Genetika, 1978 Jun, 14(6), 947 - 56 {Gene relA function in the expression of amino acid operons . I . Effect of the allelic state of gene relA on phenotypic manifestations of auxotrophic threonine and isoleucine mutations in Escherichia coli K-12}; Livshits VA et al.; The substitution of the relA gene mutant allele with wild type allele of this gene in strictly auxotrophic strain of Escherichia coli K-12 GT25, carrying thr B1007 mitation, results in the appearance of the partial dependence of the bacterial growth upon threonine . On the other hand, the introduction of relA mutation into genome of incomplete threonine auxotroph, which was isolated as pseudorevertant from the strict threonine auxotroph CP78, recovered the strict dependency of the growth on the presence of threonine in the medium . The introduction of relA mutation into genome of partial isoleucine auxotroph, carrying a mutation in ilvA gene, reduces the residual activity of threonine deaminase under the conditions of derepression and results in the appearance of strict dependency of bacterial growth on the presence of isoleucine . These data indicate that operons, which control the biosynthesis of threonine and isoleucine, are positively regulated by the product of relA gene . The possibility of using leaky mutations, which lead to incomplete block of these amino acids synthesis, for testing allelic state of relA gene is discussed. Br J Exp Pathol, 1978 Jun, 59(3), 292 - 7 Escherichia coli infection in mice and impaired fetal development; Coid CR et al.; Investigations were undertaken, using the mouse as an animal model, to study the effect of Escherichia coli on fetal development . The i.v . injection of 7.5 X 10(6) bacteria, originally obtained from a suspected case of human pyelonephritis, caused only a mild and transient disturbance of maternal health but caused severe fetal wastage . Groups of mice were examined 4, 7 and 11 days after infection and the numbers of organisms were determined in the spleen, liver, kidneys, placentas and resorptions . From the findings obtained, it was concluded that the Esch . coli grew preferentially in the placentas . By the 7th day the placentas showed marked degenerative and necrotic changes and the bacteria could be recovered from the majority of fetuses at this time . Histologically, no significant changes were seen in the spleen, liver and kidneys . As a result of these findings in an animal model, and taking into consideration the observations of other workers, it is suggested that coliform bacteraemia in human pregnancies may also cause infections of the placenta and bring about abortion or premature delivery. Br J Cancer Suppl, 1978 Jun, 37(3), 124 - 8 Activation of radiosensitizers by hypoxic cells; Olive PL et al.; Hypoxic cells can metabolize nitroheterocyclic compounds to produce toxic intermediates capable of affecting the survival of neighbouring oxygenated cells . Mutagenesis experiments with E . coli WP-2 343 (deficient in nitroreductase) indicated that reduction of nitroheterocyclics outside bacteria causes killing and mutations within bacteria, presumably due to the transfer of the "active" specie (s) . Using animal tissue slices to reduce nitrofurans, cultured L-929 cells incubated under aerobic conditions were far more sensitive to the toxic and DNA damaging effects of these drugs . Transfer of the active species also occurs in a tissue-like environment in multicell spheroids where the presence of a hypoxic central core served to convert the nitroheterocyclics to intermediates which also damaged the neighbouring oxygenated cells. Appl Environ Microbiol, 1978 Jun, 35(6), 1003 - 7 Comparison of the toxicities of patulin and patulin adducts formed with cysteine; Lindroth S et al.; The toxicities of patulin and of the patulin adducts formed with cysteine were compared using the mutation-sensitive strain Escherichia coli W3110 thy polA1 and its polA1+ revertant . The acute toxicities of patulin and of the adduct mixture were also compared using NMRI mice . The adduct mixture was shown by thin-layer chromatography to consist of one ninhydrin-positive, one ninhydrin- and MBTH (3-methyl-2-benzothiazolinone hydrazone)-positive, three MBTH-positive, and two ninhydrin- and MBTH-negative components . The results showed that patulin was over 100 times more toxic to E . coli than the adduct complex . Neither patulin nor the adduct mixture was found to induce the repair effect in E . coli . In the mouse feeding tests, the oral 50% lethal dose for patulin was 29 mg/kg, while that of the adduct mixture was greater than 2,370 mg/kg. Zh Mikrobiol Epidemiol Immunobiol, 1978 Jun, (6), 61 - 6 {Biochemical and serological characteristics of Escherichia belonging to the serological group 01}; Drobyshevskaia EI et al.; A circulation at the territory of the country of various biochemical and serological variants of escherichia belonging to serological group O1, isolated in acute intestinal diseases of children and adults, was revealed . Nonhomogeneousness of the partial composition of the O-antigen was demonstrated; K-antigens were determined; new H-antigens were described . Of the 10 serological types of escherichia there proved to prevail O1 : K? : Hp and O1 : K1 : Hp; in group and sporadic acute intestinal diseases there were for the first time isolated O1 : K1 : H34, O1 : K1 : H20, O1 : K1 : Hp, O1 : K51 : H7, and O1 : K? : H20. Zh Mikrobiol Epidemiol Immunobiol, 1978 Jun, (6), 22 - 6 {Comparative assessment of the efficacy in humoral response of T-cells of various organ origin and their substitutes}; Iarilin AA et al.; A linear dependence of the response to the thymus-dependent antigen (log of the plaque-forming cell count) on the T cell dose at the initial curve section was observed in syngeneic transfer of T and B cells mixture . The exponential slope differed for T cells of different origin and could serve as the measure of helper activity . In case of an excess of T-lymphocytes the response reaches the maximum, whose level is independent of the organic origin of T cells . By the helper activity T cells are distributed in the following order: T cells of the spleen and cortisone-resistant thymocytes greater than T cells of the lymph nodes greater than cortisone-sensitive thymocytes . There was established a quantitative equivalence by the capacity to activate B cells between the T-lymphocytes and E . coli lipopolysaccharide. Nucleic Acids Res, 1978 Jun, 5(6), 2073 - 94 Studies on nucleic acid reassociation kinetics: V . Effects of disparity in tracer and driver fragment lengths; Chamberlin ME et al.; Measurements are described of the kinetics of nucleic acid strand pair reassociation where the complementary strands are of different lengths and are present in different concentrations . Rate constants for the reaction of labelled fragments ("tracer") with excess complementary strands ("driver") were determined, both for driver fragment length greater than tracer fragment length and for the reverse case . Second order reactions and pseudo-first order reactions utilizing strand separated drivers and tracers were studied . The nucleic acids which served for this investigation were phiX174 DNA and RNA, plasmid RSF2124 DNA and E . coli DNA . Approximate empirical expressions relating driver and tracer fragment lengths with the observed rate constants were obtained for practical use . In long tracer-short driver reactions the observed rate constant for the tracer reaction increases proportionately with tracer length . In long driver-short tracer reactions the rate of tracer reaction is retarded . The latter result is unexpected and appears to represent a departure from standard interpretations of the renaturation reaction. Nucleic Acids Res, 1978 Jun, 5(6), 1971 - 8 Formation of O2-methylthymine in poly(dA-dT) on methylation with N-methyl-N-nitrosourea and dimethyl sulphate . Evidence that O2-methylthymine does not miscode during DNA synthesis; Saffhill R et al.; The alternating co-polymer has been methylated with either N methyl-N-nitrosourea (MNU) or dimethyl sulphate (DMS) and the levels of the various methylated thymidines (O2-methylthymidine, 3-methylthymidine and O4-methylthymidine) measured . MNU produced all three compounds whereas DMS only produced 3-methylthymidine and O2-methylthymidine at detectable levels . These results have been combined with our earlier results concerning the misincorporation of dGMP with E . coli DNA polymerase using MNU-methylated poly(dA-dT) . These results indicate that O2-methylthymidine does not miscode during DNA synthesis. Nucleic Acids Res, 1978 Jun, 5(6), 1801 - 20 In vitro construction of deletion mutants of the bacteriocinogenic plasmid Clo DF13; Stuitje AR et al.; The isolation and characterization of deletion mutants of the bacteriocinogenic plasmid Clo DF13 is described . To construct these deletion mutants, DNA of Clo DF13::Tn901 and Clo DF13-rep3::Tn901 plasmids was digested with restriction endonucleases, ligated with T4 ligase and introduced by transformation into Escherichia coli . The presence of the ampicilline transposon Tn901 facilitated the selection of plasmids . The resulting Clo DF13::Tn901 deletion mutants were analyzed by digestion with restriction endonucleases and electron microscopy . From the properties of the various deletion mutants it was concluded that a Clo DF13 DNA region, extending from 5 to 11.5% on the physical map, is essential for the replication of Clo DF13 . This region, comprising about 600 base pairs, contains in addition to an origin of replication, DNA sequences which are involved in the regulation of Clo DF13 DNA replication . Furthermore it was observed that in case of the Clo DF13 copy mutant, Clo DF13-rep3, deletion of the 43% to 63% part of the plasmid genome, resulted in the generation of multimeric plasmid structures, accompanied with an impaired segregation of the plasmids to daughter cells. Nucleic Acids Res, 1978 Jun, 5(6), 1767 - 77 Construction of recombinant plasmid carrying the lambda DNA fragment responsible for prophage integration; Strizhov N et al.; The recombinant DNA molecules were constructed from plasmid RSF2124 and the EcoRI fragment of lambda DNA containing the genes responsible for prophage integration . The presence of these genes in recombinant plasmids was detected genetically . lambda int-gene was shown to be expressed in either orientation of insertion in the plasmid . We found that recombinant plasmid was able to integrate into chromosome of lambda lysogens . The integration of plasmid into host chromosome was demonstrated by contransduction of chromosome and plasmid markers using generalized transducer P1 and by specialized transduction with lambda phages. Nucleic Acids Res, 1978 Jun, 5(6), 1753 - 66 Fragment of protein L18 from the Escherichia coli ribosome that contains the 5S RNA binding site; Newberry V et al.; A fragment of ribosomal protein L18 was prepared by limited trypsin digestion of a specific complex of L18 and 5S RNA . It was characterised for sequence and the very basic N-terminal region of the protein was found to be absent . No smaller resistant fragments were produced . 