane protein, the lactose permease; Eash J et al.; A monoclonal IgG antibody directed against the lactose permease was produced from animals inoculated with membranes of a lac Y plasmid strain . The appropriate antibody was selected by a series of ELISA assays in which membranes, purified permease, or a lac Y-Z chimeric protein was the immobilized antigen . The antibody recognizes a portion of the permease exposed on the surface of membrane vesicles but does not inhibit lactose transport.

Arch Biochem Biophys, 1983 Feb 1, 220(2), 435 - 43
Photoaffinity labeling of the indole sites on the Escherichia coli tryptophan synthase alpha-subunit; Brock PW Jr et al.; The alpha subunit of the Escherichia coli tryptophan synthase catalyzes the reversible aldolytic reaction: Indole-3-glycerol phosphate in equilibrium indole + glyceraldehyde 3-phosphate . The use of 5-azidoindole as a photoaffinity label has made the generation of a number of enzyme-substrate complexes possible, each with a given degree of saturation of the two postulated indole sites . When assayed in the reverse reaction (indole-3-glycerol phosphate synthesis), samples of alpha subunit treated at concentrations of 5-azidoindole less than or equal to 2 mM show a progressive 30-40% activation . A gradual inactivation occurs only in samples irradiated at concentrations in excess of 2 mM 5-azidoindole, and this inactivation is complete at 8-10 mM . A quantitatively similar activation occurs in the forward reaction (indole synthesis), however inactivation in this case is incomplete, with complexes treated at 8-12 mM 5-azidoindole retaining 30-40% relative activity in this reaction . When treated alpha subunits were assayed for their abilities to complement the beta 2-subunit in the reactions indole + L-serine leads to L-tryptophan + H2O and indole-3-glycerol phosphate + L-serine leads to L-tryptophan + glyceraldehyde 3-phosphate, quantitatively lesser amounts of activation followed by total inactivation are observed over a similar range of 5-azidoindole concentrations.

Am J Physiol, 1983 Feb, 244(2), H178 - 85
Influence of central nervous system on arterial pressure during endotoxin shock; Janssen HF et al.; Some investigators have suggested that the hypotensive effects of endotoxin are exerted at the level of central nervous system (CNS) . Others feel the effects are exerted peripherally and that the CNS is involved in the regulation of the cardiovascular system during the shock state . Still other data suggest that endotoxin shock is entirely a peripheral phenomenon . The present study used ventriculocisternal perfusion of endotoxin, a pretrigeminal brainstem transection, two midcollicular decerebrate preparations, and Cushing's reflex to investigate the involvement of the CNS during endotoxin shock . The results suggest the following: 1) endotoxin perfused centrally at a concentration equivalent to the maximum obtainable after peripheral injection will not alter mean arterial pressure (MAP); 2) either the forebrain is not involved in the MAP response or the remaining regions can compensate for its absence; and 3) Cushing's reflex will block the initial endotoxin-induced hypotension.

Surgery, 1983 Feb, 93(2), 289 - 96
Effect of thermal injury on endotoxin-induced lung injury; Nerlich M et al.; We studied the effects of a burn injury on the response of the lung to endotoxin . Seventeen unanesthetized sheep with lung lymph fistulas were studied . Eight were given Escherichia coli endotoxin (1.5 micrograms/kg) alone and nine were given the same dose 72 hours after a 25% total body surface burn injury . At this time after burn, all physiologic parameters were at baseline levels . A characteristic two-phase lung injury was seen after administration of endotoxin with an initial hypertension phase, characterized by pulmonary artery hypertension, and a second or permeability phase, characterized by an increase in protein-rich lymph flow . all eight animals that underwent only endotoxin administration survived, whereas four of the nine burned animals died during the permeability phase in pulmonary edema . Major physiologic differences between the groups were noted during the permeability phase, including a more severe hypoxia, pulmonary hypertension, and increased postburn lymph flow . Major biochemical changes included significant increases in lymph thromboxane, thromboxane B2, and beta-glucuronidase activity in the burn group . We conclude that the lung is more sensitive to endotoxin after burn, probably as a result of an increased release of products of arachidonic acid metabolism and products of leukocyte activation caused by the body burn.

Surgery, 1983 Feb, 93(2), 273 - 8
Metabolism of branched-chain amino acids in dogs with Escherichia coli endotoxin shock; Groves AC et al.; The arterial-femoral venous difference of phenylalanine concentration is proportional to net proteolysis in the leg . In ten fasting dogs receiving Escherichia coli endotoxin (2 mg/kg) intravenously, the mean systolic blood pressure decreased from 141.1 +/- 25 to 71.5 +/- 17 mm Hg . The absolute net release from the leg of valine, isoleucine, and leucine and their net release relative to net proteolysis (arterial-femoral venous difference in concentration of each branched-chain amino acid relative to that of phenylalanine) were decreased, indicating increased transamination of these amino acids in skeletal muscle . However, the net release of the branched-chain alpha-keto acids formed by transamination, relative to net proteolysis (arterial-venous difference in concentration of each alpha-keto acid relative to that of phenylalanine), was not increased . The findings indicate that in dogs with E . coli endotoxin shock, there is increased oxidative decarboxylation in muscle of the alpha-keto acids derived from valine, isoleucine, and leucine.

J Bacteriol, 1983 Feb, 153(2), 771 - 81
Growth rate-dependent regulation of 6-phosphogluconate dehydrogenase level in Escherichia coli K-12: beta-galactosidase expression in gnd-lac operon fusion strains; Baker HV 2nd et al.; The level of 6-phosphogluconate dehydrogenase is positively correlated with the cellular growth rate . To determine whether growth rate-dependent regulation of expression of gnd, which encodes this enzyme, is carried out by a transcriptional mechanism, the structural genes of the lactose operon were fused to and brought under the control of the gnd promoter through the use of phage Mu d1(Apr lac) . Four independent gnd::Mu d1(Apr lac) operon fusion strains were isolated . After the Mu d1 prophage was replaced with lambda p1(209), Lac+ specialized transducing phages carrying the gnd-lac fusions were prepared . These phages were used to demonstrate that the lac genes were fused to the gnd promoter by crossing them with gnd promoter deletion mutants and with polar phage Mu cts-induced gnd mutants . A genetic map of the fusion joints was deduced . The level of beta-galactosidase in each fusion strain was the same in cells growing on acetate as in cells growing on glucose (with specific growth rate constants of 0.1 and 0.55 h-1, respectively) and was unaffected by the presence of a gnd+ gene in trans . Our results suggest that a post-transcriptional mechanism mediates growth rate-dependent regulation of gnd and that this regulation is not autogenous . Models for regulation are discussed with respect to these results and the physiology and DNA sequence of gnd.

J Bacteriol, 1983 Feb, 153(2), 739 - 46
Thiamine pyrophosphate requirement for o-succinylbenzoic acid synthesis in Escherichia coli and evidence for an intermediate; Meganathan R et al.; Cell-free extracts of various strains of Escherichia coli synthesize the menaquinone biosynthetic intermediate o-succinylbenzoic acid (OSB) when supplied with chorismic acid, 2-ketoglutaric acid, and thiamine pyrophosphate (TPP) . To assay for OSB synthesis, 2-{U-14C}ketoglutaric acid was used as substrate, and the synthesized OSB was examined by radiogas chromatography (as the dimethyl ester) . {U-14C}Shikimic acid also gave rise to radioactive OSB if the cofactors necessary for enzymatic conversion to chorismic acid were added . Use of 2-{1-14C}ketoglutaric acid does not give rise to labeled OSB . In the absence of TPP during the incubations, OSB synthesis was much reduced; these observations are consistent with the proposed role for the succinic semialdehyde-TPP anion as the reagent adding to chorismic acid . Extracts of cells from menC and menD mutants did not form OSB separately, but did so in combination . There was evidence for formation of a product, X, by extracts of a menC mutant incubated with chorismic acid, TPP, and 2-ketoglutaric acid; X was converted to OSB by extracts of a menD mutant . It appears that the intermediate, X, is formed by one gene product and converted to OSB by the second gene product.

J Bacteriol, 1983 Feb, 153(2), 693 - 9
Identification and characterization of the TolC protein, an outer membrane protein from Escherichia coli; Morona R et al.; We used the cloned tolC gene to identify, locate, and purify its gene product . Strains carrying pPR13 or pPR42 overproduced a cell envelope protein (molecular weight, 52,000) . A protein of the same molecular weight was identified in radioactively labeled minicells carrying pPR13; this protein was absent in pPR11-carrying minicells . This protein was the tolC gene product, since pPR11 differed from pPR13 in having a Tn10 insertion in the tolC gene . The protein seen in cell envelopes of whole cells (TolC protein) was found to exist in an aggregated state in the outer membrane; under conditions in which OmpC and OmpF were peptidoglycan associated, TolC protein was not likewise associated . Using these properties, we purified the TolC protein and determined the sequence of twelve amino acids from the amino-terminal end . The location of the TolC protein in the outer membrane was consistent with the proposed function for the tolC gene product as a processing protein in the outer membrane.

J Bacteriol, 1983 Feb, 153(2), 597 - 603
Molecular cloning and expression of a gene that controls the high-temperature regulon of Escherichia coli; Neidhardt FC et al.; The high-temperature production (HTP) regulon of Escherichia coli consists of a set of operons that are induced coordinately by a shift to a high temperature under the control of a single chromosomal gene called htpR or hin . To identify more components of this regulon, the rates of synthesis of many polypeptides resolved on two-dimensional polyacrylamide gels were measured in various strains by pulse-labeling after a temperature shift-up . A total of 13 polypeptides were found to be heat inducible only in cells bearing a normal htpR gene on the chromosome or on a plasmid; on this basis these polypeptides were designated products of the HTP regulon . Several hybrid plasmids that contain segments of the E . coli chromosome in the 75-min region were found to carry the htpR gene . A restriction map of this region was constructed, and selected fragments were subcloned and tested for the ability to complement an htpR mutant . The polypeptides encoded by these fragments were detected by permitting expression in maxicells, minicells, and chloramphenicol-treated cells . Complementation was accompanied by production of a polypeptide having a molecular weight of approximately 33,000 . This polypeptide, designated F33.4, was markedly reduced in amount in an htpR mutant expected to contain very little htpR gene product . Polypeptide F33.4 is postulated to be the product of htpR and to be an effector that controls heat induction of the HTP regulon.

J Bacteriol, 1983 Feb, 153(2), 1120 - 3
Indolmycin-mediated inhibition and stimulation of transcription at the trp promoter of Escherichia coli; Bogosian G et al.; Escherichia coli cells harboring a non-attenuated trp-lac operon fusion were used to evaluate the effects of indolmycin on the initiation of transcription at the trp promoter . Indolmycin caused repression in trpR+ strains and in trpR deletion mutants, although higher effector concentrations were required in the latter situation . Plasmid-mediated elevation in tryptophanyl-tRNA synthetase reversed the inhibitory effect of indolmycin . Indolmycin did not facilitate the binding of purified Trp repressor protein to trp operator DNA.

J Bacteriol, 1983 Feb, 153(2), 1079 - 82
Escherichia coli xth mutants are hypersensitive to hydrogen peroxide; Demple B et al.; Escherichia coli mutants lacking exonuclease III (xthA) are exceptionally sensitive to hydrogen peroxide . They are killed by H2O2 at 20 times the rate of wild-type bacteria and at 3 to 4 times the rate of recA cells . This is the first clear phenotypic sensitivity reported for xth- E . coli and should aid in clarifying peroxide-induced lethality and the in vivo role of exonuclease III.

Gastroenterology, 1983 Feb, 84(2), 276 - 80
Brief prophylaxis with doxycycline for the prevention of traveler's diarrhea; Freeman LD et al.; A prospective, randomized double-blind trial of doxycycline prophylaxis for traveler's diarrhea was conducted on 145 volunteers during a 2.5-day visit to Mexico . Traveler's diarrhea occurred in 15 (21%) of the placebo group and in 3 (4%) of the doxycycline group (p = 0.002) . There was no rebound increase in the incidence of acute diarrhea after departure from the high risk area in the doxycycline-treated group . A variety of bacterial pathogens were isolated from individuals symptomatic with traveler's diarrhea . Nausea alone (8%) or nausea with vomiting (4%) occurred in the doxycycline-treated group only and were the only side effects observed (p = 0.003) . We conclude that doxycycline is safe and efficacious for the prophylaxis of traveler's diarrhea for short-term exposure in a high risk area.

Proc Natl Acad Sci U S A, 1983 Feb, 80(4), 906 - 10
Purification of biologically active simian virus 40 small tumor antigen; Bikel I et al.; The simian virus 40 small tumor antigen (t antigen) gene has been cloned downstream from a hybrid Escherichia coli trp-lac promoter and a suitable ribosome binding site . A bacterial clone (865i) transformed by such a plasmid (pTR865) expresses this gene and, under optimal conditions, can produce greater than or equal to 5% of its total protein as t antigen . Soluble extracts of such a clone were relatively depleted in t antigen, which was found in the initial pellet fraction . The protein was recovered from this fraction in a significantly purified form by extraction with urea-containing buffer . After gel filtration of such t antigen-enriched solutions, highly purified protein was obtained . When either this fraction (freed of urea) or NaDodSO4 gel-purified 865i t antigen (rendered free of detergent) was injected into untransformed rat cells, dissolution of intracellular actin cable networks was observed.

Proc Natl Acad Sci U S A, 1983 Feb, 80(4), 1058 - 62
Construction of a shuttle vector for the filamentous fungus Neurospora crassa; Stohl LL et al.; We have constructed a recombinant plasmid, pALS-1, that replicates autonomously in both Neurospora and Escherichia coli . pALS-1 consists of the mitochondrial plasmid from Neurospora strain P405-Labelle, the Neurospora qa-2+ gene, and E . coli plasmid pBR325 . pALS-1 transforms the Neurospora qa-2+ gene at frequencies 5- to 10-fold higher than those for plasmids that transform mainly by integration . When E . coli was transformed with DNA from Neurospora transformants, we recovered not only pALS-1 but also a smaller plasmid, pALS-2, which had undergone deletion of most and possibly all Labelle sequences, and the immediately flanking sequences in pBR325 . pALS-2 also appears to replicate autonomously in Neurospora, but less efficiently than does pALS-1 . Southern blots show that free pALS-1 and pALS-2 are present in nuclear and cytosolic (supernatant from high-speed centrifugation) fractions of Neurospora transformants and that small, variable proportions of the plasmids also can be detected in mitochondria . pALS-1 and pALS-2 constitute putative shuttle vectors for Neurospora.