5S RNA binding experiments indicated that the basic N-terminal region, from amino acid residues 1 to 17, was not important for the L18-5S RNA association . Under milder trypsin digestion conditions three resistant fragments were produced from the free protein . The largest corresponded to that isolated from the complex . The smaller ones were trimmed slightly further at both N- and C-terminal ends . These smaller fragments did not reassociate with 5S RNA . It was concluded on the basis of the trypsin protection observations and the 5S RNA binding results that the region extending from residues 18 to 117 approximates to the minimum amount of protein required for a specific and stable protein-RNA interaction . The accessibility of the very basic N-terminal region of L18, in the L18-5S RNA complex, suggests that it may be involved, in some way, in the interaction of 5S RNA with 23S RNA. J Virol, 1978 Jun, 26(3), 630 - 45 Genome organization of RNA tumor viruses II . Physical maps of in vitro-synthesized Moloney murine leukemia virus double-stranded DNA by restriction endonucleases; Verma IM et al.; Physical maps of the genome of Moloney murine leukemia virus (M-MLV) DNA were constructed by using bacterial restriction endonucleases . The in vitro-synthesized M-MLV double-stranded DNA was used as the source of the viral DNA . Restriction endonucleases Sal I and Hind III cleave viral DNA at only one site and, thus, generate two DNA fragments . The two DNA fragments generated by Sal I are Sal IA (molecular weight, 3.5 x 10(6)) and Sal IB (molecular weight, 2.4 x 10(6)) and by Hind III are Hind IIIA (molecular weight, 3.6 x 10(6) and Hind IIIB (molecular weight, 2.3 x 10(6)) . Restriction endonuclease Bam I generates four fragments of molecular weights of 2.1 x 10(6) (Bam IA), 2 X 10(6) (Bam IB), 1.25 X 10(6) (Bam IC), and 0.24 x 10(6) (Bam ID), whereas restriction endonuclease Hpa I cleaves the M-MLV double-stranded DNA twice to give three fragments of molecular weights of 4.4 x 10(6) (Hpa IA), 0.84 X 10(6) (Hpa IB), and 0.74 x 10(6) (Hpa IC) . Digestion of M-MLV double-stranded DNA with restriction endonuclease Sma I produces four fragments of molecular weights of 3.9 x 10(6) (Sma IA), 1.3 X 10(6) (Sma IB), 0.28 X 10(6) (Sma IC), and 0.21 x 10(6) (Sma ID) . A mixture of restriction endonucleases Bgl I and Bgl II (Bgl I + II) cleaves the viral DNA at four sites generating five fragments of approximate molecular weights of 2 x 10(6) (Bgl + IIA), 1.75 X 10(6) (Bgl I + IIB), 1.25 X 10(6) (Bgl I + IIC), 0.40 X 10(6) (Bgl I + IID), and 0.31 x 10(6) (Bgl I + IIE) . The order of the fragments in relation to the 5' end and 3' end of the genome was determined either by using fractional-length M-MLV double-stranded DNA for digestion by restriction endonucleases or by redigestion of Sal IA, Sal IB, Hind IIIA, and Hind IIIB fragments with other restriction endonucleases . In addition, a number of other restriction endonucleases that cleave in vitro-synthesized M-MLV double-stranded DNA have also been listed. J Biochem (Tokyo), 1978 Jun, 83(6), 1699 - 705 Relaxation effect of chloramphenicol on the stringent control in Escherichia coli; Sokawa J et al.; In 10B601 (rel+) strain possessing a temperature-sensitive valyl-tRNA synthetase, chloramphenicol prevented the formation of guanosine-3'-diphosphate-5'-diphosphate (ppGpp) as well as the stringent control of stable RNA synthesis, under the conditions where the incorporation of valine into protein was still detectable i.e . at the lower restrictive temperatures . On the other hand, the effect of chloramphenicol was not observed at higher restrictive temperatures above 42 degrees C where the incorporation of valine was completely absent . Pretreatment of 10B601 cells with chloramphenicol before transfer to a high restrictive temperature (43.5 degrees C) did retard the onset of accumulation of ppGpp after the shift-up . Duration of the lag period was dependent on the concentration of chloramphenicol added . In parallel with the inability of the cells to accumulate ppGpp, stable RNA synthesis was permitted to continue at that high temperature . These results suggest that chloramphenicol traps aminoacyl-tRNA at the A-sites of ribosomes by damming-up the small flow of aminoacyl-tRNA under the restrictive supply of amino acids . Unchanged tRNA which has been located at the A-site is replaced by the charged one, thus resulting in the suppression of ppGpp formation and in the restoration of stable RNA synthesis. Infect Immun, 1978 Jun, 20(3), 867 - 8 Coupling of Escherichia coli lipopolysaccharide to epoxy-activated Sepharose 6B; Fox J et al.; Escherichia coli O111:B4 lipopolysaccharide was coupled to epoxy-activated Sepharose 6B . The bound lipopolysaccharide was immunogenic and immunoadsorptive although at less efficiency than free lipopolysaccharide. Infect Immun, 1978 Jun, 20(3), 744 - 51 Mechanisms of lipopolysaccharide-initiated rabbit platelet responses: alternative complement pathway dependence of the lytic response; Morrison DC et al.; Experiments were performed to examine the relationship of endotoxin-initiated complement activation and rabbit platelet lysis . The results of these experiments supported the concept that activation of the alternative pathway is required for endotoxin-initiated complement-dependent rabbit platelet lysis . Our data demonstrated that preparations of endotoxin or isolated lipid A, which activate selectively the classical pathway, are incapable of initiating platelet lysis . Essentially equivalent results were obtained in citrated or heparinized plasma, although the latter anticoagulated plasma appeared to be more efficient in supporting lysis . Additional data support the concept that natural antibody to either the polysaccharide or the lipid A region of the lipopolysaccharide, which might be present in rabbit plasma, probably did not play a prominent role in the complement-mediated lytic response. Hoppe Seylers Z Physiol Chem, 1978 Jun, 359(6), 683 - 90 Release of granulocyte elastase in lethal canine endotoxin shock; Aasen AO et al.; The release of granulocyte elastase and its interaction with plasma protease inhibitors was studied in dogs receiving a slow infusion of a lethal dose of Escherichia coli endotoxin . During endotoxin infusion a marked decline in leucocyte counts was parallelled by a rapid increase in plasma granulocyte elastase concentrations . Maximal values were reached after 3 h, when the infusion was ended . Crossed immunoelectrophoresis with antiserum against granulocyte elastase did not reveal the presence of elastase components with the electrophoretic mobility of free elastase, but elastase-alpha1-antitrypsin complexes were detected . A gradually decreasing plasma concentration of alpha2-macroglobulin was noted during the experiments . Crossed immunoelectrophoresis, however, did not reveal any electrophoretic heterogeneity . It is concluded that the release of granulocyte proteases might be of significance for several pathophysiological changes seen in endotoxin shock. Gann, 1978 Jun, 69(3), 331 - 7 Immune response of mice in immunotherapy of tumors with syngeneic antitumor serum plus lipopolysaccharide; Saito M et al.; Humoral and cellular immune responses were investigated after combination therapy with syngeneic antitumor serum and bacterial lipopolysaccharide (LPS) . The titer of antitumor antibody determined by using a macrophage-mediated system was very high in mice cured by the combination therapy, and this high titer lasted for a long time . In contrast, no significant titer was detected using an antibody-dependent lymphocyte-mediated system . Thus, antibody-dependent macrophage-mediated cytolysis was a more sensitive method for detecting antitumor antibody . The cellular immune response was measured as the delayed hypersensitivity reaction to tumor cells . In mice that had been cured by combination therapy, this reaction appeared at an early stage, before any antitumor antibody was detectable, but it soon decreased . On the other hand, tumor-bearing mice showed a low level of antibody and no significant delayed hypersensitivity reaction. Eur J Biochem, 1978 Jun 1, 87(1), 131 - 6 Consequences of a specific cleavage in situ of 16-S ribosomal RNA for polypeptide chain initiation; Baan RA et al.; The effect of bacteriocin (cloacin DF13) treatment of Escherichia coli ribosomes on initiation of protein synthesis has been studied in detail . In agreement with our previous findings {Baan et al . (1976) Proc . Natl Acad . Sci . U.S.A . 73, 702--706} it is shown that 70-S initiation complexes can be formed with cloacin-treated ribosomes, but that the initiation factor IF-1 does not function properly . The following pleiotropic effects of this factor have been studied: (a) the acceleration of ribosomal subunit exchange with 70-S couples; (b) the stimulation of the IF-3-mediated dissociation of 70-S ribosomes; (c) the stimulation of 30-S initiation complex formation; (d) the enhancement of the rate of release of IF-2 from 70-S initiation complexes . The effects (a) and (b) are virtually abolished after cleavage of 16-S rRNA . The effect (d) is only partially reduced whereas effect (c) seems to be unimpaired . It is concluded that 70-S initiation complex formation with cloacin-treated ribosomes suffers from improper functioning of IF-1 in the generation of active subunits from 70-S tight couples . This is the only effect on initiation . It can be compensated for by adding more IF-3 . The data provide functional evidence that 16-S rRNA is involved in ribosomal subunit interaction. Cell, 1978 Jun, 14(2), 211 - 9 A yeast mutant which accumulates precursor tRNAs; Hopper AK et al.; It has been proposed that the conditional yeast mutant ts136 is defective in the transport of mRNA from the nucleus to the cytoplasm (Hutchinson, Hartwell and McLaughlin, 1969) . We have examined ts136 to determine whether it is defective in tRNA biosynthesis . At the restrictive temperature, the mutant accumulates twelve new species of RNA . These species co-migrate on polyacrylamide gels with some of the pulse-labeled precursor tRNAs . Three of the new RNAs (species 1a, 1b and 1c are large enough to contain two tandom tRNAs . Although RNAs 1a, 1b, and 1c do not contain detectable levels of modified and methylated bases, at least one of them hybridizes to DNA