J Gen Microbiol, 1983 Feb, 129 (Pt 2), 337 - 48
Phosphotransferase-mediated regulation of carbohydrate utilization in Escherichia coli K12: the nature of the iex (crr) and gsr (tgs) mutations; Parra F et al.; Mutants of Escherichia coli K12 defective in the gene iex (crr) no longer utilize glucose or N-acetylglucosamine in preference to lactose, but competition between either of these sugars and another that also enters by a phosphotransferase (PT) mechanism is not affected . In this they differ from gsr (tgs) mutants . In gsr mutants, glucose does not exclude any other sugar, though N-acetylglucosamine still does so . In gsr mutants that are also ptsM the phosphoenolpyruvate-dependent phosphorylation of glucose or methyl alpha-glucoside is reduced by 90%: N-acetylglucosamine phosphorylation is not affected . The iex mutation does not affect the phosphorylation of either of these compounds . The wild-type alleles iex+ and gsr+ are dominant in lambda heterozygotes . Glucose inhibits the lactose permease of wild-type cells, but only when the permease is present in low amounts . The inhibition is also relieved (1) by induction of another transport system that is subject to regulation by the iex system or (2) by an iex mutation . We suggest that the iex gene specifies a protein that, in cells transporting certain sugars by a PT mechanism, acts to inhibit active transport systems . The protein is present in limiting concentration in the cell, sufficient only to inhibit the basal, uninduced, level of the active transport systems . In consequence the inducer (or its precursor) may be excluded from the cell and induction thus prevented.

J Biochem (Tokyo), 1983 Feb, 93(2), 339 - 47
New 434-specific DNA binding protein copurified with the 434 tof protein from lambda imm434cI dv carrier cells of Escherichia coli; Aono J et al.; A tof-like protein that has 434-specific DNA binding activity has been copurified with the 434 tof protein from lambda imm434cI dv carrier cells . The apparent molecular weight of the new 434-specific DNA binding protein is 9,000 to 9,500, a little higher than that of the 434 tof protein, as estimated by SDS gel electrophoresis . Amino acid analysis revealed the protein to be an arginine-rich component whereas the 434 tof protein is a lysine-rich component . The specific binding reaction of the new protein to lambda imm434dv DNA is distinct from that of the 434 tof protein in respect to the sigmoid shape of the binding curve and to the temperature dependency . This suggests that the specific binding to lambda imm434dv DNA observed with the new protein is due not to a trace of the 434 tof protein contaminating the new protein preparation but rather to the new protein itself . The NH2-terminal 11 residues of the new 434-specific DNA binding protein were sequenced by manual Edman degradation . This technique revealed that the new protein is not a fragment of the 434 tof, cII, or O protein or an NH2-terminal fragment of the cI repressor . The origin and the physiological roles of the new 434-specific DNA binding protein remain unknown.

Mol Cell Biol, 1983 Feb, 3(2), 280 - 9
A cDNA cloning vector that permits expression of cDNA inserts in mammalian cells; Okayama H et al.; This paper describes a plasmid vector for cloning cDNAs in Escherichia coli; the same vector also promotes expression of the cDNA segment in mammalian cells . Simian virus 40 (SV40)-derived DNA segments are arrayed in the pcD vector to permit transcription, splicing, and polyadenylation of the cloned cDNA segment . A DNA fragment containing both the SV40 early region promoter and two introns normally used to splice the virus 16S and 19S late mRNAs is placed upstream of the cDNA cloning site to ensure transcription and splicing of the cDNA transcripts . An SV40 late region polyadenylation sequence occurs downstream of the cDNA cloning site, so that the cDNA transcript acquires a polyadenylated 3' end . By using pcD-alpha-globin cDNA as a model, we confirmed that the alpha-globin transcript produced in transfected cells is initiated correctly, spliced at either of the two introns, and polyadenylated either at the site coded in the cDNA segment or at the distal SV40 polyadenylation signal . A cDNA clone library constructed with mRNA from SV40-transformed human fibroblasts and this vector (about 1.4 X 10(6) clones) yielded full-length cDNA clones that express hypoxanthine-guanine phosphoribosyltransferase (Jolly et al., Proc . Natl . Acad . Sci . U.S.A., in press).

J Virol, 1983 Feb, 45(2), 773 - 81
Expression of a recombinant DNA gene coding for the vesicular stomatitis virus nucleocapsid protein; Sprague J et al.; A cDNA clone containing the entire vesicular stomatitis virus nucleocapsid gene was assembled by fusing portions of two partial clones . When the cDNA clone was inserted into a new general-purpose eucaryotic expression vector and introduced into appropriate host cells, abundant N-protein synthesis ensued . The expressed protein was indistinguishable from authentic N protein produced during vesicular stomatitis virus infections . The recombinant N protein was recognized by a polyclonal antibody and two different monoclonal antibodies and could not be resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis from authentic N . Our results suggest that the recombinant N protein produced in transfected cells rapidly aggregates into high-molecular-weight complexes in the absence of vesicular stomatitis virus genomic RNA.

J Gen Virol, 1983 Feb, 64 (Pt 2), 415 - 9
Antiviral effects of human fibroblast interferon from Escherichia coli against encephalomyocarditis virus infection of squirrel monkeys; Weck PK et al.; Recombinant DNA methodology has allowed the production of human fibroblast interferon (IFN-beta) from Escherichia coli and this material, in highly purified form, has been shown to reduce viraemia and mortality in encephalomyocarditis (EMC) virus-infected squirrel monkeys . These effects are dose related: six treatments over 4 days at 10(6) U/kg and 3 x 10(3) U/kg have comparable efficacy, whereas treatments at 10(3) U/kg are ineffective . The recombinant DNA-derived IFN-beta appears to be as effective as natural fibroblast cell-derived IFN-beta and both materials are effective by the intramuscular or intravenous routes . Thus, even though previous studies have shown that low circulating concentrations of IFN-beta are observed after intramuscular injections, the present data indicate that this slow release from the muscle can still confer protection.

Infect Immun, 1983 Feb, 39(2), 559 - 64
Enhanced superoxide anion release from phagocytes by muramyl dipeptide or lipopolysaccharide; Kaku M et al.; Muramyl dipeptide (MDP) and lipopolysaccharide (LPS) from Escherichia coli were tested for the ability to influence superoxide anion (O2-) release from guinea pig phagocytes . Both MDP and LPS alone did not, by themselves, stimulate O2- release by macrophages and polymorphonuclear leukocytes . However, the preincubation of macrophages with MDP or LPS primed the macrophages to release an enhanced amount of O2- when stimulated by cytochalasin E and wheat germ agglutinin . When polymorphonuclear leukocytes were treated in the same way, only LPS showed an enhancing effect . MDP enhanced NADPH oxidase activity of macrophages, which is probably the reason for enhanced O2- release by MDP.

Proc Natl Acad Sci U S A, 1983 Feb, 80(3), 792 - 6
Structural requirement for IS50-mediated gene transposition; Berg DE; Replicative transposition is signaled by the formation of cointegrates in which donor and target replicons are joined by direct repeats of a transposable element . Elements not generating such cointegrates may move by a conservative breaking and joining process . The IS50 elements forming the terminal repeats of Tn5 {which carries the determinant for kanamycin resistance (Kanr)} contain genes and sites necessary for transposition and mediate the movement of any DNA segment they bracket . To determine if IS50 generates cointegrates, the products of transposition from pBR322::Tn5 plasmids to an F factor in recA-Escherichia coli were examined . With monomeric pBR322::Tn5 plasmids, transposition of Kanr (from Tn5) was generally not accompanied by movement of the determinant for ampicillin resistance (Ampr) (from the pBR322 vector) . With dimeric pBR322::Tn5 plasmids, by contrast, half of the transpositions of kanr were accompanied by transposition of ampr . Restriction endonuclease analyses indicated that these F-Kanr Ampr chimeras contained inserts of a single copy of the pBR322 vector sequence bracketed by one Tn5 element and one IS50 element or by a pair of Tn5 elements . None of 79 chimeras tested was a true cointegrate . Because IS50 seems to move only a segment of the donor replicon it is proposed that IS50 transposition is conservative.

Cell, 1983 Feb, 32(2), 351 - 60
Partition mechanism of F plasmid: two plasmid gene-encoded products and a cis-acting region are involved in partition; Ogura T et al.; Plasmids that replicate using the replication origin (oriC) of the E . coli chromosome are not stably inherited through cell division, but can be stabilized by joining with a particular segment of F plasmid that presumably provides the partition function . The segment necessary for stabilization has been located within a 3.0 kb segment outside of the region essential for autonomous replication of the F plasmid . This segment contains three functionally distinct regions: two of them (designated sopA and sopB) specify gene products that act in trans, whereas the third region (sopC) acts in cis . All three functions seem to be essential for normal partition of the plasmid into daughter cells during cell division . The cis-acting region also specifies plasmid incompatibility.

J Bacteriol, 1983 Feb, 153(2), 921 - 9
Analysis of tetracycline resistance encoded by transposon Tn10: deletion mapping of tetracycline-sensitive point mutations and identification of two structural genes; Coleman DC et al.; Deletions in the tet genes derived from Tn10 were formed from different tet::Tn5 insertion mutations by removing DNA sequences located between a HindIII site in Tn5 and a HindIII site adjacent to the tet genes . Tetracycline-sensitive point mutations were mapped in recombination tests with the deletions and were thus aligned with the genetic and physical map of the tet region . Plasmids carrying point mutations were tested for complementation with derivatives of pDU938, a plasmid carrying cloned tet genes derived from Tn10 which had been inactivated by Tn5 insertions . Complementation occurred between promoter-proximal tet point mutations and distal tet::Tn5 insertions, suggesting the existence of two structural genes, tetA and tetB . These results, together with the analysis of polypeptides in minicells harboring pDU938tet::Tn5 mutants, suggested that tetA and tetB are expressed coordinately in an operon . The tetB gene encodes the previously characterized 36,000-dalton cytoplasmic membrane TET protein, but the product of tetA was not identified . Point mutations in either tetA or tetB led to the defective expression of the resistance mechanism involving tetracycline efflux . It is suggested that the tetA and tetB products interact cooperatively in the membrane to express resistance.

J Bacteriol, 1983 Feb, 153(2), 903 - 8
Physical and genetic characterization of the cloned sbcB (exonuclease I) region of the Escherichia coli genome; Prasher D et al.; A 17-kilobase (kb) HindIII fragment containing the structural gene for exonuclease I (sbcB) from Escherichia coli K-12 was physically and genetically characterized . The monomeric molecular weight of exonuclease I was 53,700, based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis of 35S-labeled E . coli mini- and maxicells . The gene was in close proximity to two unidentified proteins with molecular weights of 15,200 and 13,100 . No other polypeptides appeared to be constitutively synthesized from the 17-kb fragment . Genetically, no portion of the histidine operon or the shikimic acid transport gene (shiA) was detected on the fragment . Although the entire 17-kb fragment in the vector pMB9 was too unstable to be useful, a 7.6-kb BamHI-EcoRI fragment inserted into a variety of vectors was stable . A detailed restriction map of the fragment is presented . Several derivatives in the runaway-replication vectors pMB06 and pMOB45 yielded 20- to 52-fold increases in exonuclease I activity after a switch in growth temperature to 40 degrees C . Of six exonuclease I mutants examined by DNA-DNA hybridization, one (xonA6) appeared to have arisen from a 1.2-kb insertion into the structural gene for exonuclease I.

J Bacteriol, 1983 Feb, 153(2), 872 - 7
Identification and control of synthesis of the dsdC activator protein; McFall E et al.; Operon fusions between the D-serine deaminase regulatory and structural genes and lacZ were constructed and used to examine the control of expression of the positive regulatory gene, dsdC . Merodiploid strains containing both dsdCp::Mu d (lac Apr) and dsdC+A+ produced only one-fourth as much beta-galactosidase as did the haploid dsdCp::Mu d (lac Apr) strains, indicating that the dsdC+ product repressed its own synthesis . The repression was reversed by D-serine . dsdC expression was not depressed in a cya background . The basal level of D-serine deaminase was the same in wild-type and dsdCp fusion strains . The dsdC gene product was identified in maxicell strains harboring dsd plasmids as a 34,000-dalton protein . dsdC gene transcription proceeded clockwise; thus, its promoter is adjacent to that of dsdA.

J Bacteriol, 1983 Feb, 153(2), 747 - 55
Construction of hybrid plasmids containing the Escherichia coli uxaB gene: analysis of its regulation and direction of transcription; Blanco C et al.; The uxaB gene of Escherichia coli, encoding for altronate oxidoreductase involved in the hexuronate degradative pathway, was isolated on a ColE1-uxaB hybrid plasmid from the Clarke and Carbon bank . The restriction map of this plasmid was established . The uxaB gene was mapped on a 1.5-megadalton HindIII-KpnI DNA fragment . Use of an in vitro gene fusion between uxaB and lacZ genes led to the determination that uxaB is transcribed from the KpnI towards the HindIII restriction sites . Gene amplification in cells containing various uxaB hybrid plasmids allowed us to show a gradation in the level of repression of exu operator sites by the exuR regulatory gene product.

J Bacteriol, 1983 Feb, 153(2), 731 - 8
pH-sensitive CDP-diglyceride synthetase mutants of Escherichia coli: phenotypic suppression by mutations at a second site; Ganong BR et al.; In Escherichia coli, mutations which lower the level of CDP-diglyceride synthetase are designated cds and map at min 4 . The cds-8 mutation resulted in strikingly defective enzyme activity and also rendered cells pH sensitive for growth . Both the inhibition of growth and the massive accumulation of phosphatidic acid which occur in a cds-8 mutant at pH 8 were suppressed by mutations at a second locus, designated cdsS, which mapped between argG and gltB near min 68 . The cdsS3 mutation by itself did not affect CDP-diglyceride synthetase activity in wild-type cells, but it caused a twofold stimulation of the residual activity present in strains harboring cds-8 . Both the insensitivity to pH and the twofold stimulation of residual activity were lost by introduction of an F' strain carrying cdsS+ into a recA1 cds-8 cdsS3 host . When a culture of a cds-8 cdsS+ strain was shifted to pH 8, the residual specific activity of synthetase dropped by 75% within 100 min . In a cds-8 cdsS3 double mutant under the same conditions, the activity declined appreciably less, about to the level found in the cds-8 cdsS+ strain under permissive conditions (pH 6) . Thus, it appears that mutations in the cdsS gene suppress the pH sensitivity of cds mutants by inhibiting the decay of residual CDP-diglyceride synthetase activity at the nonpermissive pH . The cdsS locus appears to be distinct from any known nonsense or missense suppressor.