from an E . coli plasmid containing a yeast tRNA gene . All the remaining RNAs (2--8) contain modified and methylated bases typical of tRNA . Three of these species were tested and were found to hybridize to tRNA genes . Ribosomal RNA synthesis is also defective in ts136 . It is suggested that ts136 may be defective in a nucleolytic activity, which is a prerequisite to RNA transport. Can J Microbiol, 1978 Jun, 24(6), 761 - 4 Effect of amino acid deprivation and chloramphenicol treatment on cell sizes of rel+ and relA- strains of Escherichia coli; Ishiguro EE et al.; The effects of inhibition of protein synthesis on the cell size distributions of rel+ and relA- derivatives of Escherichia coli K-12 were determined . Amino acid deprivation resulted in a reduction in the cell sizes of rel+ strains but not of relA- strains . Treatment with chloramphenicol (CAM) did not alter the size distributions of either rel+ or relA- strains except when they were rel+ dap- . CAM treatment of rel+ dap- strains resulted in an increase in cell size . It is proposed that these results reflect differences in the structures of the cell envelopes of rel+ and relA- bacteria. Can J Biochem, 1978 Jun, 56(6), 654 - 8 Structure and function of aspartate transcarbamoylase studied using chymotrypsin as a probe; Chan WW et al.; Aspartate transcarbamoylase from Escherichia coli is composed of six catalytic (c) and six regulatory (r) polypeptides . We have studied the structure and function of this enzyme using chymotrypsin as a probe . The protease inactivates the isolated catalytic subunit (c3) but has not effects on the native enzyme (c6r6) . Under identical conditions, the c3r6 complex is inactivated at a much slower rate than c3 . The presence of the substrate analogue succinate together with carbamoyl phosphate reduces substantially the rate of inactivation . Extended exposure to chymotrypsin converts the catalytic subunit into a partially active derivative with a fourfold higher Michaelis constant . This derivative is indistinguishable from the unmodified catalytic subnit in gell electrophoresis under nondenaturing conditions . However, in the presence of sodium dodecyl sulfate, the major fragment in the electropherogram is smaller than that of the intact catalytic polypeptide . The results could be explained by postulating the presence of a chymotrypsin-sensitive peptide bond at or near the active site . Since X-ray crystallographic studies have indicated that the active sites are located in a central cavity, the resistance of the native enzyme towards inactivation may be due to the inability of chymotrypsin to enter this cavity. Can J Biochem, 1978 Jun, 56(6), 528 - 33 The relationship between the spoT gene, the synthesis of stable RNA, ribosomal proteins, and the beta beta' subunits of RNA polymerase following a nutritional shiftup of Escherichia coli; Boyle SM et al.; The level of ppGpp and rates of synthesis of stable RNA, ribosomal protein, and the beta and beta' subunits of RNA polymerase were measured following a nutritional shiftup in Escherichia coli strains, NF 929 (spoT+) and NF 930 (spoT-) . In the spoT+ strain, ppGpp levels decreased 50% within 2 min following shiftup, and the rates of synthesis of stable RNA, ribosomal proteins, and the beta and beta' subunits of RNA polymerase increased with little or no lag . In contrast, in the spoT- strain, ppGpp levels transiently increased 40% during the first 6 min following shiftup . An inhibition in the rate of stable RNA synthesis and a delay in the increased synthesis of ribosomal proteins and beta and beta' subunits occurred concurrently with the transient increase in ppGpp . In addition, the DNA-dependent synthesis in vitro of the beta and beta' subunits of RNA polymerase was inhibited by physiological levels of ppGpp . Because of the timing and magnitude of the changes in ppGpp levels in the spoT- strain versus the timing when the new rates of stable RNA, ribosomal protein, and beta and beta' subunits synthesis are reached, it is concluded that ppGpp is not the sole element regulating the expression of these genes. Biophys J, 1978 Jun, 22(3), 431 - 8 Induced radioresistance in four strains of Escherichia coli, two with lambda lysogens; Pollard EC; Cells of E . coli that are recA+ and lex+ show a phenomenon of induced radioresistance . A preexposure to ultraviolet light, or ionizing radiation followed by incubation to allow protein synthesis, followed by treatment with rifampin to prevent further induction, renders the cells resistant to further doses of radiation . When this is attempted with lambda lysogens of the same strains, no radioresistance is seen, even though the preexposure is too small to induce lambda itself . If the lysogens are ind-, namely lambda C1857, about the normal radioresistance can be developed by pretreatment . These findings suggest that the lambda repressors can bind to single-strand breaks caused by the inducing agent and can modify the course of induction. Biochem J, 1978 Jun 1, 171(3), 719 - 23 Kinetic studies with the use of proton-magnetic-resonance spectroscopy of the specific alpha-deuteration of amino acids by Escherichia coli aspartate aminotransferase; Gout E et al.; Escherichia coli aspartate aminotransferase was exposed to aspartate or phenylalanine without oxo acid in buffered 2H2O . The alpha-hydrogen of the amino acids underwent first-order exchange with respect to both substrate and enzyme . P.m.r . spectroscopy gave consistent reaction-rate constants . The deuterium-exchange rate was only moderately increased by addition of oxo acids and was of the same order as the transamination rate . No beta-deuteration was observed . The C(alpha)-H-bond-breaking step is discussed as a part of the entire transamination mechanism. Proc Natl Acad Sci U S A, 1978 Jun, 75(6), 2569 - 73 Reconstitution of an Escherichia coli repair endonuclease activity from the separated uvrA+ and uvrB+/uvrC+ gene products; Seeberg E; An in vitro complementation assay has been used for partial purification of uvrA+, uvrB+, and uvrC+ gene products from Escherichia coli . The uvrB+ and uvrC+ products cochromatograph on DEAE-cellulose and are completely resolved from the uvrA+ product, which has been further purified by phosphocellulose chromatography of the nonadsorbed protein fraction from the DEAE-cellulose . Neither the uvrB+/uvrC+ nor the uvrA+ product shows appreciable endonuclease activity on UV-irradiated DNA when tested separately . However, these factors complement each other to yield and ATP-dependent endonuclease activity specific for UV-irradiated DNA . Gel filtration experiments with the partially purified proteins indicate that the functional uvrA+ gene product has a molecular weight of 100,000 . The uvrB+ gene product has an apparent molecular weight of 70,000, but it is presently unclear if this is the size of the uvrB+ product alone or the size of a complex of the uvrB+ and uvrC+ gene products. Nature, 1978 Jun 1, 273(5661), 354 - 8 A mutation affecting the sigma subunit of RNA polymerase changes transcriptional specificity; Travers AA et al.; The RNA polymerase mutation, alt-1, affects the sigma subunit and alters the in vitro selectivity of RNA polymerase to parallel the in vivo phenotype . We propose that the mutation changes the distribution of functionally distinct polymerase isomers. J Gen Virol, 1978 Jun, 39(3), 531 - 5 Transfection of Escherichia coli spheroplasts: infectious lambda prophage DNA; Benzinger R et al.; High mol . wt . DNA was extracted from Escherichia coli lambda lysogens and was shown to be infectious . Its infectivity was due to prophage DNA integrated into the host chromosome rather than to DNA released from mature phage particles, as established by the following criteria: the titre of infectious DNA exceeded by 100-fold the titre of infectious units present before DNA extraction; mild shear selectively reduced prophage DNA infectivity to 2% of the unsheared DNA while lambda phage DNA infectivity retained 50% of its infectivity; DNA extracted from an E . coli (lambda c857 tsxisam6) lysogen yielded 200 times as many plaques on sup+ than on sup- spheroplasts . Thus lambda prophage DNA infectivity depends on expression of the excision gene while the infectivity of non-integrated forms of lambda does not . About 10(4) genome equivalents of E . coli DNA yielded one infectious centre unit in this assay system; this high infectivity should make prophage DNA a useful marker in genetic transformation experiments. J Bacteriol, 1978 Jun, 134(3), 958 - 66 Recombinant levels of Escherichia coli K-12 mutants deficient in various replication, recombination, or repair genes; Zieg J et al.; Escherichia coli strains containing mutations in lexA, rep, uvrA, uvrD, uvrE, lig, polA, dam, or xthA were constructed and tested for conjugation and transduction proficiencies and ability to form Lac+ recombinants in an assay system utilizing a nontandem duplication of two partially deleted lactose operons (lacMS286phi80dIIlacBK1) . lexA and rep mutants were as deficient (20% of wild type) as recB and recC strains in their ability to produce Lac+ progeny . All the other strains exhibited increased frequencies of Lac+ recombinant formation, compared with wild type, ranging from 2- to 13-fold . Some strains showed markedly increased conjugation proficiency (dam uvrD) compared to wild type, while others appeared deficient (polA107) . Some differences in transduction proficiency were also observed . Analysis of the Lac+ recombinants formed by the various mutants indicated that they were identical to the recombinants formed by a wild-type strain . The results indicate that genetic recombination in E . coli is a highly regulated process involving multiple gene products. J Bacteriol, 1978 Jun, 134(3), 944 - 9 3-hydroxypyruvate substitutes for pyridoxine in serC mutants of Escherichia coli K-12; Shimizu S et al.; Escherichia coli K-12 mutants with serC genotype required pyridoxine and serine for normal growth, as do E . coli B mutants of this type . Mutants of the K-12 strain, however, reverted easily to pyridoxine independence without regaining activity in the 3-phosphoserine oxoglutarate transaminase coded for by the serC gene . Both these revertants and the parental type synthesized pyridoxine in normal amounts when 3-hydroxypyruvate was used as a supplement, although neither of these mutants could use this compound to satisfy their serine requirement . Since serine alone was inadequate to provide the nutritional requirement of serC mutants, these mutants must have been unable to synthesize 3-hydroxypyruvate from serine . We suggest that 3-phosphoserine oxoglutarate transaminase in normal E . coli serves as a catalyst for transaminating small amounts of serine to 3-hydroxypyruvate, which is then used in pyridoxine biosynthesis . In serC mutants, this activity is blocked, and these mutants then show a double requirement for serine and pyridoxine. J Bacteriol, 1978 Jun, 134(3), 929 - 35 Hyper-recombination in Escherichia coli K-12 mutants constitutive for protein X synthesis; Lloyd RG; Genetic recombination was studied in Escherichia coli F- strains in which synthesis of the recA gene product protein X is increased due to mutation in either recA (tif-1) or lexA (spr) . When a single donor marker was selected, the recombination proficiency of these strains was not significantly altered in Hfr crosses . However, linkage of unselected, proximal Hfr markers was found to be much reduced among the progeny tested, and more of the progeny showed evidence of multiple exchanges between donor and recipient DNA . These effects were much more apparent when the recipient carried both tif-1 and spr mutations, but in this case recombination proficiency was reduced compared with those strains carrying either mutation alone, particularly in crosses with Hfr Cavalli . A lexA mutation was found to suppress the effect of tif-1 on the recombinant genotype. J Bacteriol, 1978 Jun, 134(3), 913 - 9 Rich culture medium for the radiochemical labeling of proteins and nucleic acids; Oeschger MP; Yeast extract was treated with tyrosine decarboxylase and used to prepare a rich, complex medium virtually free of tyrosine . The medium supported maximal growth rates for Escherichia coli prototrophs, as well as for defined and undefined auxotrophs . It has made possible the efficient radiochemical labeling of cells growing optimally in complex medium and the characterization of mutants with undefined requirements . Similarly prepared media may be useful for the study of fastidious organisms and organisms for which no defined medium has been described. J Bacteriol, 1978 Jun, 134(3), 902 - 12 Chromosome replication in an Escherichia coli dnaA mutant integratively suppressed by prophage P2; Kuempel PL et al.; Escherichia coli CRT4624-P2sig5 is a dnaA mutant in which integration of the prophage P2sig5 has occurred at the attP2II site (min 85) . This strain was integratively suppressed, and when cells were shifted to 42 degrees C replication was initiated at a site in or near the P2 prophage . Initially, this replication occurred primarily in the direction that corresponds to the clockwise direction on the genetic map . Replication also occurred in the counterclockwise direction, but the initiation of replication in this direction occurred approximately 40 min later than the initiation of replication in the other direction . Because of this delay, the replication forks that traveled in the clockwise direction were the first to arrive in the region of the replication terminus . These replication forks ceased replication near the aroD locus (min 37), and it is proposed that the replication terminus is between the aroD and rac loci (min 31) . A model is proposed for the cycle of chromosome replication in this strain at 42 degrees C. J Bacteriol, 1978 Jun, 134(3), 808 - 20 Dependence of Escherichia coli hyperbaric oxygen toxicity on the lipid acyl chain composition; Harley JB et al.; This study examines certain membrane-related aspects of oxygen poisoning in Escherichia coli K1060 (fabB fadE lacI) and its parent strain, K-12 Ymel . Cells were grown to exponential or stationary phase in a minimal medium and exposed to air plus 300 lb/in2 of O2 as a suspension in minimal salts . After an initial lag, both strains lost viability with apparent first-order kinetics . Hypebaric oxygen was more toxic to cells harvested during the exponential phase of growth than to cells harvested from the stationary phase of growth for both strains K-12 Ymel and K1060 . Control suspensions exposed to air plus 300 lb/in2 of N2 did not lose viability during a 96-h exposure . The sensitivity of the unsaturated fatty acid auxotroph, strain K1060, to hyperbaric oxygen increased as the degree of unsaturation of the fatty acid supplement increased . Cells grown with a cyclopropane fatty acid (9,10=methylenoctadecanoate) were the most resistant; cells grown with a monounsaturated fatty acid (oleate) were intermediate; and those grown with polyunsaturated fatty acids (linoleate and linolenate) were most sensitive to hyperbaric oxygen . The parent strain, K-12 Ymel, lost viability in hyperbaric oxygen most similarly to strain K1060 supplemented with oleate . To determine the relative effect of hyperbaric oxygen on the survival of E . coli with saturated membranes, substrains of K1060 were selected for growth on 12-methyltetrade-canoate or on 9 or 10-monobromostearate . Substrains grown with a saturated fatty acid supplement were equally or more sensitive to hyperbaric oxygen than when the same substrains were grown with a cyclopropane fatty acid supplement . The lipid acyl chain composition was determined in E . coli K1060 before and after exposure to hyperbaric oxygen or hyperbaric nitrogen . The proportion of nonsaturated acyl chain lipid of either the oleate- or the 9,10-methyleneoctade-canoate-supplemented K1060 remained unchanged after hyperbaric gas exposure . In strain K1060 supplemented with linoleate and grown to stationary phase, however, the relative unsaturated acyl chain content after hyperbaric exposure decreased in both gases . This finding prompted an investigation of the role of lipid oxidation in hyperbaric oxygen toxicity . Assays of potential lipid oxidation products were performed with linoleate-grown cells . The lipid hydroperoxide and peroxide content of the lipid extract increased by 6.9 times after 48 h of air plus 300 lb/in2 of O2; malondialdehyde and fluorescent complex lipid oxidation products showed much smaller or no changes . Lipid extracts from hyperbaric oxygen-exposed cells were not toxic to viable E . coli K1060, nor did they increase the rate of loss of viability in cells simultaneously exposed to hyperbaric oxygen . Linoleic acid hydroperoxide at 1.0 mM had no effect on the viability of E . coli K-12 Ymel and only marginally decreased the viability of E . coli K1060 supplemented with linoleate . We conclude that the kinetics of oxygen toxicity in E... J Bacteriol, 1978 Jun, 134(3), 801 - 7 Variations among glyV-derived glycine tRNA suppressors of glutamic acid codons; Murgola EJ et al.; Glutamic acid codon suppressors in 18 isogenic strains of Escherichia coli have been further characterized as to map location, dominance, growth rates in various media, suppression of the GAG codon, and tRNA profiles after reversed-phase column chromatography . In general the evidence supports the conclusion that all of these suppressors are due to mutations in glyV55, the gene for a GGA/G-reading mutant form of glyV tRNA, and that they represent several different classes that may correspond to at least as many different nucleotide changes . Furthermore, 17 of the 18 suppressors can coexist in a haploid genome with a glyT suppressor that is devoid of GGA-reading ability . This result indicates the retention by those glyV suppressors of some ability to respond to GGA as well as the acquisition of the ability to read GAA, and suggests the possibility of "wobble" in the middle position of the anticodons of those tRNA's. J Bacteriol, 1978 Jun, 134(3), 795 - 800 Electron microscope heteroduplex studies of sequence relations among plasmids of Escherichia coli: isolation of a new F-prime factor, F80, and its implication for the mechanism of F integration into the chromosome; Ohtsubo E et al.; A new F-prime factor, F80, was isolated from an Escherichia coli strain harboring the F-prime factor F8 by selecting for transfer of the supE marker to a RecA- recipient . Genetic analysis shows that F80 carries a segment of the chromosomal DNA between lip and suc in addition to the tol-gal region normally in F8 . Physical analysis by the electron microscope heteroduplex method suggests that the formation of F80 from F8 involves recombination between the alphabeta segment of F, which is present in F8, and the homologous sequence of F present in the E . coli chromosome at the site where F is supposed to integrate to form HfrP3 . The implications of this result for the general mechanisms of F integration to form Hfr's are discussed. J Bacteriol, 1978 Jun, 134(3), 778 - 94 Electron microscope heteroduplex studies of sequence relations among plasmids of Escherichia coli: structure of F100, F152, and F8 and mapping of the Escherichia coli chromosomal region fep-supE-gal-attlambda-uvrB; Ohtsubo E et al.; The genetic and physical structures of commonly used F-prime factors carrying the galactose region of the Escherichia coli chromosome were analyzed . Deletions in the chromosomal DNA sequences in the F-prime factors were found to be frequent events . A genetic method was developed to reconstruct the original F-prime factors from deletion variants . Heteroduplex analysis of the reconstructed F-prime factors confirmed the derivation of the F-prime factors F100 and F152, from the same Hfr, and finally determined the normal E . coli chromosomal sequence in the region between fep and uvrB, containing about 5 min in genetic units and about 246.5 in kilobase units (kb) . This sequence could be connected with the DNA sequences of the lac-purE region, which had been physically determined previously . Together they constituted a total of 528.6 kb . From these combined sequences, the distance from lacPO to galK was calculated to be 412.9 kb, which corresponds to 8.8 min in genetic units. J Bacteriol, 1978 Jun, 134(3), 765 - 70 Analogs of the dnaB gene of Escherichia coli K-12 associated with conjugative R plasmids; Wang PY et al.; The dnaB266(Am) mutation in Escherichia coli K-12 is an amber mutation such that strains carrying this mutation are not viable in a sup+ strain . With five different R plasmids, it has been possible to construct viable R+ derivatives of this amber mutant and show that the plasmids themselves do not carry amber suppressors . This is interpreted as evidence for the presence of dnaB analog genes associated with these plasmids . Plasmid-positive strains carrying these genes often showed some degree of cryosensitivity of DNA synthesis and colony-forming ability . These observations indicate that the presence of dnaB analog genes in association with R plasmids must be relevant to the plasmid state or to some aspect of conjugative ability. J Bacteriol, 1978 Jun, 134(3), 1195 - 8 Survival of recombination-deficient mutants of Escherichia coli during incubation with nalidixic acid; McDaniel LS et al.; The ability of several Escherichia coli strains deficient in recombination (rec) to survive in the presence of nalidixic acid was