J Bacteriol, 1983 Feb, 153(2), 722 - 30
Multiple genes for membrane-bound phosphatases in Escherichia coli and their action on phospholipid precursors; Icho T et al.; We have devised a coupled radiochemical assay for detecting phosphatidylglycerolphosphate (PGP) phosphatase activity in Escherichia coli colonies immobilized on filter paper . There appeared to be at least two enzymes capable of dephosphorylating PGP, as judged by the characterization of mutations in two genes designated pgpA and pgpB . The former is located near min 10 and is cotransducible with proC and dnaZ . The latter is situated near min 28 and is closely linked to cysB . The available mutant alleles of pgpA reduced the specific activity of PGP phosphatase in crude extracts by about 30%, but they had no effect on phosphatidic acid (or lysophosphatidic acid) phosphatase . Mutants altered in the pgpB locus inactivated most of the residual PGP phosphatase activity present in single-step pgpA mutants, and the level of phosphatidic acid phosphatase was also reduced 20-fold . The available mutations in pgpA and pgpB elevated the cellular PGP pool by 10- to 50-fold . The maximal PGP levels never exceeded 5%, and these strains were not conditionally lethal . The simplest interpretation of our findings is that there are at least two membrane-associated phosphatases in E . coli, both distinct from alkaline phosphatase . The pgpA gene product is specific for PGP, whereas the pgpB gene product also acts on phosphatidic acid and lysophosphatidic acid.

J Bacteriol, 1983 Feb, 153(2), 610 - 5
Origin of Escherichia coli K-12 Hfr B7; Virolle MJ et al.; Several F' plasmids encoding resistance to tetracycline have been derived from a trg::Tn10 Hfr B7 strain of Escherichia coli K-12 . One of these plasmids, JGF312, was analyzed by restriction endonuclease digestion and Southern blot hybridization to cloned chromosomal fragments . This analysis revealed that JGF312 was formed by Tn10-promoted deletion from the Tn10 insertion (31.4 min) to within the prophage rac at 30.1 min . Hfr B7 was shown to result from recombination between IS2 of F delta (33-43) and a chromosomal IS2 located within the rac-man region at 30.9 min on the genetic map.

J Bacteriol, 1983 Feb, 153(2), 604 - 9
Molecular cloning of the region of the terminus of Escherichia coli K-12 DNA replication; Bejar S et al.; A series of plasmids have been isolated either by ligation of defined restriction fragments to plasmid pBR325 or by screening of a cosmid bank by in situ colony hybridization . Together with one previously isolated plasmid, they spanned 86% of the 30.5- to 34-min region of the genetic map of Escherichia coli K-12 . Physical analysis of these plasmids and hybridizations to Southern blots confirmed the endonuclease map of this region, with the exception of a 9.3-kilobase pair inversion.

J Bacteriol, 1983 Feb, 153(2), 588 - 96
Cloning, mapping, and expression of the fumarase gene of Escherichia coli K-12; Guest JR et al.; Two classes of fumarase-transducing phages, lambda fumA and lambda fumB, were isolated from populations of recombinant phages containing HindIII fragments of Escherichia coli DNA; they were isolated by virtue of their ability to complement the metabolic lesion of a fumarase-negative mutant . The strongly complementing lambda fumA phages contained a 6.2-kilobase HindIII fragment encoding: the fumA gene, located at 35.5 min in the E . coli linkage map and expressing the major fumarase activity; the mannosephosphate isomerase gene, manA; and an unidentified gene, g48 . The three genes were located relative to the restriction map of the cloned fragment and the genetic linkage map (terC-g48-fumA-manA-uidAoR), their transcription polarities were defined as anticlockwise in the chromosome, and the molecular weights of the corresponding gene products were established: fumA, 61,500; manA, 42,000; g48, 48,000 . Organisms containing the fumA gene sub-cloned in multicopy plasmids overproduced fumarase up to 50-fold . The weakly complementing class of transducing phages, lambda fumB, contained several genes in an 8.2-kilobase HindIII fragment, including one (fumB) that determines a minor fumarase activity . Complementation by fumB was only observed in high-copy situations such as transduction plaques and in strains containing a multicopy plasmid in which 40% of normal fumarase activity was detected . The basis for the complementation by fumB was not defined.

J Bacteriol, 1983 Feb, 153(2), 1111 - 3
Cloning of the aerobactin-mediated iron assimilation system of plasmid ColV; Bindereif A et al.; The high-affinity iron assimilation system of plasmid ColV-K30 was cloned on the vector plasmid pPlac . Plasmid pABN1 was isolated by means of sensitivity to cloacin, a bacteriocin using the same outer membrane receptor as ferric aerobactin . Restriction maps were determined for this plasmid and for a subclone, pABN5 . Plasmid pABN1 codes for the complete gene complex, whereas plasmid pABN5 encodes only the biosynthetic genes for aerobactin . Regulation of the uptake system by iron is retained in cloned sequences of pABN1.

J Bacteriol, 1983 Feb, 153(2), 1072 - 4
Role of the sfiA-dependent cell division regulation system in Escherichia coli; Huisman O et al.; Several authors have suggested that the SOS-associated (sfiA-dependent) system of division inhibition, normally induced by perturbations of DNA replication, also regulates steady-state (unperturbed) cell division . The present work shows that mean cell mass is identical in sfiA+ and sfiA mutant cultures during steady-state growth, that mass adjustment is identical after shift up, that sfiA expression is not induced by shift up, and that a sfiA mutation does not cause aberrant chromosome segregation.

Acta Pathol Microbiol Immunol Scand {B}, 1983 Feb, 91(1), 43 - 7
The use of frozen erythrocytes in macrophage studies . 2 . Attachment and phagocytosis mediated by the two immunological receptors of the macrophages; Myhrvold V et al.; Sheep erythrocytes opsonized with IgG or C3b were frozen in various cryoprotective agents, thawed, and compared to corresponding unfrozen erythrocytes exposed to the cryoprotectants and to unfrozen erythrocytes not exposed to the cryoprotectants (controls) as test particles in macrophage attachment and phagocytosis assays . Fc-receptor-mediated attachment and phagocytosis were not influenced by the use of any cryoprotective agent tested or by freezing the erythrocytes . This was also the case with C3b-receptor-mediated attachment . Phagocytosis via this receptor was negligible in normal macrophages, but tended to be slightly more effective when the test particles had been treated with cryoprotective agents . In vitro stimulation of the macrophages with Escherichia coli endotoxin, however, triggered the capacity to internalize treated and untreated erythrocytes equally.

Immunopharmacology, 1983 Feb, 5(3), 183 - 95
Comparison of the effects of glucocorticoid and indomethacin treatment on the acute inflammatory reaction in rabbits; Issekutz AC; We have recently shown that indomethacin and ASA diminish the elevated blood flow, protein exudation, and leukocyte infiltration during acute inflammation induced by killed Escherichia coli, the reversed Arthus reaction, or zymosan-activated plasma (ZAP; C5ades-arg) in rabbit skin . All of these effects were likely due to the inhibition by these drugs of prostaglandin (PG) synthesis in the lesions . Because glucocorticoids are also reported to inhibit PG production and, in large doses, to suppress inflammation accompanying various clinical conditions, we investigated the effects of hydrocortisone (HC), and methylprednisolone (MP), administered in large doses (100 mg/m2/d of MP or 2.5 g/m2/d of HC) on the above three forms of acute inflammation in rabbits . The effect of indomethacin treatment was studied in parallel for comparison . Blood flow, protein exudation, and leukocyte infiltration were quantitated simultaneously with 86Rb Cl, 125I-labelled rabbit albumin and 51Cr labelled blood leukocytes . Systemic indomethacin therapy decreased the blood flow and permeability, while local indomethacin (2.5 micrograms) significantly inhibited leukocyte infiltration into the lesions . In contrast, HC and MP caused only a mild decrease in blood flow, without altering protein exudation or leukocyte influx . However, HC and MP did inhibit protein exudation induced by bradykinin or histamine injection . These results indicate that, at least in rabbits, HC and MP, in contrast to indomethacin, have very weak anti-inflammatory actions on three complement- and neutrophil-mediated inflammatory responses, i.e., E . coli, ZAP (C5ades-arg) and reversed Arthus reactions.

Arch Biochem Biophys, 1983 Feb 1, 220(2), 398 - 404
Conformational change of the alpha subunit of Escherichia coli F1 ATPase: ATP changes the trypsin sensitivity of the subunit; Senda M et al.; Conformational change in the alpha subunit of Escherichia coli proton-translocating ATPase was studied using trypsin . The subunit was cleaved with a small amount of trypsin (1 microgram/mg subunit) to peptides of less than 8000 daltons . On the other hand, the subunit was cleaved to two main polypeptides (30,000 and 25,000 daltons) in the presence of sufficient ATP (1 mM-0.5 microM) to saturate the high-affinity site of the subunit . Analysis of digests of the subunit combined with fluorescent maleimide suggested that the subunit was digested in the middle of the polypeptide chain in the presence of the nucleotide . ADP and adenylyl imidodiphosphate had the same effect as ATP . These results suggest that the conformation of the subunit changed to form two trypsin-resistant domains upon binding of ATP to the high-affinity site.

J Bacteriol, 1983 Feb, 153(2), 716 - 21
Cloning and expression of the cloacin DF13/aerobactin receptor of Escherichia coli (ColV-K30); Krone WJ et al.; A DNA fragment derived from the ColV-K30 plasmid and coding for both sensitivity to cloacin DF13 and Fe3+-aerobactin uptake was cloned into pBR322 . The cloned fragment coded for two polypeptides with molecular masses of 74,000 (the cloacin DF13/aerobactin receptor protein) and 50,000 daltons, respectively . When grown with sufficient iron, cells harboring pFS8 (with this fragment) possessed about 10 times as many receptor protein molecules as compared with cells of Escherichia coli (ColV-K30) . The synthesis of the receptor protein specified by pFS8, however, was independent of the availability of iron, in contrast to strains harboring the intact ColV-K30 plasmid . Aerobactin was taken up but not synthesized by cells harboring pFS8 . No growth occurred when iron-starved cultures of these cells were incubated with Fe3+-aerobactin, suggesting that expression of other ColV-K30-encoded genes is necessary to remove the iron from the Fe3+-aerobactin complex.

J Bacteriol, 1983 Feb, 153(2), 1027 - 37
Identification and localization of enzymes of the fumarate reductase and nitrate respiration systems of escherichia coli by crossed immunoelectrophoresis; van der Plas J et al.; Crossed immunoelectrophoresis was used to analyze the components of membrane vesicles of anaerobically grown Escherichia coli . The number of precipitation lines in the crossed immunoelectrophoresis patterns of membrane vesicles isolated from E . coli grown anaerobically on glucose plus nitrate and on glycerol plus fumarate were 83 and 70, respectively . Zymogram staining techniques were used to identify immunoprecipitates corresponding to nitrate reductase, formate dehydrogenase, fumarate reductase, and glycerol-3-phosphate dehydrogenase in crossed immunoelectrophoresis reference patterns . The identification of fumarate reductase by its succinate oxidizing activity was confirmed with purified enzyme and with mutants lacking or overproducing this enzyme . In addition, precipitation lines were found for hydrogenase, cytochrome oxidase, the membrane-bound ATPase, and the dehydrogenases for succinate, malate, dihydroorotate, D-lactate, 6-phosphogluconate, and NADH . Adsorption experiments with intact and solubilized membrane vesicles showed that fumarate reductase, hydrogenase, glycerol-3-phosphate dehydrogenase, nitrate reductase, and ATPase are located at the inner surface of the cytoplasmic membrane; on the other hand, the results suggest that formate dehydrogenase is a transmembrane protein.

Anal Biochem, 1983 Feb 1, 128(2), 415 - 21
Spot blot: a hybridization assay for specific DNA sequences in multiple samples; Cunningham M; Small aliquots of DNA-containing samples from gradient or column fractions are spotted onto agarose plates containing ethidium bromide, and the DNA is visualized qualitatively . After it is overlaid with a thin agarose cover, the DNA is denatured and transferred to nitrocellulose filters for hybridization . This method, called the "spot-blot assay," is particularly convenient when dealing with large numbers of samples.

J Virol, 1983 Feb, 45(2), 859 - 63
Production and characterization of monoclonal antibodies against avian retrovirus reverse transcriptase; Weis J et al.; Monoclonal antibodies were prepared against the avian myeloblastosis virus reverse transcriptase . These monoclonal antibodies specifically immunoprecipitated the alpha and beta subunits of the reverse transcriptase molecule, as well as the Pr180gag-pol precursor protein present in virus-infected cells . In addition, these monoclonal antibodies inhibited the DNA polymerase activity associated with the reverse transcriptase molecule but not the RNase H activity . The monoclonal antibody preparations were specific for the amino-terminal portion of the protein, as determined by the immunoprecipitation of a reverse transcriptase-beta-galactosidase fusion protein produced in Escherichia coli by molecular cloning procedures.

Infect Immun, 1983 Feb, 39(2), 623 - 9
Serological, chemical, and structural analyses of the Escherichia coli cross-reactive capsular polysaccharides K13, K20, and K23; Vann WF et al.; The Escherichia coli K13, K20, and K23 capsular polysaccharide antigens are serologically related . All of these polysaccharides contain ribose and 2-keto-3-deoxyoctonate in equimolar quantities . The K13 and K20 polysaccharides are partially O-acetylated . A comparison of these polysaccharides after O-deacetylation, by nuclear magnetic resonance and permethylation analysis, showed that these polysaccharides contained the disaccharide repeat unit leads to)-beta-ribofuranosyl-(1 leads to 7)-beta-2-keto-3-deoxyoctonate . They differed in the presence and location of an acetyl moiety . The K13 polysaccharide was O-acetylated at C-4 of the 2-keto-3-deoxyoctonate . The K20 antigen was O-acetylated at C-5 of the ribose moiety . The K23 polymer was nonacetylated . The cross-reactivity of these antigens was demonstrated by tandem-crossed immunoelectrophoresis . Antibodies to K23 could be completely absorbed from OK K23 serum by K13, K20, and K23 antigenic extracts . The K13 and K20 antibodies could be completely absorbed from their respective antisera only by homologous antigenic extracts . Monoclonal antibodies were prepared against a protein conjugate of the K13 polysaccharide . Analyses of the reactions of these antibodies with the three polysaccharides suggest that the K13 polysaccharide has at least three antigenic sites, one of which is common to the K13, K20, and K23 polysaccharides.

Cell, 1983 Feb, 32(2), 361 - 9
Mutations in the rpIJ leader of Escherichia coli that abolish feedback regulation; Friesen JD et al.; We have isolated mutants that fail to exhibit biosynthetic feedback regulation of a rpIJ-lacZ fusion . Analysis of these mutants and of others that were isolated earlier indicates that crucial sequences for both translational feedback regulation and efficient translation lie closely intermingled in the central region of the rpIJ mRNA leader 70-195 bases upstream from the translation start of rpIJ . We suggest that our point mutations define a region of the rpIJ leader mRNA to which L10 binds in effecting autogenous translational regulation.