determined . Genetic blocks of the RecBC or the RecF pathways resulted in increased sensitivity to nalidixic acid when compared with the wild-type strain . Mutants lacking functional recA, recL, or recB recC recF genes showed the most rapid decrease in colony-forming ability when incubated with nalidixic acid . However, the uvrB gene also plays a role in maintaining cell viability. J Bacteriol, 1978 Jun, 134(3), 1192 - 4 Mutator activity of a short Okazaki fragment mutant of Escherichia coli; Frisch SM et al.; A mutant of Escherichia coli (sof) which was previously shown to have increased recombination frequency, to produce abnormally short "Okazaki fragments," and to be deficient in deoxyuridine triphosphatase has now been found also to possess mutator activity for several genes; point mutation rates and deletion rates are affected . The mutational stimulation effects are consistent with the hypothesis that incorporation of uracil into DNA is directly or indirectly responsible for the observed mutator activity. J Bacteriol, 1978 Jun, 134(3), 1181 - 3 Apparent molecular weights of a heat-modifiable protein from the outer membrane of Escherichia coli in gels with different acrylamide concentrations; Heller KB; The apparent molecular weights of the two forms of a heat-modifiable protein from the outer membrane of Escherichia coli K-12, estimated in gels with different concentrations of acrylamide, indicate that the protein binds excess amounts of sodium dodecyl sulfate, possibly due to large beta structures before boiling. J Bacteriol, 1978 Jun, 134(3), 1117 - 22 Replication of the nonconjugative plasmid RSF1010 in Escherichia coli K-12; de Graaff J et al.; Replicating DNA molecules of the nonconjugative R plasmid RSF1010 (Smr Sur) were cleaved with the EcoRI restriction endonuclease and examined with the electron microscope . Results of this analysis indicated that replication is initiated from an origin located at about 19% of total genome size from one of the EcoRI ends . Replication proceeded either unidirectionally or bidirectionally with equal frequency . Results of the analysis of replicative intermediates of RSF1010 containing the Apr-transposable sequence (Tn) are also presented. J Bacteriol, 1978 Jun, 134(3), 1020 - 9 Outer membrane-dependent transport systems in Escherichia coli: turnover of TonB function; Kadner RJ et al.; Recent reports demonstrated that the energy-dependent step of vitamin B12 uptake into cells of Escherichia coli rapidly declines after cessation either of the expression of the tonB gene or of general protein synthesis . It is shown here that inhibition of protein synthesis results in the decline, with similar kinetics, of all tonB-dependent processes, including sensitivity to colicins B and Ia, irreversible adsorption of phage phi80, and siderophore-mediated iron uptake . The role of ongoing TonB-dependent reactions on this lability of TonB function was investigated . Ferrichrome and the enterochelin precursor, 2,3-dihydroxybenzoate, caused both a moderate depression of B12 uptake activity in growing cells (reversed upon removal of the siderophore) and an acceleration of the loss of activity following inhibition of protein synthesis by addition of spectinomycin . Strains lacking the tonB-dependent siderophore uptake systems did not show these responses . The results suggest the consumption of tonB product during its action. J Bacteriol, 1978 Jun, 134(3), 1002 - 12 In vitro synthesis and and regulation of the biotin enzymes of Escherichia coli K-12; Prakash O et al.; The synthesis and regulation of two of the enzymes of the biotin operon of Escherichia coli, 7,8-diaminopelargonic acid aminotransferase and dethiobiotin synthetase, were studied in vitro in a coupled transcription-translation system . These enzymes are encoded by genes located on opposite strands of the divergently transcribed operon (A . Guha, Y . Saturen, and W . Szybalski, J . Mol . Biol . 56:53-62, 1971) . The kinetics of synthesis of both the enzymes were determined and the efficiency of the system was 0.3 to 0.4% that of the in vivo rate of synthesis in derepressed cells . Guanosine 3'-diphosphate 5'-diphosphate at 0.2 mM concentration stimulated the synthesis of 7,8-diaminopelargonic acid aminotransferase two- to threefold but had no effect on dethiobiotin synthetase synthesis . Biotin, which was most effective as the corepressor in vivo, also functioned in vitro at physiological concentrations in conjunction with a crude repressor protein isolated from a lysogen carrying the bioR gene . However, the two strands showed differential repression . At a repressor concentration where 7,8-diaminopelargonic acid aminotransferase synthesis was completely repressed, the repression of dethiobiotin synthetase was only 20% and did not exceed 50% with increasing repressor concentrations . Although the exact reason for the partial repression remains to be resolved, our data clearly suggest that the biotin operon is regulated from two separate operators. Am J Pathol, 1978 Jun, 91(3), 595 - 606 Prevention by aspirin of the classic generalized Shwartzman reaction; Latour JG et al.; The classic generalized Shwartzman reaction induced in the rabbit was prevented with large doses of aspirin (250 mg/kg) when administered at the time of the first and preparing injection of endotoxin . Such a result was not observed when the drug was given at the time of the second and provoking injection of endotoxin . Our investigations on platelet and coagulation indicate that aspirin prevents disseminated intravascular coagulation through an interference with the blood coagulation and Hageman factor activation rather than by the inhibition of platelet aggregation and availability of platelet procoagulant activity. Surgery, 1978 Jun, 83(6), 717 - 25 Evaluation of the mechanism of zymosan-induced resistance to experimental peritonitis; Joyce LD et al.; Three injections of intraperiotoneal (IP) zymosan-induced profound resistance to E . coli peritonitis in Sprague-Dawley rats . IP zymosan had minimal effects on organ weights and systemic phagocytic clearance ability, suggesting that this mode of administration had few systemic reticuloendothelial system (RES) effects . Hemoglobin (a known inhibitor of local phagocytosis) reduced the protection induced by zymosan, giving further evidence that IP zymosan acts locally . IP zymosan stimulation results in an initial marked influx of polymorphonuclear cells followed by a greater percentage replacement of mononuclear cells by the third day . Examination of these cells via chemiluminescence studies demonstrated that the phagocytic capacity of zymosan-stimulated peritoneal cells was markedly greater than the control group on a cell-for-cell basis . IP zymosan also gave some protection against intravenous (IV) E . coli, but IV zymosan did not significanly protect against IP E . coli . Possible mechanisms of action are discussed . These findings suggest that a technique of local RES stimulation could have a place in preparation of certain high-risk patients for elective abdominal surgery where peritoneal contamination is likely. Biochem J, 1978 Jun 1, 171(3), 601 - 6 Chemical modification as a probe of conformational changes in transfer ribonucleic acid on aminoacylation; Lowdon M et al.; Treatment of Escherichia coli CA265 phenylalanyl-tRNA with 3M-NaHSO3, pH6.0, at 25 degrees C resulted in modification of four bases and in the deacylation of the charged tRNAphe . The similarity of the rates of base modification and of the deacylation of the phenylalanyl-tRNA permitted the isolation of partially modified phenylalanyl-tRNAphe and partially modified deacylated tRNAphe . The sites and extents of base modification in these fractions were determined and found to be the same as those in uncharged tRNAphe modified under identical conditions . These findings are discussed in relation to previous evidence for and against a conformational change in tRNA on its aminoacylation . The methods described should prove adaptable to study of other aminoacyl-tRNA species. Nucleic Acids Res, 1978 Jun, 5(6), 2113 - 31 Binding of phosphorylated histone H1 to DNA; Knippers R et al.; A chromatin associated protein kinase was used to add 3 moles of phosphate to seryl side chains of 1 mole of histone H1 . The DNA binding properties of this in vitro phosphorylated H1 were compared with those of unmodified H1 . Considerably more radioactive superhelical DNA was retained on nitrocellulose filters at 20mM-40mM NaCl by phosphorylated H1 than by unmodified H1 . However, zone velocity sedimentation analysis of histone-DNA complexes indicated that similar amounts of phosphorylated and unmodified H1 are bound to DNA . It is therefore concluded that phosphorylated H1 binds distributively to many or all DNA molecules available (depending on the histone/DNA ratio) while unmodified H1 binds cooperatively to a fraction of the DNA molecules in the reaction mixture. Mol Gen Genet, 1978 Jun 1, 162(1), 89 - 94 Catabolite modulator factor: physiological properties and in vivo effects; Dessein A et al.; Catabolite modulator factor (CMF) specifically inhibits the expression of operons sensitive to catabolite repression . Systems known to be catabolite independent are not affected by CMF . The rate of metabolism of CMF depends on the extent of catabolite repression: it is slow under conditions of strong repression and high in catabolically derepressed cells . Cyclic AMP does not interfere with the rate of CMF metabolism . It has been found that a certain class of crp mutants are partially resistant to the repressive effect of CMF . Our results provide considerable support for the existence of an additional negative control in the regulation of catabolite repression. Mol Gen Genet, 1978 Jun 1, 162(1), 83 - 7 Catabolite repression in Escherichia coli mutants lacking cyclic AMP; Dessein A et al.; The regulation of catabolite repression of beta-galactosidase has been studied in Escherichia coli mutants deleted for the adenyl cyclase gene (cya delta), and thus unable to synthesize cyclic AMP . It has been found that, provided a second mutation occurs either in the crp gene coding for the catabolite gene activator protein (CAP) or in the Lactose region, these mutants exhibit catabolite repression.If the catabolite repression seen in the mutant strains corresponds to the mechanism operating in wild-type cells the results would suggest that the intracellular concentration of cyclic AMP cannot be the unique regulator of catabolite repression. J Virol, 1978 Jun, 26(3), 615 - 29 Genome organization of RNA tumor viruses . I . In vitro synthesis of full-genome-length single-stranded and double-stranded viral DNA transcripts; Verma IM; Genome-length complementary DNA (cDNA) transcripts were synthesized in vitro by using purified virions of avian myeloblastosis virus . Moloney murine leukemia virus, and clone 124 mouse sarcoma virus . The size of the genomelenth cDNA transcripts was measured on either alkaline sucrose gradients or alkaline agarose gels . The longest cDNA transcripts synthesized by using avian myeloblastosis virus, Moloney murine leukemia virus, and clone 124 mouse sarcoma virus were 7, 9 and 6 kilobases (kb), respectively . The in vitro system used was capable of synthesizing double-stranded DNA, but the plus strands (same polarity as the viral RNA) were only 0.5 to 1.5 kb long . Lone Moloney murine leukemia virus cDNA transcripts were used as templates to synthesize the second plus strand . Essentially two strategies were employed as follows . (i) The 3' ends of the cDNA transcripts were extended by addition of 50 to 100 dAMP residues by terminal deoxynucleotidyl transferase . The (dA)n-tailed cDNA transcripts were used as templates along with an oligomer of dT as primer and Escherichia coli DNA polymerase to synthesize the plus strands . (ii) DNase-digested calf thymus DNA was used to prime the synthesis of plus strands on long cDNA with E . coli DNA polymerase I . In both cases, the synthesis of the plus strands was monitored by increased resistance of the cDNA templates to single-strand-specific S1 nuclease . The double-stranded DNA was fractionated on neutral sucrose gradients . Analysis of the double-stranded DNA synthesized by using oligo(dT) primer showed the plus strands to be about 5 to 6 kb long, whereas the plus strands synthesized by using DNase-digested calf thymus DNA primers were only 0.3 to 0.5 kb long . Double-stranded DNA synthesized by either method has an average size of 6 x 10(6) daltons . Double-stranded DNA was also synthesized by using cDNA transcripts as templates without the addition of any primers . In this case, the plus strands were covalently linked to the template strand and were not representative of the whole parent strand. J Bacteriol, 1978 Jun, 134(3), 992 - 1001 Acetate kinase production by Escherichia coli during steady-state and transient growth in continuous culture; Koplove HM et al.; The synthesis of acetate kinase by Escherichia coli ATCC 9637 was studied during growth in anaerobic continuous cultures under steady-state and transient conditions . During growth in anaerobic, glucose-limited chemostats, acetate kinase synthesis was linearly associated with growth . Two types of non-steady-state transients were studied: the perturbation in one was the addition of glucose alone, and, in the second, glucose plus Casamino Acids . During the nutritional shift-up in the second case, but not in the first, the instantaneous specific acetate kinase activities and specific synthesis rates exceeded pre- and postshift values . Trajectory curves demonstrated that the increase in specific activity remained within the bounds of values obtainable under steady-state conditions with minimal and Casamino Acids media . Specific synthesis rates, however, greatly exceeded steady-state values . Enzyme yield values on glucose after the transient nutritional shift-up increased up to fivefold . Active protein synthesis is shown to be necessary to achieve the enhanced specific synthesis rates and enzyme yields . The results from these transient responses are discussed in terms of a conceptful model for metabolic regulation. Acta Med Okayama, 1978 Jun, 32(2), 159 - 67 Endotoxin receptor site . I . Binding of endotoxin to platelets; Washida S; Binding of bacterial endotoxin to platelets, erythrocytes, lymphocytes and granulocytes was examined by using diffusion dialysis . Platelets, erythrocytes, lymphocytes and granulocytes were fractionated from normal human blood and the binding of endotoxin (LPS: Lipopolysaccharide of E . coli) to each cell fraction was measured at 4 degrees C and the binding efficiency was expressed as a binding index (%d4degreesC +/- SD) . The binding index for each cell fraction was as follows; 10.2 +/- 1.6 for platelets, 1.0 +/- 0.9 for erythrocytes, 4.3 +/- 1.6 for lymphocytes and 10.0 +/- 1.5 for granulocytes (n = 11) respectively . Since a platelet possesses a small cell surface area compared with other cells, it was clear that the endotoxin bound preferentially to platelets in vitro . The binding mechanism to the platelet cell surface was suggested to be direct binding of endotoxin to the receptor on platelet cell membrane rather than through an immunologically activated mechanism. Acta Med Okayama, 1978 Jun, 32(2), 147 - 58 A study of endotoxemia in ulcerative colitis and Crohn's disease . I . Clinical study; Aoki K; Endotoxin (lipopolysaccharide, LPS) and LPS antibody in the blood were studied in 61 cases of ulcerative colitis (U.C.) by radioimmunoassay . Lysozyme (LZM) concentration was also studied by the turbidimetric method . As a result, it was found that the blood LPS value as well as serum LZM concentration reflects the clinical observations . The case of endotoxemia in the active phase group showed a positive correlation between the LPS value and LZM concentration . LPS antibody which could not be detected in many cases of the active phase, had a high titer in cases of remission with a long history of the disease . These results would suggest that in U.C . with damaged intestinal mucosal barrier, LPS originating from intestinal flora enters into the blood and aggravates the disease and further that this invading LPS releases LZM into the blood . The same studies were performed on 7 cases of Crohn's disease and the same result was obtained. Can J Biochem, 1978 Jun, 56(6), 559 - 64 Binding of the Ca2+,Mg2+-activated adenosine triphosphatase of Escherichia coli to phospholipid vesicles; Bragg PD et al.; Incubation of the Ca2+,Mg2+-activated adenosine triphosphatase of Escherichia coli with phospholipid vesicles resulted in binding of the enzyme to the lipid . Binding was observed with vesicles of soybean phospholipid (asolectin), phosphatidyglycerol, phosphatidylserine, phosphatidylcholine, and cardiolpin . Binding was not affected by alterations in pH in the range of pH 6.5 to 8.5, by ionic strength, or by the presence of Mg2+ . Loss of the delta subunit from the enzyme had no effect on binding . However, removal of the delta and epsilon subunits by treatment of the enzyme with trypsin prevented binding to phospholipid . This treatment also removed a small portion (less than 2000 daltons) of the alpha subunit . It is concluded that the ATPase of E . coli binds to phospholipid vesicles mainly by nonpolar interactions through the alpha and (or) epilson subunits of the enzyme. J Bacteriol, 1978 Jun, 134(3), 728 - 36 Mu-induced polarity in the unc operon of Escherichia coli; Gibson F et al.; Mutant strains of Escherichia coli were isolated in which mutator (Mu) phage was inserted into various unc genes . Partial diploid strains were prepared from each of the Mu-induced unc mutants by using F-plasmids carrying mutations in one of the known unc genes (uncA, uncB, uncC, or uncD) . The partial diploid strains and the corresponding segregant strains were examined for their ability to grow on succinate . The aerobic growth yields on limiting concentrations of glucose were also determined . Magnesium-stimulated adenosine triphosphatase activities, ATP-dependent transhydrogenase activities, and Atebrin fluorescence quenching activities were determined by using membrane preparations from each strain . Genetic complementation was assessed from the results obtained, and it was concluded that the four unc genes examined are part of a single transcriptional unit and that they are transcribed in the order uncBADC. J Bacteriol, 1978 Jun, 134(3), 1133 - 40 Transient regulation of protein synthesis in Escherichia coli upon shift-up of growth temperature; Yamamori T et al.; Synthesis of total cellular proteins of Escherichia coli was studied upon transfer of a log-phase culture from 30 (or 37) to 42 degrees C . Cells were pulse-labeled with {3H}leucine, and the labeled proteins were analyzed by gel electrophoresis in the presence of sodium dodecyl sulfate . The rates of synthesis of at least five protein chains were found to increase markedly (5- to 10-fold) within 5 min after temperature shift-up and gradually decrease to the new steady-state levels, in contrast to the majority of proteins which gradually increase to the steady-state levels (about 1.5-fold the rate at 30 degrees C) . Temperature shift-down did not cause any appreciable changes in the pattern of protein synthesis as detected by the present method . Among the proteins greatly affected by the temperature shift-up were those with apparent molecular weights fo 87,000 (87K), 76K, 73K, 64K, and 61K . Two of them (64K and 61K) were found to be precipitated with specific antiserum against proteins that had previously been shown to have an adenosine triphosphatase activity . The bearings of these findings on bacterial adaptation to variation in growth temperature are discussed. J Bacteriol, 1978 Jun, 134(3), 1030 - 8 Energy transduction in Escherichia coli: new mutation affecting the Fo portion of the ATP synthetase complex; Rosen BP et al.; A mutation affecting the intrinsic membrane portion (BFo) of the ATP synthetase complex is described . The phenotype is different from previously reported BFo mutants . This mutation results in the ability of membranes lacking the extrinsic membrane portion (BF1) of the ATP synthetase complex to maintain a transmembrane pH gradient . Unlike other BFo mutants, this strain, NR71, is capable of utilizing ATP hydrolysis for the formation of a transmembrane pH gradient. Br J Cancer Suppl, 1978 Jun, 37(3), 60 - 3 Hypoxic radiosensitizers: prospects for effective compounds with fewer toxic side-effects; Rupp WD et al.; Several radiosensitizing chemicals, including a family of simple nitroimidazoles, were examined in E . coli and compared with misonidazole for toxic side-effects on endpoints such as mutagenesis, cell killing and inhibition of the synthesis of the inducible enzyme beta-galactosidase . While all the compounds were similar to misonidazole or better in radiosensitization, marked differences in the various side effects were found . There results show that for E . coli it is possible to find compounds that sensitize as well as misonidazole but which have decreased mutagenicity and fewer other side-effects . Of the compounds examined, KA121 (2,5-dinitroimidazole) is the most promising for future study because it combines good radiosensitization with low mutagenicity and toxicity. Mol Gen Genet, 1978 Jun 1, 162(1), 95 - 100 New regulatory mutations affecting the expression of the threonine operon in Escherichia coli K-12; Saint-Girons