J Bacteriol, 1983 Feb, 153(2), 955 - 61
Localization of the major antigenic determinant of EDP208 pili at the N-terminus of the pilus protein; Worobec EA et al.; Trypsin digestion of pilin monomers from EDP208 conjugative pili causes cleavage of Lys12 to yield an N-terminal dodecapeptide, ET1 (Mr approximately equal to 1,500), and the remaining C-terminal fragment, ER (Mr approximately equal to 10,000) . Using the amino acid sequence for ET1 provided by Frost et al . (J . Bacteriol . 153:950-954), we synthesized the N-terminal dodecapeptide chemically, conjugated it to bovine serum albumin, and subjected it to immunological studies . Antisera prepared against intact EDP208 pili as well as against the synthetic ET1-BSA conjugate were used in experiments involving an enzyme-linked immunosorbant assay and electrophoretic transfer of proteins from sodium dodecyl sulfate-polyacrylamide gels to nitrocellulose sheets . Both experimental approaches showed strong reactivity between the synthetic dodecapeptide and antiserum raised against whole pili . It was also found that antiserum raised against the synthetic peptide was reactive against intact pilus protein, indicating that the N-terminal dodecapeptide is an important antigenic determinant of the EDP208 pilus protein . Additional studies showed that the C-terminal fragment, ER, may contain one or two additional antigenic sites.

J Bacteriol, 1983 Feb, 153(2), 672 - 84
Efficient read-through of Tn9 and IS1 by RNA polymerase molecules that initiate at rRNA promoters; Siehnel RJ et al.; Transcription and translation are coupled in most Escherichia coli operons . As a consequence, ribosomes must be present on an mRNA molecule while transcription of the mRNA is in progress or else premature termination of transcription may result . This requirement is most clearly manifested when premature nonsense codons result in polarity in multicistronic operons . Polarity can also result from insertions of transposons and insertion sequences . However, since rRNA operons are not translated, some property of these operons must allow transcription to be uncoupled from translation . In this paper we demonstrate that transposon Tn9 and insertion sequence IS1 are nonpolar or incompletely polar in rRNA operons during normal growth . We also show that essentially all expression of rrn sequences distal to IS1 and Tn9 results from transcripts that originate at rRNA promoters . These results suggest either that rRNA operons possess some mechanism which reduces or prevents termination within rRNA operons or that Tn9 and IS1 can be very inefficient at blocking normal transcription . Insertions of Tn10 in rRNA operons are substantially but incompletely polar . We could not determine whether the residual downstream transcription observed results from promoters within Tn10 or from read-through of Tn10 . We discuss the meaning of read-through of Tn9 and IS1 and the residual expression of genes downstream from Tn10 with regard to rRNA operon structure and previous experiments in which polarity of transposons or insertion sequences was observed in protein-encoding operons.

J Bacteriol, 1983 Feb, 153(2), 616 - 26
Characterization of RNA synthesis in an Escherichia coli mutant with a temperature-sensitive lesion in stable RNA synthesis; Williams DE et al.; Previous experiments with Escherichia coli strain 2S142 have shown that the synthesis of stable RNA is preferentially blocked at the restrictive temperature . In this paper, we have examined the capacity of this mutant strain to synthesize RNA in vitro . Growth of the strain for as short a period as 10 min at 42 degrees C resulted in a 40 to 60% loss of RNA synthetic capacity and a fourfold decrease in percent rRNA synthesized in toluenized cell preparations . The time course for the loss and recovery of this RNA synthetic capacity correlated very well with the changes in RNA synthesis observed in vivo . We found no difference in temperature sensitivity of the purified RNA polymerase from the mutant and the parental strains . Moreover, there was no detectable alteration in the amount of enzyme, specific activity of the enzyme, or electrophoretic mobility of the subunits when the mutant strain was grown at 42 degrees C . The capacity for rRNA synthesis was also measured with the Zubay in vitro system (Reiness et al., Proc . Natl . Acad . Sci . 72:2881-2885, 1975) . Supernatant fractions (S-30) prepared from cells grown at 30 degrees C were capable of up to 31.2% rRNA synthesis, using phi 80d3 DNA as template . S-30 fractions from cells grown at 42 degrees C synthesized 8.6% rRNA . The bottom one-third of the S-100 fraction and the ribosomal salt wash from 30 degrees C cells contained one or more factors which partially restored preferential rRNA synthesis in S-30 fractions from cells grown at 42 degrees C . Preliminary evidence suggests that the factor(s) is protein in nature.

Infect Immun, 1983 Feb, 39(2), 889 - 97
Colonization factor antigens I and II and type 1 somatic pili in enterotoxigenic Escherichia coli: relation to enterotoxin type; Levine MM et al.; Enterotoxigenic Escherichia coli (ETEC) isolates from 36 persons with acute traveler's diarrhea from whom no other pathogens were recovered were tested (after no more than three subcultures) for the presence of colonization factor antigens I and II (CFA/I and CFA/II) and type 1 somatic pili . CFA/I or CFA/II was identified in 7 of 10 strains with heat-labile and heat-stable enterotoxins (LT+/ST+), but in only 2 of 12 LT-/ST+ (P less than 0.05) and 0 of 14 LT+/ST- (P less than 0.02) strains . CFA pili were not found among 74 non-enterotoxigenic E . coli strains . Type 1 somatic pili were demonstrable in 42% of the 36 ETEC and in 49% of the 74 non-enterotoxigenic E . coli isolates . The nine ETEC isolates bearing a CFA were serially subcultured on 10 consecutive days and retested for CFA and toxin . After five subcultures only one strain had lost a CFA, but after 10 passages three strains were negative: two lost CFA/I and one lost CFA/II . The strain that lost CFA/II became negative for both LT and ST as well and was found to lack a 48- and a 60-megadalton plasmid . The two strains that lost CFA/I also became negative for ST, but plasmid analysis revealed no plasmid loss . Disappearance of the CFA/I phenotype without loss of a plasmid can be explained by phase variation, as exhibited by type 1 somatic pili, or by rearrangement of base sequences in the CFA/I plasmid genome . If purified pili vaccines are to provide broad-spectrum protection against ETEC diarrhea, the search must be intensified to identify the antigens responsible for adhesion to intestinal mucosa in the many ETEC strains that lack CFA/I and CFA/II.

J Bacteriol, 1983 Feb, 153(2), 950 - 4
N-terminal amino acid sequencing of EDP208 conjugative pili; Frost LS et al.; EDP208 conjugative pili contain a single polypeptide subunit of 11,500 daltons with a blocked N-terminus . This N-terminal blocking moiety was identified as an N-acetyl group by 1H nuclear magnetic resonance analysis of an N-terminal tripeptide isolated from pronase digests of EDP208 pilin . Limited acid hydrolysis of the tripeptide allowed its sequence to be determined as acetyl-NH-Thr-Asp-Leu . Trypsin digestion of EDP208 pilin resulted in the quantitative release of a fragment containing 12 residues from the N-terminus of the protein . The sequence of this dodecapeptide was determined to be acetyl-NH-Thr-Asp-Leu-Leu-Ala-Gly-Gly-Lys-Asp-Val-Asp-Lys.

J Bacteriol, 1983 Feb, 153(2), 1038 - 44
Properties of pili from Escherichia coli SS142 that mediate mannose-resistant adhesion to mammalian cells; Mett H et al.; We isolated pili from Escherichia coli SS142 . These pili had a diameter of 6 nm and an average length of 400 nm . They were composed of subunits with a molecular weight of 18,000 . Their amino acid composition was determined; methionine and proline were not detected . The isolated pili retained mannose-resistant hemagglutinating activity . Proteolytic digestion and glutaraldehyde fixation led to partial or complete loss of the hemagglutinating activity of the pili without causing any detectable damage to their supramolecular structure, which was only disintegrated by treatment with hot sodium dodecyl sulfate . The hemagglutinating activity of E . coli SS142 was inhibited by the glycoproteins fetuin and Tamm-Horsfall protein, as well as by the glycolipids phytyl lactoside, dansyl-sphingosine lactoside, and digalactosyl diglyceride . Isolated pili inhibited the adhesion of the homologous strain E . coli SS142 to Intestine 407 cell monolayers, but did not inhibit the adhesion of E . coli strain B-413, B-506, or 2699 . This indicates that E . coli SS142 binds to a receptor different from those recognized by the other strains and that mannose-resistant adhesion to tissue culture cells can be classified into different subtypes.

C R Seances Acad Sci III, 1983 Jan 31, 296(4), 177 - 80
{Characterization of Escherichia coli esterase B after separation by chromatography}; Goullet P et al.; Esterase B of Escherichia coli has been purified 56 fold with recovery of 39% . The apparent molecular weight as determined by gel filtration was approximately 57000 . The pI as determined by isoelectric focusing was 4.6 . This enzyme exhibited Michaelis-Menton kinetics with apparent Km of 0.25 mM for l-naphtyl acetate . It remained stable at 60 degrees C but was sensitive to pH values below 6 . The esterase activity was completely inhibited by Di-isopropyl-fluorophosphate (DFP) but was resistant to iodoacetamide and to EDTA.

Biochem Biophys Res Commun, 1983 Jan 27, 110(2), 625 - 31
Identification of N5-methyl-N5-formyl-2,5,6-triamino-4-hydroxypyrimidine as a major adduct in rat liver DNA after treatment with the carcinogens, N,N-dimethylnitrosamine or 1,2-dimethylhydrazine; Beranek DT et al.; A major and previously undetected carcinogen-DNA adduct was found in the livers of rats given N,N-dimethylnitrosamine or 1,2-dimethylhydrazine . This adduct, which accounted for 55% of the total methyl residues in DNA at 72 hours after carcinogen treatment, was chromatographically identical to a synthetic purine ring-opened derivative of 7-methylguanine and could be released from the isolated hepatic DNA by a specific E . coli glycosylase . The synthetic ring-opened adduct was characterized by mass and NMR spectroscopy as N5-methyl-N5-formyl-2,5,6-triamino-4-hydroxypyrimidine and appears to exist in two rotameric forms.

Biochem Biophys Res Commun, 1983 Jan 27, 110(2), 609 - 15
The modulating influence of the fluidity of cell membrane on excision repair of DNA in UV-irradiated Escherichia coli; Todo T et al.; When the extent of liquid holding recovery (LHR) was measured as a function of the temperature at the time of liquid holding and the Arrhenius plot was made, two distinctive phases for the LHR were demonstrated in UV-irradiated RecA- derivative of E . coli ole28E1, which are unable to synthesize and degrade unsaturated fatty acids . The inflection temperatures were 17-18 degrees C, 23-24 degrees C and 28-30 degrees C for linoleate-, oleate- and elaidate-grown cells, respectively . These temperatures well corresponded to the phase transition temperatures of the cell membrane supplemented with the fatty acid . It is therefore concluded that at least a component involved in in vivo excision repair in E . coli is associated with cell membrane.

Biochem Biophys Res Commun, 1983 Jan 27, 110(2), 552 - 8
Imidazole open ring 7-methylguanine: an inhibitor of DNA synthesis; Boiteux S et al.; Guanine methylated at the N7 position (me7G) is susceptible to cleavage of the imidazole ring yielding: 2,6-diamino-4-hydroxy-5N-methyl-formamidopyrimidine (rom7G) . DNA synthesis catalysed by E.coli DNA polymerase I, using as templates poly(dGC) containing either me7G or rom7G, show that rom7G blocks DNA chain elongation . It implies a potential killing effect . Furthermore rom7G does not induce mispairing with either dAMP or dTMP . me7G does not affect DNA synthesis . The results suggest that, beside AP-sites, rom7G is a potential killing lesion in cells treated by alkylating agents.

Biochim Biophys Acta, 1983 Jan 26, 742(2), 391 - 8
Proline biosynthesis in Escherichia coli . Kinetic and mechanistic properties of glutamate semialdehyde dehydrogenase; Hayzer DJ et al.; The kinetics of the NADP+- and phosphate-dependent oxidation of glutamic acid 5-semialdehyde are consistent with a rapid-equilibrium random order mechanism . The Km for DL-pyrroline-5-carboxylic acid is 2.5 mM, for NADP+ is 0.05 mM and for phosphate is 0.35 mM . The Vmax is approx . 8.0 units per mg protein . The reaction is highly specific for the DL-pyrroline-5-carboxylic acid and NADP+, but a number of divalent anions can substitute for phosphate . NADPH is competitive with respect to all three substrates and an analog of gamma-glutamyl phosphate, 3-(phosphonoacetylamido)-L-alanine, is competitive with respect to DL-pyrroline-5-carboxylic acid and non-competitive with respect to NADP+ and phosphate, suggesting dead-end complex formation.

Biochim Biophys Acta, 1983 Jan 26, 742(2), 374 - 83
Kinetic studies on the mechanism of chorismate mutase/prephenate dehydratase from Escherichia coli K12; Baldwin GS et al.; The effect of pH on chorismate mutase/prephenate dehydratase (chorismate pyruvate mutase/prephenate hydro-lyase (decarboxylating) EC 5.4.99.5/EC 4.2.1.51) from Escherichia coli K12 has been studied . While the maximum velocity of both activities is independent of pH, Km for chorismate or prephenate shows a complex pH dependence . Differences in mutase activity in acetate/phosphate/borate and citrate/phosphate/borate buffers were traced to inhibition by citrate . When a variety of analogues of citrate were tested as possible inhibitors of the enzyme, several were found to inhibit mutase and dehydratase activities to different extents, and by different mechanisms . Thus citrate competitively inhibits mutase activity, but inhibits dehydratase activity by either a non-competitive or an uncompetitive mechanism . Conversely, cis- and trans-aconitate competitively inhibit dehydratase activity, but are partially competitive inhibitors of mutase activity . The differential effects of these inhibitors on the two activities are consistent with the existence of two distinct active sites, but additionally suggest some degree of interconnection between them . The implications of these results for possible mechanisms of catalysis by chorismate mutase/prephenate dehydratase are discussed.

J Mol Biol, 1983 Jan 25, 163(3), 409 - 30
Three-dimensional reconstruction and averaging of 30 S ribosomal subunits of Escherichia coli from electron micrographs; Knauer V et al.; From the micrographs of a tilt series, several particles of negatively stained 30 S ribosomal subunits of Escherichia coli were three-dimensionally reconstructed . Three of them showing similar orientation with respect to the supporting foil were averaged after alignment by newly developed three-dimensional correlation methods . As a main result we found a stained channel-like structure inside the particle . We tentatively propose that this corresponds, at least partially, to positively stained segments of the 16 S RNA.

J Mol Biol, 1983 Jan 25, 163(3), 395 - 408
Use of deletions created in vitro to map transcriptional regulatory signals in the malA region of Escherichia coli; Raibaud O et al.; The malA region of Escherichia coli contains one of the three maltose operons, namely malPQ, and the positive regulatory gene, malT . Gene malT and the malPQ operon are transcribed in opposite directions, in a divergent manner . The distance separating the transcription start-points in the two directions was previously shown to be 513 base-pairs . We are now presenting a deletion analysis of this unexpectedly long intergenic region . Two sets of deletions were created in vitro, by using exonuclease BAL31 . One set comprised deletions centered on a HincII restriction site located in the malPQ promoter, and extending towards gene malT . The other set was centered on an EcoRI site, which had been introduced close to the beginning of the malT cistron, and extended towards gene malP . These deletions, initially created on plasmids, were transferred onto the bacterial chromosome . By studying the phenotype resulting from the presence of these deletions, we concluded that: (1) all of the DNA sequences required for expression of malT and malPQ are within 100 base-pairs of the respective transcription start-points for these genes; (2) a sequence located more than 120 base-pairs upstream from the malT transcription start-point plays a role in limiting malT expression; and (3) a remaining DNA segment, 150 to 300 base-pairs in length, and centrally located in the inter-promoter region, seems to play no role in the expression of malT or malPQ.

Nucleic Acids Res, 1983 Jan 25, 11(2), 515 - 24
Crosslinking of Escherichia coli 50S ribosomal subunits with chlorambucilyl oligoprolyl phenylalanyl-tRNA molecular rulers; Parker KK et al.; A series of P-site probes, chlorambucilyl-(Pro)n-Phe-tRNAPhe, were prepared and reacted with poly(U)-directed Escherichia coli MRE 600 ribosomes . Upon binding of the probes to ribosomes, 90% of the cpm bound were not released following subsequent interaction with puromycin . In the absence of poly(U) or in the presence of poly(C), binding was limited to the amount of cpm bound if ribosomes were incubated in the presence of puromycin before adding modified tRNA and poly(U) . AcPhe-tRNAPhe was a competitive inhibitor of chlorambucilyl Phe-tRNAPhe . Binding to 50S subunits was strongly stimulated by poly(U), while binding to 30S subunits was not . Crosslinked 50S proteins were analyzed by two-dimensional gel electrophoresis . Crosslinking with molecular rulers containing zero prolines led to poly(U)-dependent labeling of L1 and L27 . With rulers containing five prolines, L6, L25, L28, and the group L18,23,24 were labeled . Analysis of crosslinked ribosomal RNA on sucrose density gradients revealed almost no cpm in the 16S or 23S peaks, but only in the 5S peaks . This was observed with molecular rulers containing either zero or five proline residues.

Nucleic Acids Res, 1983 Jan 25, 11(2), 265 - 75
Amplification of ribonuclease II (rnb) activity in Escherichia coli K-12; Donovan WP et al.; A 7.1 kb HindIII-XhoI fragment of E . coli DNA which contains the structural gene for ribonuclease II (rnb) has been cloned in the recombinant plasmid pDK24 . At least two constitutively expressed genes are encoded on the fragment as shown by maxicell analysis . On denaturing polyacrylamide gels RNase II appears as a single 72,000 dalton species . The approximate site of transcription initiation of the rnb gene has been mapped . Although derivatives of E . coli harboring pDK24 contained 10-fold more RNase II activity that wild type strains without the plasmid, the degradation rate of mRNA was similar in all strains tested . Strains deficient in both RNase II and polynucleotide phosphorylase appear inviable.

J Biol Chem, 1983 Jan 25, 258(2), 1343 - 51
Purification and characterization of transfer RNA (guanine-1)methyltransferase from Escherichia coli; Hjalmarsson KJ et al.; The tRNA modifying enzyme, tRNA (guanine-1)methyltransferase has been purified to near homogeneity from an overproducing Escherichia coli strain harboring a multicopy plasmid carrying the structural gene of the enzyme . The preparation gives a single major band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis . The enzyme is probably a single polypeptide chain of molecular weight 32,000 . The amino acid composition is presented and the NH2-terminal amino acid sequence was established to be H2N-Met-Trp-Ile-Gly-Ile-Ile-Ser-Leu-Phe-Pro . The enzyme has a pI of 5.2 . The tRNA (guanine-1)-methyltransferase has a pH optimum of 8.0-8.5, an apparent Km of 5 microM for S-adenosylmethionine . S-adenosylhomocysteine is a competitive inhibitor for the enzyme with an apparent Ki of 6 microM . Spermidine or putrescine are not required for activity, but they stimulate the rate of methylation 1.2-fold with optima at 2 and 6 mM, respectively . Ammonium ion is not required and is inhibitory at concentrations above 0.15 M . Magnesium ion inhibited the activity at a concentration as low as 2 mM . Sodium and potassium ions were inhibitory at concentrations above 0.1 M . The molecular activity of tRNA (guanine-1)-methyltransferase was calculated to 10.0 min-1 . It was estimated that the enzyme is present at 80 molecules/genome in cells growing with a specific growth rate of 1.0.

J Biol Chem, 1983 Jan 25, 258(2), 1297 - 8
Crystallographic studies of Escherichia coli citrate synthase; Rubin BH et al.; The citrate synthase from Escherichia coli B has been crystallized in a cubic space group with a unit cell spacing of 220 A . X-ray diffraction, electron microscopy, symmetry considerations, and low resolution projection Patterson syntheses are consistent with a model proposed in which 24 tetrameric molecules of Mr = 188,000 +/- 12,000 occupy the unit cell . The space group is apparently P23, although at low resolution the observed systematic absences in reflections are consistent with the space group P43n, a space group not allowed for asymmetric molecules . Estimates of VM suggest that in the true space group, P23, two tetrameric molecules occupy the asymmetric unit.

J Biol Chem, 1983 Jan 25, 258(2), 1215 - 20
Purification of the colicin I receptor; Bowles LK et al.; The colicin I outer membrane receptor was solubilized from the cell envelope of Escherichia coli K12 by extraction with Triton X-100 and purified to homogeneity by a combination of ion exchange and gel filtration chromatography as well as isoelectric focusing . The receptor was isolated as a single polypeptide and retained capacity to form a complex with pure colicin . The apparent molecular weight of the receptor as determined by polyacrylamide gel electrophoresis in sodium dodecy sulfate was 74,000 or 54,000 depending on whether the preparation was boiled or not in sodium dodecyl sulfate, respectively, prior to electrophoresis . Isoelectric focusing of the receptor in the presence of Triton X-100 revealed that the protein was slightly acidic (pI 4.75).

Nucleic Acids Res, 1983 Jan 25, 11(2), 349 - 68
Kilo-sequencing: an ordered strategy for rapid DNA sequence data acquisition; Barnes WM et al.; A strategy for rapid DNA sequence acquisition in an ordered, nonrandom manner, while retaining all of the conveniences of the dideoxy method with M13 transducing phage DNA template, is described . Target DNA 3 to 14 kb in size can be stably carried by our M13 vectors . Suitable targets are stretches of DNA which lack an enzyme recognition site which is unique on our cloning vectors and adjacent to the sequencing primer; current sites that are so useful when lacking are Pst, Xba, HindIII, BglII, EcoRI . By an in vitro procedure, we cut RF DNA once randomly and once specifically, to create thousands of deletions which start at the unique restriction site adjacent to the dideoxy sequencing primer and extend various distances across the target DNA . Phage carrying a desired size of deletions, whose DNA as template will give rise to DNA sequence data in a desired location along the target DNA, may be purified by electrophoresis alive on agarose gels . Phage running in the same location on the agarose gel thus conveniently give rise to nucleotide sequence data from the same kilobase of target DNA.

J Biol Chem, 1983 Jan 25, 258(2), 1276 - 81
DNA sequence of the gene coding for Escherichia coli ribonuclease H; Kanaya S et al.; The gene for Escherichia coli ribonuclease H has been studied by use of a plasmid which contains a segment of the E . coli chromosome . The genomic DNA was subcloned from pLC28-22 to pBR322 by use of various restriction enzymes . Such subcloning limited the RNase H gene to a piece of DNA no longer than 760 base pairs . Cells bearing plasmids containing the RNase H gene produce as much as 10-15 times the normal amount of RNase H without any drastic effect on maintenance of the plasmid or cell growth . DNA sequence analysis has permitted the prediction of a protein whose molecular weight is 17,559 (155 amino acid residues) . The predicted sequence was confirmed by amino acid analysis, NH2-terminal amino acid sequence, and size determination of highly purified RNase H.

J Biol Chem, 1983 Jan 25, 258(2), 1235 - 41
Cloning of the MspI modification enzyme . The site of modification and its effects on cleavage by MspI and HpaII; Walder RY et al.; The gene for the MspI modification enzyme from Moraxella was cloned in Escherichia coli using the plasmid vector pBR322 . Selection of transformants carrying the gene was based on the resistance of the modified plasmid encoding the enzyme to cleavage by MspI . Both chromosomal and plasmid DNA were modified in the selected clones . None of the clones obtained produced the cognate restriction enzyme which suggests that in this system the genes for the restriction enzyme and methylase are not closely linked . Crude cell extracts prepared from the recombinant strains, but not the host (E . coli HB101), contain an S-adenosylmethionine-dependent methyltransferase specific for the MspI recognition site, CCGG . Production of the enzyme is 3-4-fold greater in the transformants than in the original Moraxella strain . 5-Methylcytosine was identified as the product of the reaction chromatographically . The outer cytosine of the recognition sequence, *CCGG, was shown to be the site of methylation by DNA-sequencing methods . This modification blocks cleavage by both MspI and its isoschizomer HpaII . HpaII, but not MspI, is able to cleave the unmethylated strand of a hemimethylated substrate . The relevance of these results to the use of MspI and HpaII to analyze patterns of methylation in genomic DNA is discussed.

J Biol Chem, 1983 Jan 25, 258(2), 995 - 1000
Different regions of aminoacyl-tRNA regulate the function of elongation factor Tu; Parlato G et al.; In this work we show that intact aminoacyl-tRNA (aa-tRNA) and its 3' half-molecule, but not its 3' C-C-A-aa fragment, require selective ionic conditions for stimulating the mRNA-independent GTPase of elongation factor Tu (EF-Tu) in the presence of ribosomes.l Stimulation by aa-tRNA and its 3' half-molecule is only observed at 20 and 30 mM Mg2+ and not at 10 mM, where they exert inhibitory activity; by contrast, C-C-A-aa enhances the GTPase activity at all three of these Mg2+ concentrations . Ammonium ion is needed for stimulation by C-C-A-aa, whereas it inhibits the stimulation by aa-tRNA and its 3' half-molecule . The concentration of aminoacylated fragments needed for half-maximum stimulation follows this order: A-Val much greater than C-A-Val greater than C-C-A-Val much greater than 3' Val-tRNA1Val half-molecule greater than Val-tRNA1Val . The extent of maximum stimulation of the EF-Tu GTPase in the presence of ribosomes varies moderately depending on the aa-tRNA species; a clear dependence on the nature of the aminoacyl side chain is observed in the effects of their respective C-C-A-aa fragments tested (C-C-A-Arg, C-C-A-Val, C-C-A-Phe, C-C-A-Met, C-C-A-Lys) . In the absence of ribosomes and at low {Mg2+}, the one-round GTP hydrolysis by EF-Tu is enhanced by C-C-A-aa fragments, whereas it is inhibited by the corresponding aa-tRNAs . Our results suggest that besides the 3' aminoacylated extremity another region(s) of the aa-tRNA molecule controls the GTPase of EF-Tu . The "unspecific" stimulation by C-C-A-aa and the "specific," aa-tRNA-like effect of the 3' aa-tRNA half-molecule point to the importance of the T chi C loop and stem, as well as of the adjacent regions for the regulation of this function.

FEBS Lett, 1983 Jan 24, 151(2), 237 - 42
Mechanism of caffeine-induced inhibition of DNA synthesis in escherichia coli; Sandlie I et al.; Caffeine inhibited DNA synthesis in toluene-treated Escherichia coli K12 strains to the same extent as in intact cells using the incorporation of {3H}thymidine as a measure of DNA synthesis . The inhibition was found to be competitive with ATP, and it was not influenced by the concentrations of deoxynucleoside triphosphates to any extent . When caffeine was added together with other DNA synthesis inhibitors such as novobiocin, nalidixic acid or actinomycin D, the inhibition in all cases was non-additive . It is suggested that caffeine inhibits one of the ATP-requiring enzymes in the DNA replication machinery, possibly DNA polymerase III or one of the DNA helicases.

FEBS Lett, 1983 Jan 24, 151(2), 189 - 92
Effects of solubilisation on some properties of the membrane-bound respiratory enzyme D-amino acid dehydrogenase of Escherichia coli; Jones H et al.; Solubilisation, delipidation and partial purification of the membrane-bound enzyme D-amino acid dehydrogenase of Escherichia coli K12 produced significant changes in several of its properties . Solubilised enzyme showed a broader substrate specificity, increased affinity for at least three substrates, and a lower pH optimum with D-alanine as substrate . Solubilised enzyme was more heat-labile than native enzyme, particularly at 37 degrees C, and re-binding to envelope preparations restored protection against heat denaturation . Activity of delipidated enzyme could be increased by addition of pure phospholipids . Native enzyme showed biphasic Arrhenius kinetics associated with phase changes of membrane lipids.

Biochim Biophys Acta, 1983 Jan 20, 739(1), 8 - 16
Characterization of the DNA-cellulose-binding proteins from Escherichia coli K 12; Sjastad K et al.; The various {35S}DNA-binding proteins present in lysates of Escherichia coli K 12 cells have been analyzed by means of two-dimensional SDS-polyacrylamide gel electrophoresis . The proteins were isolated by the DNA-cellulose technique and eluted by increasing concentrations of NaCl (0.15, 0.4, 0.6 and 2 M) . Only 2% of the total 35S radioactivity in the lysate became bound to the DNA-cellulose column . A total of 237 polypeptides were detected and the distribution among the salt eluates were 85, 83, 40 and 29 polypeptides, respectively . The 40 major polypeptides with regard to concentrations were also identified from gels stained with a protein-specific reagent . The polypeptides could be divided into two main groups according to pI values, namely, acidic polypeptides (total number, 174) and basic polypeptides (total number, 63) . The ratio between acidic and basic polypeptides decreased with increasing salt concentrations in the eluates . The majority of the basic polypeptides had molecular weights in the range 10 000-30 000, whereas the acidic polypeptides had molecular weights from 10 000 to 165 000.