I; The promoter of the threonine operon was joined to the structural genes of the lac operon in Escherichia coli K 12 . The synthesis of beta-galactosidase was thus repressed by threonine plus isoleucine in the fusion strains . To isolate mutations which affect the expression of the threonine operon, alterations in the level of expression of the lacZ gene were selected . A new type of regulatory mutation was discovered. Infect Immun, 1978 Jun, 20(3), 811 - 5 Demonstration of K88ac and K88ab antigens of Escherichia coli by means of immunoelectrophoresis and immunodiffusion; Cahill EE et al.; Five strains of Escherichia coli were tested for the presence of the K88ac or K88ab antigens by immunoelectrophoresis and immunodiffusion . The K88ac antigen of 0A2 and Sojka Abbotstown gave an anodic line in the immunoelectrophoresis test and a line in immunodiffusion with homologous K88ac antisera . The K88ab antigens of 0G7, 0E68, and Moon 263 also gave anodic lines in immunoelectrophoresis, and were detectable by immunodiffusions . The 0 groups of these strains were also demonstrated by immunoelectrophoresis and immunodiffusion with homologous 0 antisera . Lack of complete inactivation at 100 degrees C of both the K88ac and K88ab antigens was noted in this study. Can J Microbiol, 1978 Jun, 24(6), 693 - 702 Studies on transfer RNA from mycobacteria; Deobagkar DN et al.; Active preparations of tRNA and aminoacyl-tRNA synthetases have been isolated from exponentially growing cells of Mycobacterium smegmatis and Mycobacterium tuberculosis H37Rv . Though the aminoacyl-tRNA synthetases of older cells retain their activity, the tRNAs seem to undergo modification and show poorer activity . The mycobacterial enzyme preparations catalyse homologous and heterologous aminoacylation between tRNA from the two species (M . smegmatis and M . tuberculosis H37Rv) or from Escherichia coli, with equal efficiency; tRNA samples from eukaryotic cells (yeast and rat liver) do not serve as substrates for the mycobacterial synthetases . The analytical separation of the different amino acid specific tRNAs from M . smegmatis resembles the pattern found in other bacteria . Purification of valine- (three species) and methionine-specific tRNA (two species) to 70-80% purity has been accomplished by using column-chromatographic techniques . Of the two species of tRNAMet, one can be formylated in the presence of formyl tetrahydrofolate and the transformylase from mycobacteria. Proc Natl Acad Sci U S A, 1978 Jun, 75(6), 2746 - 9 Immunological screening method to detect specific translation products; Broome S et al.; We describe a very sensitive method to detect as antigens the presence of specific proteins within phage plaques or bacterial colonies . We coat plastic sheets with antibody molecules, expose the sheet to lysed bacteria so that a released antigen can bind, and then label the immobilized antigen with radioiodinated antibodies . Thus, the antigen is sandwiched between the antibodies attached to the plastic sheet and those carrying the radioactive label . Autoradiography then shows the positions of antigen-containing colonies or phage plaques . A few molecules of antigen released from each bacterial cell generatean adequate signal. Eur J Biochem, 1978 Jun 1, 87(1), 155 - 60 Complementation in vitro of mutant and wild-type ATPase of Escherichia coli using isolated subunits; Vogel G et al.; 1 . The inactive ATPases of four different mutant strains of Escherichia coli have been purified to homogeneity . 2 . Molecular weights, subunit patterns in sodium dodecylsulfate electrophoresis and immunological properties of mutant and wild-type proteins are identical . The mutant enzymes compete with the wild-type enzyme for the binding sites on the membrane . 3 . On freezing and thawing in salt solutions, the ATPase is split into subunits IA (alpha, gamma, epsilon), IB (delta; alpha, gamma, epsilon), and II (beta) . By complementation in vitro of the isolated subunits, it is shown that subcomplex IA (alpha, gamma, epsilon) is altered in the mutant strains described here. J Biochem (Tokyo), 1978 Jun, 83(6), 1779 - 82 EPR investigation of the Mn(II) binding sites in glutamine synthetase (Escherichia coli W) . II . Intermediate-affinity binding sites; Hofmann GE et al.; The nature of the intermediate-affinity (n2) Mn(II) binding sites in glutamine synthetase {EC 6.3.1.2} has been studied as a function of adenylylation in a variety of enzyme-metal complexes by EPR . In the absence of nucleotide the n2 Mn(II) environment is nearly isotropic, the Mn(II) bonds are highly ionic, and the interaction distance R greater than or equal to 12-14 A . Nucleotide binding at the n2 Mn(II) site renders the n2 Mn(II) signal unobservable and causes a reduction in signal amplitude (approximately 30%) and line broadening (approximately 6 G) at the high-affinity (n1) Mn(II) site . This behavior indicates that nucleotide binding induces a conformational change in the enzyme which brings the previously distant n1 and n2 sites into closer proximity (R less than or equal to 8-11 A), possibly for the purpose of activating the nucleotide for direct phosphoryl transfer to L-glutamate . In line with this suggestion, the broad, unresolved resonances in complexes containing both L-methionine SR-sulfoximine (MSOX) and nucleotide may result from the phosphorylation of MSOX . The n2 Mn(II) site is not affected by adenylylation in all the enzyme-metal complexes studied, which suggests that the regulatory effects of adenylylation may only act at the n1 Mn(II) sites. Can J Biochem, 1978 Jun, 56(6), 647 - 53 The control of pyruvate kinases of Escherichia coli: further studies of the enzyme activated by ribose-5-phosphate; Mort JS et al.; The pyruvate kinase activated by ribose-5-phosphate from Escherichia coli has been purified to homogeneity, taking advantage of the stabilization of the enzyme by its inhibitor phosphate and by thiol reagents . The native enzyme has a tetrameric quaternary structure which, while prone to dissociation under many conditions, remains intact in the presence of the above reagents . The enzyme was found to reactivate on dilution out of 8 M urea . Interestingly, the recovery of activity is greatly increased by phosphate, an allosteric inhibitor, but markedly reduced by the allosteric activator, ribose-5-phosphate, implying that it is harder for the enzyme to refold to a 'relaxed state.' Proteolysis studies indicate a more open structure in the presence of the activator. Biochem J, 1978 Jun 1, 171(3), 567 - 73 The effects of acridine orange on deoxyribonucleic acid in Escherichia coli; Barker GR et al.; 1 . Acridine Orange inhibits growth of Escherichia coli K12 when incubated at pH 7.9, but not at pH 7.4.2 . At a non-permissive temperature for DNA polymerase I, Acridine Orange inhibits growth of a temperature-sensitive strain and also increases the rate of elimination of the F'-Lac plasmid . 3 . DNA isolated from cells treated with Acridine Orange under conditions that inhibit growth contains material of low molecular weight, which is absent from DNA isolated from cells treated under conditions in which growth is not impaired . 4 . Cells incubated with Acridine Orange at both pH 7.4 and 7.9 suffer degradation of DNA, as shown by loss of labelled DNA from the acid-insoluble fraction, which is not observed with untreated cells at either pH . 5 . The results suggest that elimination of the F'-Lac plasmid by Acridine Orange requires inactivation of repair processes. Proc Natl Acad Sci U S A, 1978 Jun, 75(6), 2800 - 4 Heat-stable enterotoxin of Escherichia coli: in vitro effects on guanylate cyclase activity, cyclic GMP concentration, and ion transport in small intestine; Field M et al.; A partially purified preparation of the heat-stable enterotoxin of Escherichia coli caused a rapid and persistent increase in electric potential difference and short-circuit current when added in vitro to the luminal surface of isolated rabbit ileal mucosa . As little as 1 ng/ml produced an easily detectable response . Under short-circuit condition, the enterotoxin abolished net Cl- absorption; this change was half that produced by theophylline, which stimulated net secretion . The enterotoxin did not change cyclic AMP concentration but caused large and persistent increases in cyclic GMP concentration . The electrical and nucleotide responses exhibited similar and unusually broad concentration-dependences and maximal effects could not be demonstrated . Theophylline elevated cyclic GMP concentration 3-fold both in the presence and absense of the enterotoxin, suggesting no effect of the toxin on cyclic GMP phosphodiesterase . Guanylate cyclase {GTP pyrophosphatelyase(cyclizing); EC 4.6.1.2} activity in a crude membrane fraction from intestinal epithelial cells was stimulated 7-fold by the enterotoxin . These results suggest that guanylate cyclase stimulation is the basis for the toxin's diarrheagenic effect. Proc Natl Acad Sci U S A, 1978 Jun, 75(6), 2654 - 8 Elimination of cooperativity in aspartate transcarbamylase by nitration of a single tyrosine residue; Landfear SM et al.; In a previous report {Landfear, S . M., Lipscomb, W . N . & Evans, D.R . (1978) J . Biol . Chem . 