Biochim Biophys Acta, 1983 Jan 20, 739(1), 42 - 7
Expression of ultraviolet-induced restriction alleviation in Escherichia coli K-12 . Detection of a lambda phage fraction with a retarded mode of DNA injection; Thoms B et al.; Ultraviolet-induced restriction alleviation is an SOS function which partially relieves the K-12-specific DNA restriction in Escherichia coli . Restriction alleviation is determined by observing elevated survival of unmodified phage lambda in cells irradiated with ultraviolet prior to infection . We demonstrate that restriction of lambda is also relieved when log-phase cells are irradiated as late as 50 min after adsorption of lambda . At this time more than 60% of the lambda DNA is already released as acid-soluble material from the cells . Experiments involving reextraction of lambda DNA from infected cells and a mild detergent treatment removing absorbed phages from the cellular surface showed that only a small specific fraction of all lambda infections is destined to escape restriction due to restriction alleviation . This fraction (10-20%) has a retarded mode of DNA injection (60 min or longer) after adsorption which allows the expression of the restriction alleviation function before the phage DNA is exposed to restriction endonucleases . This behaviour of a fraction of lambda phages explains why the SOS function restriction alleviation could initially be discovered . We show that the retarded mode of DNA injection is not required for another SOS function acting on lambda DNA, the increased repair of ultraviolet-irradiated DNA (Weigle reactivation).

Biochim Biophys Acta, 1983 Jan 20, 739(1), 27 - 34
The roles of RNA polymerase and RNAase I in stable RNA degradation in Escherichia coli carrying the srnB+ gene; Ito R et al.; In Escherichia coli cells carrying the srnB+ gene of the F plasmid, rifampin, added at 42 degrees C, induces the extensive rapid degradation of the usually stable cellular RNA (Ohnishi, Y., (1975) Science 187, 257-258; Ohnishi, Y., Iguma, H., Ono, T., Nagaishi, H . and Clark, A.J . (1977) J . Bacteriol . 132, 784-789) . We have studied further the necessity for rifampin and for high temperature in this degradation . Streptolydigin, another inhibitor of RNA polymerase, did not induce the RNA degradation . Moreover, the stable RNA of some strains in which RNA polymerase is temperature-sensitive did not degrade at the restrictive temperature in the absence of rifampin . These data suggest that rifampin has an essential role in the RNA degradation, possibly by the modification of RNA polymerase function . A protein (Mr 12 000) newly synthesized at 42 degrees C in the presence of rifampin appeared to be the product of the srnB+ gene that promoted the RNA degradation . In a mutant deficient in RNAase I, the extent of the RNA degradation induced by rifampin was greatly reduced . RNAase activity of cell-free crude extract from the RNA-degraded cells was temperature-dependent . The RNAase was purified as RNAase I in DEAE-cellulose column chromatography and Sephadex G-100 gel filtration . Both in vivo and with purified RNAase I, a shift of the incubation mixture from 42 to 30 degrees C, or the addition of Mg2+ ions, stopped the RNA degradation . Thus, an effect on RNA polymerase seems to initiate the expression of the srnB+ gene and the activation of RNAase I, which is then responsible for the RNA degradation of E . coli cells carrying the srnB+ gene.

Biochemistry, 1983 Jan 18, 22(2), 281 - 4
Neutron scattering study of the binding of tRNAPhe to Escherichia coli phenylalanyl-tRNA synthetase; Dessen P et al.; Escherichia coli phenylalanyl-tRNA synthetase has been characterized by small-angle neutron scattering . In solution (20 mM imidazole hydrochloride, pH 7.6, 10 mM 2-mercaptoethanol, and 0.1 mM ethylenediaminetetraacetic acid), this enzyme has a molecular weight of 227K +/- 20K with a radius of gyration of 48.3 +/- 0.6 A, independent of the presence of MgCl2 up to 50 mM . The change of the scattering upon adding tRNAPhe to the enzyme has been followed with 10 mM MgCl2 present in the buffer . One enzyme molecule is capable of binding two tRNAPhe molecules with affinity constants larger than 10(6) M-1 . Parallel titration experiments in 73% 2H2O, close to the matching point of tRNA, show that the RG of the enzyme is not changed by the binding of one or two tRNAPhe molecules . These results are compared with quasi-electric light scattering studies {Holler, E., Wang, C . C., & Ford, N.C., Jr . (1981) Biochemistry 20, 861-867} where the addition of either MgCl2 or tRNAPhe was shown to cause dramatic changes of the apparent translational diffusion constant of phenylalanyl-tRNA synthetase.

Biochemistry, 1983 Jan 18, 22(2), 504 - 15
Electron paramagnetic resonance and optical evidence for interaction between siroheme and Fe4S4 prosthetic groups in complexes of Escherichia coli sulfite reductase hemoprotein with added ligands; Janick PA et al.; Janick & Siegel {Janick, P . A., & Siegel, L . M . (1982) Biochemistry 21, 3538-3547} showed that the EPR spectrum of the reduced Fe4S4 center (S = 1/2) in fully reduced native ("unligated") Escherichia coli NADPH-sulfite reductase hemoprotein subunit (SiR-HP) is perturbed by interaction with paramagnetic ferrous siroheme (S = 1 or 2) to yield several novel sets of EPR signals: one set with all g values between 2.0 and 2.8, termed "S = 1/2" type, and two sets with the lowest field g value between 4.7 and 5.4, termed "S = 3/2" type . The present study has shown that EPR spectra of fully reduced SiR-HP are nearly quantitatively converted to the classical "g = 1.94" type typical of S = 1/2 Fe4S4 clusters when the heme has been ligated by strong field ligands such as CO, CN-, S2-, and AsO2-, converting the ferroheme to S = 0 . However, the exact line shapes and g values of the g = 1.94 differ markedly when different ligands are bound to the heme . Also, optical difference spectra taken between enzyme species in which the heme is kept in the same (Fe2+) oxidation state while the Fe4S4 center is reduced or oxidized show that the optical spectrum of the ligated siroheme is sensitive to the oxidation state of the Fe4S4 cluster . These results indicate that the heme-Fe4S4 interaction of native SiR-HP persists even when the heme Fe is bound to exogenous ligands . We have also found that the g values of the exchange-coupled S = 1/2 and S V 3/2 type signals of native reduced SiR-HP can be significantly shifted by addition of potential weak field heme ligands--halides and formate--or low concentrations of certain chaotropic agents--guanidinium salts and dimethyl sulfoxide--to the fully reduced enzyme . Such agents can also promote interconversion of the S = 1/2 and S = 3/2 type signals . These effects are reversed on removal of the agent . Treatment of reduced SiR-HP with relatively large concentrations of chaotropes, e.g., 60% dimethyl sulfoxide or 2 or 3 M urea, leads to abolition of the S = 1/2 and S = 3/2 EPR signals and their replacement by signals of the g = 1.94 type.

Eur J Biochem, 1983 Jan 17, 130(1), 123 - 30
Studies on energy supply for genetic processes . Requirement for membrane potential in Escherichia coli infection by phage T4; Kalasauskaite EV et al.; In this study the hypothesis considering the requirement for an electrochemical proton gradient in the injection of phage T4 DNA into Escherichia coli cell has been verified experimentally . The phage caused a reversible depolarization of cell membrane, while phage 'ghosts' induced an irreversible depolarization . The phage infection was strictly dependent on E . coli membrane potential value when phage/cell ratio was 5 and higher . When the ratio was close to 1, the decrease in the membrane potential up to -100 mV caused practically no effect on the phage infection . The infection inhibition was observed when the membrane potential was lowered below this 'threshold' value . On the other hand, the decrease in the membrane potential caused no effect on the phage infection under conditions promoting a concomitant increase in the value of the transmembranous pH gradient . The phage DNA transfer through the membrane of ATPase-deficient cells was reversibly inhibited by switching off the respiratory chain - the sole generator of a protonmotive force in these mutant cells . The membrane should be kept in the energized state during the phage DNA entrance into the cell . Adsorption of the phage on E . coli was followed by the reversible release of the respiratory control . Thus the results presented here indicate the requirement of the electrochemical proton gradient across the plasma membrane for injection of phage T4 DNA into E . coli . They support the concept postulating an expenditure of host cell metabolic energy for phage T4 DNA transfer through the membrane.

Eur J Biochem, 1983 Jan 17, 130(1), 161 - 5
Selective expression of cloned middle-repetitive sequences in nuclear RNA of mouse organs; Kuroiwa A et al.; Mouse middle-repetitive sequences have been cloned in the bacterial plasmid pBR322 . The DNA sequences, expressed only in nuclei, were screened by filter hybridization using nuclear RNA as probes . Several clones were expressed predominantly in the nuclear RNA in certain types of organs . The copy numbers of typical middle-repetitive sequence clones in genomic DNA were analyzed quantitatively and results showed that 10(3)-10(4) copies exist per haploid DNA, indicating that they belong to a relatively abundant class of repeated sequences . Homology within the family of each middle-repetitive sequence ranged from 81% to 94% . The expression of cloned middle-repetitive sequences was analyzed by liquid-phase hybridization with nuclear RNAs from particular organs . The content of transcripts of the middle-repetitive sequence in nuclear RNA of a particular organ was at least 1000 times higher than that in other organs . In steady-state nuclear RNA these transcripts were calculated to amount to 10(2)-10(4) copies per nucleus . Most of these transcripts seem to be retained in the nuclei and do not appear in the cytoplasm.

Biochem Biophys Res Commun, 1983 Jan 14, 110(1), 243 - 9
The origin of sulfur in biotin; DeMoll E et al.; The comparative ability of Escherichia coli K-12 hpb lambda-, a biotin overproducing strain, to incorporate 35S from isotopically labeled L-methionine, L-cystine, and the sulfane sulfur of thiocystine was determined . Comparison of the specific activity of sulfur in biotin produced by the organism with that of the 35S-labeled amino acids demonstrates that the sulfur of cystine is transferred to biotin with an efficiency of at least 75%, that the sulfur of methionine does not contribute to biotin significantly, and that the sulfane sulfur of thiocystine contributes approximately one-third of the sulfur to newly synthesized biotin and is essentially equivalent in utilization to that of the other two sulfur atoms in the molecule.

Biochem Biophys Res Commun, 1983 Jan 14, 110(1), 169 - 75
Binding of lac repressor headpiece to poly{d(A-T)} . A thermal denaturation study; Durand M et al.; The binding of the lac repressor headpiece to poly{d(A-T)} has been investigated using thermal denaturation experiments . The binding of the headpiece leads to a stabilization of the double-stranded structure . Competition experiments using circular dichroism measurements confirm that the headpiece binds stronger on the double-stranded poly{d(A-T)} than on a single-stranded nucleic acid like poly(A) . Theoretical analyses of the melting curves allow the determination of the binding constant and of the site size on the poly{d(A-T)} . This latter value is found to be 4 base pairs, in good agreement with that determined by other experimental approaches, and is much smaller than the values previously found for the lac repressor.

Biochem Biophys Res Commun, 1983 Jan 14, 110(1), 176 - 80
Escherichia coli dnaJ- and dnaK-gene products: synthesis in minicells and membrane-affinity; Zylicz M et al.; Escherichia coli dnaJ- and dnaK-gene products have been identified in a system of minicells infected with the appropriate transducing lambda phages . The molecular weights of these polypeptides in dodecyl sulphate/acrylamide electrophoresis amounted to 39,000 and 77,000, respectively . Equilibrium sedimentation of minicell lysates in metrizamide density gradients has revealed that both these host proteins, indispensable for lambda DNA replication, are membrane-bound.

Biochim Biophys Acta, 1983 Jan 12, 742(1), 16 - 24
Study of binding protein-ligand interaction by ammonium sulfate-assisted adsorption on cellulose esters filters; Richarme G et al.; A technique for the study of neutral carbohydrate binding protein-ligand interaction is described in this report . It is based on filtration on cellulose esters filters of a mixture of the binding protein and the radioactive ligand, following a treatment of this mixture with ammonium sulfate; the technique is described for the galactose binding protein and for the maltose binding protein of Escherichia coli . For the galactose binding protein, an ammonium sulfate concentration far below that required for precipitation of the protein is sufficient to promote an almost complete retention of the protein on the filters . Furthermore, the addition of ammonium sulfate does not modify the amount of preexisting binding protein-ligand complex, and, in much less than one second, leads to a conformation of the protein-ligand complex which does not allow further ligand binding or dissociation . Hence, the technique is not only very useful for the detection of binding proteins in crude extracts and during purification procedures, it is also of value in the determination of the kinetic parameters of protein-ligand interactions.

Nucleic Acids Res, 1983 Jan 11, 11(1), 193 - 202
Subspecies of DNA polymerase alpha from calf thymus with different fidelity in copying synthetic template-primers; Brosius S et al.; Three different subspecies of DNA polymerase alpha from calf thymus sedimenting at 9 S, 7 S and 5.7 S have been investigated with respect to their accuracy of in vitro DNA synthesis on poly (dA) (dT)16 and poly d(AT) as template-primers . Our results indicate that the structure of DNA polymerase alpha has a strong influence on the accuracy of DNA synthesis . The 9 S enzyme shows a misincorporation frequency of about 1:100 000 . An error rate of 1:15 000 is measured for the 7 S species . The 5.7 S enzyme for which an error rate of 1:3 000 is determined, has to be considered as error prone . Lowering the rate of DNA synthesis leads to a decrease in fidelity . The single stranded DNA binding protein from E.coli increases the accuracy of the 5.7 S and the 7 S enzyme by a factor of two . Mn2+ decreases the fidelity of all three subspecies in a concentration dependent manner.

Nucleic Acids Res, 1983 Jan 11, 11(1), 127 - 39
Nucleotide sequence of the ilvB multivalent attenuator region of Escherichia coli K12; Hauser CA et al.; The ilvB gene of Escherichia coli K12 has been cloned into a multicopy plasmid . The regulation of the cloned gene by valine or leucine limitation and by catabolite repression is the same as for the chromosome encoded gene . The nucleotide sequence of a regulatory region preceding the ilvB structural gene has been determined . This DNA sequence includes a promoter, a region which codes for a putative 32 amino acid polypeptide containing multiple valine and leucine codons, and a transcription termination site . In vitro transcription of this region produces a 184 nucleotide terminated leader transcript . Mutually exclusive secondary structures of the leader transcript are predicted . On the basis of these data, a model for multivalent attenuation of the ilvB operon is presented . Data are presented which suggests that at least part of the postulated CRP-cyclic AMP binding site of the ilvB operon precedes the transcription start site by more than 71 base pairs.

Nucleic Acids Res, 1983 Jan 11, 11(1), 141 - 58
CAP and RNA polymerase interactions with the lac promoter: binding stoichiometry and long range effects; Fried MG et al.; The binding stoichiometries of the complexes formed when the E . coli cyclic AMP receptor protein (CAP) binds to 203 bp lac promoter-operator restriction fragments have been determined . Under quantitative binding conditions, a single dimer of CAP occupies each of two sites in the promoter . Different electrophoretic mobilities are observed for 1:1 complexes formed with L8-UV5 mutant, L305 mutant, and wild type promoter fragments, indicating sequence-specific structural differences between the complexes . The differences in gel mobility between L8-UV5 and wild type complexes disappear when the promoter fragments are cleaved with Hpa II restriction endonuclease . Models in which CAP alters DNA conformation or in which CAP forms a transient intramolecular bridge between two domains of a DNA molecule could account for these observations . The selective binding of RNA polymerase to CAP-promoter complexes is demonstrated: the binding of a single CAP dimer to the promoter is sufficient to stimulate subsequent polymerase binding . Functional CAP molecules are not released from the promoter on polymerase binding.

Nucleic Acids Res, 1983 Jan 11, 11(1), 105 - 25
The major transcripts of the kinetoplast DNA of Trypanosoma brucei are very small ribosomal RNAs; Eperon IC et al.; The nucleotide sequence has been determined of a 2.2 kb segment of kinetoplast DNA, which encodes the major mitochondrial transcripts (12S and 9S) of Trypanosoma brucei . The sequence shows that the 12S RNA is a large subunit rRNA, although sufficiently unusual for resistance to chloramphenicol to be predicted . The 9S RNA has little homology with other rRNAs, but a possible secondary structure is not unlike that of the 2.5-fold larger E . coli 16S rRNA . We conclude that the 12S RNA (about 1230 nucleotides) and the 9S RNA (about 640 nucleotides) are the smallest homologues of the E . coli 23S and 16S rRNAs yet observed.

J Biol Chem, 1983 Jan 10, 258(1), 108 - 18
Excision repair and DNA synthesis with a combination of HeLa DNA polymerase beta and DNase V; Mosbaugh DW et al.; The ability of HeLa DNA polymerases to carry out DNA synthesis from incisions made by various endodeoxyribonucleases which recognize or form baseless sites in DNA was examined . DNA polymerase beta carried out limited strand displacement synthesis from 3'-hydroxyl nucleotide termini made by HeLa apurinic/apyrimidinic (AP) endonuclease II at the 5'-side of apurinic sites . Escherichia coli endonuclease III incises at the 3'-side of apurinic sites to produce nicks with 3'-deoxyribose termini which did not efficiently support DNA synthesis with beta-polymerase . However, these nicks could be activated to support limited DNA synthesis by HeLa AP endonuclease II, an enzyme which removes the baseless sugar phosphate from the 3'-termini, thus creating a one-nucleotide gap . With dGTP as the only nucleoside triphosphate present, the beta-polymerase catalyzed one-nucleotide DNA repair synthesis from those gaps which lacked dGMP . In contrast, HeLa DNA polymerase alpha was unreactive with all of the above incised DNA substrates . Larger patches of DNA synthesis were produced by nick translation from one-nucleotide gaps with HeLa DNA polymerase beta and HeLa DNase V . Moreover, incisions made by E . coli endonuclease III were activated to support DNA synthesis by the DNase V which removed the 3'-deoxyribose termini . HeLa DNase V also stimulated both the rate and extent of DNA synthesis by DNA polymerase beta from AP endonuclease II incisions . In this case the baseless sugar phosphate was removed from the 5'-termini, and nick translational synthesis occurred . Complete DNA excision repair of pyrimidine dimers was achieved with the beta-polymerase, DNase V, and DNA ligase from incisions made in UV-irradiated DNA by T4 UV endonuclease and HeLa AP endonuclease II . Such incisions produce a one-nucleotide gap containing 3'-hydroxyl nucleotide and 5'-thymine: thymidylate cyclobutane dimer termini . DNase V removes pyrimidine dimers primarily as a dinucleotide and then promotes nick translational DNA synthesis.

FEBS Lett, 1983 Jan 10, 151(1), 143 - 7
Interaction of cinnamyl-tRNAPhe with Escherichia coli elongation factor Tu; Derwenskus KH et al.; The products of nitrous acid mediated-deamination of Phe-tRNAPhe from E . coli were analyzed and their capability to interact with elongation factor Tu from E . coli was investigated . Thin-layer chromatography as well as HPLC analysis revealed the existence of at least two deamination products, 3-phenyl-lactyl-tRNAPhe and cinnamyl-tRNAPhe . It could be shown that the aminoacyl-tRNA analogues were active in the formation of the ternary complex with EF-Tu X GTP, although with a lower efficiency than native Phe-tRNAPhe . For both modified acyl-tRNAs the dissociation constant was determined to be 3 X 10(-5) M.

J Biol Chem, 1983 Jan 10, 258(1), 408 - 16
31P nuclear magnetic resonance of phosphoenzyme intermediates of alkaline phosphatase; Gettins P et al.; Covalent (E-P) and noncovalent (E X P) phosphoenzyme intermediates exist on the reaction path of alkaline phosphatase of Escherichia coli . Zn(II) and Cd(II) alkaline phosphatases both form E-P and E X P from inorganic phosphate . These intermediates show well separated 31P NMR resonances in slow chemical exchange with respect to each other and to unbound phosphate . The 31P signals of E X P of all forms of the 113Cd(II) enzyme are doublets (J = approximately 30 Hz) due to 113Cd-O-31P coupling . Heteronuclear decoupling shows the phosphate of E X P to be coordinated to the A site metal of the two metal ions, A and B, approximately 3.9 A apart at each catalytic center . The chemical shifts of E X P vary from approximately 4 ppm for the Zn(II) enzyme to 12.6-13.4 ppm for forms of the Cd(II) enzyme and indicate a major influence of the metal ions on the conformation around phosphorus . The phosphoryl group of E-P is not coordinated to either of the two metal ions at the active center as shown by the absence of 113Cd-O-31P coupling on the 31P signals of E-P formed by the 113Cd(II) enzymes . The chemical shift of E-P is not sensitive to metal ion species or stoichiometry and is 8-9 ppm for all forms of the Zn(II) and Cd(II) enzymes . The E-P in equilibrium E X P in equilibrium E + Pi equilibria are described by analogous pH functions for the Zn(II) and Cd(II) enzymes . At acid pH E-P predominates and is converted to E X P as the pH is raised, following a sigmoid pH profile . For the Zn(II) enzyme the midpoint of the E-P in equilibrium E X P equilibrium occurs at pH 5, while for the Cd(II)6 and Cd(II)2 enzymes the midpoints are pH 8.7 and 10, respectively . The ionization controlling the equilibrium between E-P and E X P may be that of a metal-bound H2O (-OH nucleophile) whose pKa will depend strongly on the hardness of the coordinating metal ion . For the Zn(II)4 enzyme one of 2 mol of E-P formed by the enzyme at acid pH dissociates readily at pH 7.5-8 where dissociation of E X P (Kd approximately equal to mM) is rate-limiting . Phosphate binds more tightly to the Cd(II) enzyme and 2 mol of phosphate remain bound until above pH 9 where E X P begins to dissociate at mM concentrations . The low Kd for E X P and the alkaline shift in the E-P in equilibrium E X P pH profile probably account for the slow turnover of the Cd(II) enzyme . Precise chemical shifts of the 113Cd and 31P NMR signals as well as the ratio of E-P/E X P at one active center of the dimer are altered by metal ion binding at the other active center indicating significant subunit-subunit interactions.

J Biol Chem, 1983 Jan 10, 258(1), 396 - 407
113Cd nuclear magnetic resonance of Cd(II) alkaline phosphatases; Gettins P et al.; 113Cd NMR spectra of 113Cd(II)-substituted Escherichia coli alkaline phosphatase have been recorded over a range of pH values, levels of metal site occupancy, and states of phosphorylation . Under all conditions resonances attributable to cadmium specifically bound at one or more of the three pairs of metal-binding sites (A, B, and C sites) are detected . By following changes in both the 113Cd and 31P NMR spectra of 113Cd(II)2 alkaline phosphatase during and after phosphorylation, it has been possible to assign the cadmium resonance that occurs between 140 and 170 ppm to Cd(II) bound to the A or catalytic site of the enzyme and the resonance occurring between 51 and 76 ppm to Cd(II) bound to B site, which from x-ray data is located 3.9 A from the A site . The kinetics of phosphorylation show that cadmium migration from the A site of one subunit to the B site of the second subunit follows and is a consequence of phosphate binding, thus precluding the migration as a sufficient explanation for half-of-the-sites reactivity . Rather, there is evidence for subunit-subunit interaction rendering the phosphate binding sites inequivalent . Although one metal ion, at A site, is sufficient for phosphate binding and phosphorylation, the presence of a second metal ion at B site greatly enhances the rate of phosphorylation . In the absence of phosphate, occupation of the lower affinity B and C sites produces exchange broadening of the cadmium resonances . Phosphorylation abolishes this exchange modulation . Magnesium at high concentration broadens the resonances to the point of undetectability . The chemical shift of 113Cd(II) in both A and B sites (but not C site) is different depending on the state of the bound phosphate (whether covalently or noncovalently bound) and gives separate resonances for each form . Care must be taken in attributing the initial distribution of cadmium or phosphate in the reconstituted enzyme to that of the equilibrium species in samples reconstituted from apoenzyme . Both 113Cd NMR and 31P NMR show that some conformational changes consequent to metal ion or phosphate binding require several days before the final equilibrium species is formed.

J Biol Chem, 1983 Jan 10, 258(1), 386 - 95
65Zn(II), 115mCd(II), 60Co(II), and mg(II) binding to alkaline phosphatase of Escherichia coli . Structural and functional effects; Coleman JE et al.; Zn(II), Cd(II), Co(II) and Mg(II) binding to apoalkaline phosphatase of Escherichia coli and the relative stabilities of the resulting metalloenzyme complexes have been measured by equilibrium dialysis and metal exchange reactions using gamma-emitting isotopes of these metals . At millimolar concentrations of these metal ions the alkaline phosphatase dimer binds three pairs of metal ions (A, B, and C sites) . One of these pairs dialyzes readily without detectable change in the structure or function of the enzyme (C site) . Of the remaining two pairs, the binding affinity of both for Zn(II) and Cd(II) is increased by formation of the phosphoenzyme intermediates . Cd(II) is bound less tightly to both A and B sites than Zn(II), and at pH 6.5 Cd(II) is induced to bind to the B sites by formation of the phosphate complexes . Mg(II), 5-10 mM, competes successfully with the IIB metal ions for the second or lower affinity pair of binding sites (B sites), although Mg(II) is a relatively poor competitor on an equimolar basis, especially for Cd(II) . Binding of metal ions to the apoenzyme appears to be a cooperative process involving conformational changes in the protein which are not readily reversible . The initial binding of a pair of Zn(II) or Cd(II) ions to the apoenzyme is characterized by equilibrium constants of 10(-5) to 10(-7) M for Zn(II) and 10(-4) to 10(-5) M for Cd(II) . Following the cooperative binding of all three pairs of metal ions, however, re-establishment of equilibrium by dialysis indicates binding constants of less than 10(-8) M for Zn(II) and less than 10(-6) M for Cd(II) at the sites of greatest affinity (A sites) . Binding of Mg(II) or Cd(II) to the B site, once the A site is occupied, increases the phosphorylation rate of the Cd(II) enzyme by 20-fold . In the presence of saturating concentrations of Mg(II) complete activity is restored to the apoenzyme by 2 Zn(II) ions . In the absence of Mg(II) as many as 6 Zn(II) ions may be required before complete restoration is achieved . Roles for the A and B site metal ions in the catalytic mechanism are discussed.

J Biol Chem, 1983 Jan 10, 258(1), 31 - 4
Structure of the lac carrier protein of Escherichia coli; Foster DL et al.; Circular dichroic measurements on the lac carrier protein purified from the cytoplasmic membrane of Escherichia coli indicate that 85 +/- 5% of the amino acid residues comprising this integral membrane protein are arranged in helical secondary structures . Analysis of the sequential hydropathic character of this protein by the method of Kyte and Doolittle (J . Mol . Biol . (1982) 157, 105-132) indicates that the protein is composed of at least 12 hydrophobic segments with a mean length of 24 +/- 4 residues/segment . Approximately 70% of the 417 amino acids in the lac carrier are found in these domains . The hydropathic profile, together with the circular dichroic measurements, suggest that the 12 hydrophobic segments are largely in a helical conformation . If the segments are assumed to be alpha-helical, the mean length of each domain approximates the thickness of the most hydrophobic portion of the lipid bilayer . Based on these considerations, it is proposed that the lac carrier protein consists of at least 12 alpha-helical segments that traverse the membrane in a perpendicular sense, i.e . in a fashion similar to bacteriorhodopsin.

J Biol Chem, 1983 Jan 10, 258(1), 276 - 82
The amino acid sequence of Escherichia coli cyanase; Chin CC et al.; The amino acid sequence of the enzyme cyanase (cyanate hydrolase) from Escherichia coli has been determined by automatic Edman degradation of the intact protein and of its component peptides . The primary peptides used in the sequencing were produced by cyanogen bromide cleavage at the methionine residues, yielding 4 peptides plus free homoserine from the NH2-terminal methionine, and by trypsin cleavage at the 7 arginine residues after acetylation of the lysines . Secondary peptides required for overlaps and COOH-terminal sequences were produced by chymotrypsin or clostripain cleavage of some of the larger peptides . The complete sequence of the cyanase subunit consists of 156 amino acid residues (Mr 16,350) . Based on the observation that the cysteine-containing peptide is obtained as a disulfide-linked dimer, it is proposed that the covalent structure of cyanase is made up of two subunits linked by a disulfide bond between the single cystine residue in each subunit . The native enzyme (Mr 150,000) then appears to be a complex of four or five such subunit dimers.

J Biol Chem, 1983 Jan 10, 258(1), 215 - 21
Studies of the protein encoded by the lon mutation, capR9, in Escherichia coli . A labile form of the ATP-dependent protease La that inhibits the wild type protease; Chung CH et al.; The product of the lon (capR or deg) gene in Escherichia coli is protease La, an ATP-dependent protease with a linked ATPase activity . Unlike most lon mutations, capR9 is dominant over the wild type under certain conditions . When protease La was isolated from R9 cells and from a recessive capR- strain using DEAE-cellulose chromatography, the mutant enzymes showed about 50% of the wild type activity . Unlike the wild type, the R9 and R- proteases were inhibited by addition of NaCl (less than 0.1 M) . In addition, the R9, but not the R-, material inhibited protelysis by normal protease La, and this effect may account for its dominant phenotype . When isolated by phosphocellulose chromatography, the R9 protein lost proteolytic activity but still inhibited the wild type enzyme . This inhibitory activity was purified to near homogeneity using DEAE-cellulose and heparin-agarose chromatography, and corresponded to the 94,000-dalton R9 gene product . At different concentrations, it inhibited ATP-dependent casein degradation and casein-stimulated ATP hydrolysis to a similar extent . Thus, rates of ATP and protein cleavage remained proportional . Similar inhibition of the wild type protease was observed in the presence of DNA which stimulates both protein and ATP hydrolysis . Half-maximal inhibition was observed with approximately a 1:1 ratio of the R9 to the wild type protein . The subunit sizes of the R9 and the wild type protease were indistinguishable but they differed in isoelectric points . Upon gel filtration, both eluted as tetramers (450,000 daltons) in the absence of salt . However, with 0.1 M NaCl, the wild type protease La remained as a tetramer, but the R9 protein dissociated into dimers and monomers and became a more effective inhibitor . After mixing with R9 protein, 3H-labeled protease La remained tetrameric, though it had lost activity . These findings suggest that tetramer formation between the wild type and defective R9 subunits is responsible for the inhibition of the proteolytic and ATPase activities.

J Biol Chem, 1983 Jan 10, 258(1), 150 - 6
Chemical modification in situ of Escherichia coli 30 S ribosomal proteins by the site-specific reagent pyridoxal phosphate . Inactivation of the aminoacyl-tRNA and mRNA binding sites; Ohsawa H et al.; epsilon-Amino groups of lysines of 30 S ribosomal subunits with affinity for phosphate groups were selectively modified in situ by reaction with pyridoxal phosphate and reduction of the Schiff base with nonradioactive or radioactive sodium borohydride . This reaction modified only a limited number of ribosomal proteins and resulted in the loss of only some 30 S activities . The modified proteins were identified and the extent of their modification determined . The main targets of the reaction were S3 greater than S1 greater than S6 . The activity most severely affected by the pyridoxal phosphate reaction was mRNA-dependent aminoacyl-tRNA binding . Some inhibition of poly(U) binding was also observed, while neither binding of initiation factors nor association with 50 S subunits was inhibited . The inhibition of aminoacyl-tRNA binding showed distinct selectivity: the inhibition was far greater with NAcPhe-tRNA than with fMet-tRNA and with "A" site than with "P" site binding . In addition, initiation complex formation with some mRNAs (e.g . MS2 RNA) was affected more than with others (e.g . T7 early mRNA) . Ribosome reconstitution experiments showed that the modification of protein S3 was the primary cause of the inhibition; a role was also played by ribosomal proteins S1, S2, and S21 . Substrate protection experiments showed that the 30 S activity can be protected from pyridoxal phosphate inactivation upon formation of a ternary complex with poly(U) and tRNAPhe or NAcPhe-tRNAPhe . Accordingly, the extent of modification of ribosomal protein S3 was reduced in the ternary complex while modification of S1 was reduced in the presence of poly(U) alone.

FEBS Lett, 1983 Jan 10, 151(1), 159 - 64
DNA sequence of the promoter region of the ompC gene and the amino acid sequence of the signal peptide of pro-OmpC protein of Escherichia coli; Mizuno T et al.; The osmoregulated ompC gene of Escherichia coli was cloned and the DNA sequence of a fragment encompassing the promoter region and a portion of the coding region was determined . There were no obvious homologies in the DNA sequences of the promoter regions of the ompC and ompF genes, in contrast to those of the coding regions of the two genes, both of which code for the matrix porins (major outer membrane proteins) and form passive diffusion pores . The amino acid sequence of the signal peptide of pro-OmpC protein was also deduced from the DNA sequence.

FEBS Lett, 1983 Jan 10, 151(1), 102 - 4
Dependence on pH of substrate binding to lactose carrier in Escherichia coli cytoplasmic membranes; Yamato I et al.; Lactose permease in Escherichia mediates proton-substrate cotransport . The molecular mechanism of this process is not understood . We examined the effect of proton concentration on the binding of a substance analogue to the carrier . The dissociation constant of p-nitrophenyl-alpha-galactoside from the carrier was dependent on pH, with an apparent pKa of 9.7.

J Biol Chem, 1983 Jan 10, 258(1), 333 - 8
Location of 5.8 S rRNA contact sites in 28 S rRNA and the effect of alpha-sarcin on the association of 5.8 S rRNA with 28 S rRNA; Walker TA et al.; We have constructed phage M13mp7 clones each containing the coding strand from one of three restriction fragments which collectively span the mouse 28 S rRNA gene with the exception of the 3'-terminal approximately 500 base pairs . When hybridized to 28 S rRNA, only the fragment containing the 5'-terminal 1400 nucleotides of the gene inhibited the annealing of 5.8 S rRNA to the 28 S rRNA . The same results were obtained when either the 5'- or 3'-terminal fragment of 5.8 S rRNA was used in lieu of intact 5.8 S rRNA, each of which had been shown to contain an independent 28 S rRNA contact site . However, alpha-sarcin, a cytotoxin that inhibits protein synthesis by hydrolyzing a phosphodiester bond near the 3' end of 28 S rRNA, produces a 3'-terminal 488-nucleotide fragment which exhibits a marginal capacity to anneal to 5.8 S rRNA . These results indicate that 5.8 S rRNA interacts predominantly with a structural domain near the 5' end of 28 S rRNA . This conclusion is consistent with base-pairing interactions between 5.8 S rRNA and 28 S rRNA based on the proposed secondary structures for Escherichia coli 23 S and yeast 26 S rRNAs . However, alpha-sarcin treatment of ribosomes affects the stability of the binding of 5.8 S rRNA to the 28 S rRNA, even though the toxin hydrolyzes a phosphodiester bond several thousand nucleotides from the proposed contact regions . Finally, mouse 5.8 S rRNA was shown to lack two internal nucleotides reported to be present in rat 5.8 S rRNA.

J Biol Chem, 1983 Jan 10, 258(1), 604 - 9
H+-ATPase of Escherichia coli uncB402 mutation leads to loss of chi subunit of subunit of F0 sector; Fillingame RH et al.; The uncB402 mutation in Escherichia coli results in formation of an H+-ATPase complex that is defective in energy-transducing capacity . The mutation, originally described by Butlin et al . (Butlin, J.D., Cox, G.B., and Gibson, F . (1973) Biochim . Biophys . Acta 292, 366-375), alters the F0 sector of the H+-ATPase complex . Here, we show that uncB402 is an amber-suppressible, chain-terminating mutation that results in loss of the chi subunit from F0 . This was demonstrated in crude membrane fractions after overproduction of the ATPase complex by heat induction of a lambda transducing phage carrying the unc operon of uncB402 . The lambda-uncB402 DNA was used as a template in an in vitro transcription-translation system . A synthesis product that may correspond to the truncated form of the chi subunit was observed . Despite the absence of chi, the F1-ATPase was still bound to the membrane, although more weakly than in wild type . The omega subunit of F0 ("dicyclohexylcarbodiimide-binding protein") shows normal reactivity with dicyclohexylcarbodiimide, indicating that at least this portion of F0 integrates properly in the membrane in the absence of the chi subunit . The F0 of uncB402 was not functional in H+ translocation activity . This was shown by direct H+ flux measurements with crude membrane vesicles that were treated with guanidine to disrupt the binding of F1 to F0 . Secondly, a method was developed for isolation of F0 from F1-depleted membranes . The F0 from uncB402 was shown to have less than 5% the proton-translocase activity of wild type F0 when reconstituted into liposomes . Although the uncB402 mutant shows these defects, the question of whether the chi subunit plays a direct role in F1-binding or H+ translocation remains open, since the loss of chi may lead to subtle changes in the assembly of the other F0 subunits . Analysis of other mutants should permit a more definitive assignment of function.

Biochim Biophys Acta, 1983 Jan 7, 750(1), 134 - 40
Characterization of phospholipase A2 activity in rat aorta smooth muscle cells; Thakkar JK et al.; Phospholipase A activity was measured in homogenates and acid extracts of smooth muscle cells from rat aorta and mesenteric artery using {1-14 C}oleate-labeled autoclaved Escherichia coli and 1-{1-14C}stearyl-2-acyl-3-sn-glycerophosphorylethanolamine as substrates . The results demonstrate the presence of neutral-active phospholipase(s) A that exclusively catalyze the release of fatty acid from the 2-position of phospholipids . Optimal activity was at pH 7.5, and there was an absolute requirement for low concentrations of Ca2+ . Mg2+ did not substitute for Ca2+, and EGTA inhibited the activity . Phospholipase A2 activity was predominantly membrane-associated and was solubilized by homogenization in 0.18 N H2SO4 . Sulfuric acid extracts of rat aortic smooth muscle cells were four times more active than extracts of mesenteric artery (710 vs . 170 nmol/h per mg protein) . By comparison, acid extracts of rat lung, heart, and liver were less active (60-75 nmol/h per mg) . Indomethacin, sodium meclofenamate, mepacrine and chlorpromazine, but not dexamethasone or aspirin, inhibited acid-solubilized phospholipase(s) A2 between 10(-6) and 10(-3) M in a dose-dependent manner . Preincubation with p-bromophenacyl bromide or diethylpyrocarbonate inhibited phospholipase(s) A2, suggesting the presence of a histidine residue at the active site . An extract from the leaves of feverfew plant (Tanacetum parthenium) was also a potent inhibitor of aortic smooth muscle phospholipase(s) A2.

Biochim Biophys Acta, 1983 Jan 7, 750(1), 41 - 6
Multienzyme complexes of fatty acid oxidation from Escherichia coli K12 and from a mutant with a defective L-3-hydroxyacyl coenzyme A dehydrogenase; Pramanik A et al.; An Escherichia coli mutant (fadB64), with a defective L-3-hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35) which is unable to grow on long-chain fatty acids as the sole carbon source, was shown to possess a fatty acid oxidation complex that contains five beta-oxidation enzymes, including L-3-hydroxyacyl-CoA dehydrogenase . A comparative study of the complexes from the mutant, from its parental strain and from wild-type E . coli B demonstrated the immunological and gross structural identity of all three fatty acid oxidation complexes . A kinetic evaluation of the complexes led to the suggestion that the mutation may have affected the active site of L-3-hydroxyacyl-CoA dehydrogenase so that it is inactive with acetoacetyl-CoA as a substrate, but exhibits an increasing percentage of the parental dehydrogenase activity with increasing chain length of the substrate.

Biochemistry, 1983 Jan 4, 22(1), 94 - 8
Inhibition of ribosomal translocation by peptidyl transfer ribonucleic acid analogues; Wagner T et al.; The activity of peptidyl-tRNALys-CpCp2'dA was measured in an in vitro poly(A)-dependent polypeptide synthesizing system derived from Escherichia coli . It has already been shown that Lys-tRNALys-CpCp2'dA is active as an acceptor and Ac2-Lys-tRNALys-Cp2'dA can donate its peptidyl residue but that the overall poly(A)-dependent synthesis of polylysine does not take place with Lys-tRNALys-CpCp2'dA {Wagner, T., Cramer, F., & Sprinzl, M . (1982) Biochemistry 21, 1521-1529} . This is due to the efficient inhibition of the EF-G-dependent translocation of the peptidyl-tRNA CpCp2'dA from the ribosomal A to the ribosomal P site . In addition, the EF-G-dependent release of the deacylated tRNALys-CpCp2'dA from the ribosomes is also inhibited . The action of the elongation factor G or some other ribosomal component participating in the translocation process requires the presence of the 2'-hydroxyl group on the terminal adenosine of tRNA . If this hydroxyl group is not present on the tRNA, the ribosomes remain locked in their pretranslocational state.

Biochemistry, 1983 Jan 4, 22(1), 228 - 35
lac Repressor headpiece binds specifically to half of the lac operator: a proton nuclear magnetic resonance study; Scheek RM et al.; The complex formation of the N-terminal domain (headpiece) of the Escherichia coli lac repressor and a synthetic 14-base-pair lac operator fragment has been investigated by 1H NMR . Titration shifts in the imino-proton region of the DNA spectrum and in the aromatic region of the headpiece spectrum are examined in detail and interpreted where possible . The assignment of the resonances in the complex follows in part from the titration data and is completed by nuclear Overhauser measurements . The shift of the His-29 C-2 resonance has been used to assess the binding strength of the complex . Evidence is presented for the presence of a high-affinity site on the lac operator fragment (KD less than or equal to 2 X 10(-5) M), which shows features in common with one of the specific binding sites on the complete lac operator, and for the presence of a second, nonspecific binding site with lower affinity . The influence of this second site on the interpretation of the binding data is discussed.

Biochemistry, 1983 Jan 4, 22(1), 158 - 65
A triglobular model for the polypeptide chain of aspartokinase I-homoserine dehydrogenase I of Escherichia coli; Fazel A et al.; Limited proteolysis of aspartokinase I-homoserine dehydrogenase I from Escherichia coli by type VI protease from Streptomyces griseus yields five proteolytic fragments, three of which are dimeric, the other two being monomeric . One of the monomeric fragments (27 kilodaltons) exhibits residual aspartokinase activity, while the second one (33 kilodaltons) possesses residual homoserine dehydrogenase activity . The smallest of the dimeric species (2 X 25 kilodaltons) is inactive; the two other dimers exhibit either only homoserine dehydrogenase activity (2 X 59 kilodaltons) or both activities (hybrid fragment, 89 + 59 kilodaltons) . This characterization of the proteolytic species in terms of molecular weight, subunit structure, and activity leads to the proposal of a triglobular model for the native enzyme . In addition, the time course of the formation of the various fragments was followed by measuring enzymatic activity and performing gel electrophoretic analysis of the protein mixture at defined time intervals during proteolysis . On the basis of the results of these studies, a reaction scheme describing the succession of events during proteolysis is given.

Biochemistry, 1983 Jan 4, 22(1), 150 - 7
Stereoisomers of tetrahydrothiamin pyrophosphate, potent inhibitors of the pyruvate dehydrogenase multienzyme complex from Escherichia coli; Lowe PN et al.; Tetrahydrothiamin pyrophosphate, an analogue of thiamin pyrophosphate (TPP) in which the thiazolium ring has been reduced to a thiazolidine ring, was prepared by borohydride reduction of TPP . It consists of four stereoisomers, comprising two diastereomers each of which is a racemic mixture, generated by the introduction of two chiral centers on the thiazolidine ring . The major and minor diastereomers were separated and inferred to be of the cis and trans configurations, respectively, from a study of the nuclear Overhauser effects in the 1H NMR spectrum of the simpler tetrahydrothiamin . Tetrahydro-TPP behaves as a mixture of potent inhibitors of the pyruvate decarboxylase (E1) component of the pyruvate dehydrogenase complex from Escherichia coli . The site of binding is probably the TPP-binding site on E1, and the Kd for each of the four stereoisomers was estimated . The cis isomers of tetrahydro-TPP bind more tightly than does TPP, whereas the trans isomers appear to bind with about the same Kd as TPP . Sodium borohydride caused a rapid inhibition of E1 activity in the presence of TPP, believed to be due to reaction of borohydride with enzyme-bound TPP . The experiments are consistent with an enhancement of the reactivity of the thiazole ring of TPP when bound to the catalytic site of E1, which could be due to polarization of the greater than +N=C bond near a hydrophobic or positively charged region of the protein . A spontaneous reactivation occurred after the initial inhibition by borohydride, attributable to a weakly binding inhibitor, not tetrahydro-TPP, being formed at the catalytic site.






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