253, 3988--3996} we demonstrated that tetranitromethane can be employed to nitrate a limited number of tyrosine residues in aspartate transcarbamylase (carbamoylphosphate:L-aspartate carbamoyltransferase, EC 2.1.3.2); such modification eliminates cooperativity, feedback inhibition, and enzymatic activity, and reduces binding of the feedback inhibitor cytidine triphosphate . Cooperativity is lost more rapidly than other properties, and this loss correlates with the nitration of a single tyrosine residue . In this paper, we describe the saturation kinetics of hybrid species constructed from nitrated subunits of one type (either catalytic or regulatory) and native subunits of the other type . We conclude that the modification responsible for loss of cooperativity is on the catalytic subunit . The tryptic peptide containing this modification has been isolated and identified. Proc Natl Acad Sci U S A, 1978 Jun, 75(6), 2579 - 83 Mode of action of colicin Ia: effect of colicin on the Escherichia coli proton electrochemical gradient; Tokuda H et al.; By use of the technique of flow dialysis, the membrane potential (deltapsi) and pH gradient (deltapH) have been measured in colicin Ia-treated Escherichia coli K-12 cells and in membrane vesicles prepared from such cells . Although such cells and vesicles are able to generate a transmembrane deltapH at pH 5.5, they do not generate a transmembrane deltapsi . Glucose-6-phospate uptake by cells is shown to be stimulated at pH 5.5 and inhibited at pH 7.5 by colicin Ia treatment . On the other hand, proline uptake is demonstrated to be inhibited progressively at pH 5.5, 6.6, and 7.5 in colicin Ia-treated cells . These data provide strong evidence for a colicin Ia-induced membrane deplorization and indicate that the membrane becomes permeable to ion(s) other than protons after treatment with colicin Ia. Boll Ist Sieroter Milan, 1978 May 31, 57(2), 134 - 9 Photodynamic effect of 3,4-benzpyrene on thermo-sensitive mutants of Escherichia coli B; Molina AM et al.; Results obtained with thermo-sensitive mutants of Escherichia coli B have shown that the presence of a protein with impaired structure may be responsible for enhanced sensitivity to the lethal photodynamic effect of 3,4-benzpyrene . Thus, the evidence has been obtained of the in vivo action of excited 3,4-benzpyrene on proteins. Mol Gen Genet, 1978 May 31, 161(3), 311 - 5 Detection of specific DNA sequences in yeast by colony hybridization; Blanc H et al.; A procedure is described for the detection of specific DNA sequences in Saccharomyces cerevisiae . This method allows a rapid screening of a large number of yeast colonies . The yeast cells of each colony, grown on nitrocellulose filters, are converted, in situ, to protoplasts by snail enzyme, and are then lysed and their DNAs are denatured and fixed on the filter . The presence of the specific DNA sequence is detected directly on the filter by hybridization with a radioactive cRNA . We have used successfully this technique to detect the presence or the absence of specific mt DNA sequences in p+, p- and p0 strains, and to detect the presence or the absence of the 2 mum DNA sequences in different strains. Mol Gen Genet, 1978 May 31, 161(3), 291 - 6 Phage Mu-1 mediated transposition: a tool to study the organization of ribosomal protein genes in Escherichia coli; Cabezon T et al.; Phage Mu-1 mediated transposition has been used to map genes coding for ribosomal proteins and elongation factor G inside transcriptional units . The data indicate that 1) the str A and fusA genes belong to the same operon, 2) the spcA and strA genes are expressed independently, 3) the spcA gene is located in a different transcriptional unit to that of the eryA and eryB genes. Mol Gen Genet, 1978 May 31, 161(3), 231 - 6 Coding of stable 4S RNA molecules by the resistance factor R1; KricekF et al.; With the aid of DNA-RNA hybridization experiments it was shown that an E . coli strain harbouring the resistance factor R1 contains small, stable RNA species which are capable of annealing with the DNA of this R-factor . At a higher hybridization temperature RNA molecules form the isogenic, sensitive strain could also hybridize to the R1-DNA . These RNA-species were again detected if the RNA was extracted from P . mirabilis carrying this plasmid.The fact that sulfur-labelled RNA also hybridized to the plasmid DNA seems to indicate that some of these molecules are tRNAs. Biochemistry, 1978 May 30, 17(11), 2118 - 23 Studies of virus structure by laser-Raman spectroscopy . Turnip yellow mosaic virus and capsids; Hartman KA et al.; Laser-Raman spectroscopy of the turnip yellow mosaic virus (TYMV) and its capsid indicate the following features of the structure and assembly of the virion . The secondary structure of coat-protein molecules in TYMV is comprised of 9 +/- 5% alpha-helix, 43 +/- 6% beta-sheet, and 48 +/- 6% irregular conformation and is not altered by the removal of the RNA from the capsid . Introduction of as many as 200 chain scissions per RNA molecule also does not affect the overall secondary structure of the encapsulated RNA, which is 77 +/- 5% in the A-helix form . Tryptophan and cysteine residues of the coat protein appear to be in contact with the solvent, while only one of three tyrosines per coat protein is available for hydrogen bonding of its p-hydroxyl group with H2O molecules . Both cytosine and adenine residues of TYMV RNA are protonated in substantial numbers near pH 4.5, suggesting elevation of their respective pKa values within the virion . The Raman data are consistent with chemical evidence favoring interaction between protonated bases of RNA and amino acid side chains of coat protein in TYMV. Biochemistry, 1978 May 30, 17(11), 2110 - 8 Properties of 3-methyladenine-DNA glycosylase from Escherichia coli; Riazuddin S et al.; An Escherichia coli enzyme that releases 3-methyladenine and 3-ethyladenine in free form from alkylated DNA has been purified 2800-fold in 7% yield . The enzyme does not liberate several other alkylation products from DNA, including 7-methylguanine,O6-methylguanine, 7-methyladenine, N6-methyladenine, 7-ethylguanine, O6-ethylguanine, and the arylalkylated purine derivatives obtained by treatment of DNA with 7-bromomethyl-12-methylbenz{a}anthracene . The reaction of the enzyme with alkylated DNA leads to the introduction of apurinic sites but no chain breaks (less than one incision per ten apurinic sites), and there is no detectable nuclease activity with native DNA, depurinated DNA, ultraviolet-irradiated DNA, or X-irradiated DNA as potential substrates . The enzyme is termed 3-methyladenine-DNA glycosylase . It is a small protein, Mr = 19 000, that does not require divalent metal ions, phosphate, or other cofactors in order to cleave base-sugar bonds in alkylated DNA. Biochemistry, 1978 May 30, 17(11), 2054 - 9 Selectivity of RNA chain initiation in vitro . 1 . Analysis of RNA initiations by two-dimensional thin-layer chromatography of 5'-triphosphate-labeled oligonucleotides; Miller JS et al.; A method for the rapid and quantitative analysis of 5'-terminal oligonucleotides of RNAs made in vitro is described . The method involves synthesis of RNA in the presence of {gamma-32P}ATP or GTP, isolation of the RNA, and digestion with T1 or pancreatic ribonucleases to release labeled 5'-triphosphate termanated oligonucleotides . The oligonucleotides are then subjected to chromatography on a polyethyleniminecellulose thin-layer system using 2 M LiCl, 0.01 M EDTA (pH 6.5) in the first dimension and 1.5 M LiCl, 1.8 M formic acid, 0.005 M EDTA (pH 2.0) in the second . RNAs made with E . coli RNA polymerase and lambdacb2, T7, T4, and adenovirus 2 DNA yield characteristic fingerprint patterns . The utility of this method in studying selectivity of in vitro RNA chain initiation is discussed. Vet Rec, 1978 May 27, 102(21), 454 - 8 Pathological changes in the small intestine of neonatal calves naturally infected with reo-like virus (rotavirus); Pearson GR et al.; Nine calves, of which six had been challenged with an enteropathogenic strain of Escherichia coli, were found to be naturally infected with rotavirus . Rotavirus was recovered from the faeces of six calves and rotavirus antigen was detected in the intestinal mucosa of all calves . Stunting and fusion of villi were seen principally in the proximal and middle small intestine, where rotavirus antigen was detected by immunofluorescence . Typical lesions of enteric colibacillosis were found in the distal ileum of the challenged calves, associated with adhesion of the challenge strain of E coli to the mucosa . All samples were removed from the intestine under general anaesthesia and denudation of villi was not observed . However, following exsanguination and resampling at the same site in the small intestine of one calf, denudation was a constant feature. Lancet, 1978 May 27, 1(8074), 1119 - 22 Escherichia coli strains that cause diarrhoea but do not produce heat-labile or heat-stable enterotoxins and are non-invasive; Levine MM et al.; Three enteropathogenic Escherichia coli (E.P.E.C.) strains (O127:K63:H6, O128:K67:H2, and O142:K86:H6) isolated from outbreaks of infantile diarrhoea and one strain from the "normal" colonic flora (E . coli HS) of a healthy adult were fed in doses of 10(6), 10(8), and 10(10) organisms in NaHCO3 to adult volunteers . The strains, which had been stored for 7--9 years, gave negative results in sensitive tests for heat-labile (L.T.) enterotoxin (Y-1 adrenal-cell test), heat-stable (S.T.) enterotoxin (infant mouse assay), invasiveness (guineapig eye test), and gross fluid accumulation (infant rabbit assay) . Two strains (O142 and O127) caused diarrhoea . L.T . or S.T . enterotoxins were not found in E . coli stool isolates from individuals with diarrhoea and no one had a rise in L.T . antitoxin titre; the findings suggest that L.T . and S.T . enterotoxins were not involved in pathogenesis of the diarrhoea . Non-invasive E.P.E.C . strains probably induce diarrhoea by a mechanism (presumably an enterotoxin) distinct from L.T . or S.T . enterotoxins. Biochim Biophys Acta, 1978 May 25, 529(2), 237 - 49 Function of phospholipids on the regulatory properties of solubilized and membrane-bound sn-glycerol-3-phosphate acyltransferase of Escherichia coli; Kito M et al.; Glycerophosphate acyltransferase (acyl-CoA:sn-glycerol-3-phosphate O-acyltransferase, EC 2.3.1.15) solubilized from Escherichia coli membranes was highly activated by phosphatidylglycerol . Phosphatidylethanolamine, cardiolipin and 1,2-diacyl-sn-glycerol 3-phosphate showed no effect . The Km of the enzyme for sn-glycerol 3-phosphate was increased 20-fold by solubilization . The value could not be restored by the addition of phospholipids . Temperature-sensitive regulation of the synthesis of either 1-palmitoyl- or cis-vaccenoyl-sn-glycerol 3-phosphate by the solubilized enzyme was identical with that by the membrane-bound enzyme in vivo and in vitro . The proportion of the molecular species of 1-acyl-sn-glycerol 3-phosphate varied when the ratios of palmitoyl-CoA and cis-vaccenoyl-CoA were changed, but changes in the sn-glycerol 3-phosphate concentration had no effect on selective acylation by both the solubilized and membrane-bound enzymes. J Biol Chem, 1978 May 25, 253(10), 3717 - 20 The shape of L-arabinose isomerase from Escherichia coli; Wallace LJ et al.; L-Arabinose isomerase, EC 5.3.1.4, catalyzes the conversion of L-arabinose to L-ribulose, the first step in the catabolism of L-arabinose by Escherichia coli B/r . Patrick and Lee (1969) J . Biol . Chem . 244, 4277--4283) demonstrated that native L-arabinose isomerase is composed of six identical subunits of approximately Mr = 60,000 . In this paper we describe an electron microscopy study of the arrangement of the six identical subunits . The isomerase is seen in two distinctly different orientations . The first has three subunits visible, with a 3-fold axis of symmetry, corresponding to a face-on view of two stacked, eclipsed trimers . The second orientation is rectangular in shape with 2-fold symmetry; suggesting a side-on view of the stacked trimers . The six identical subunits are thus arranged with D3 symmetry as in a trigonal prism . Measurements were made on the maximum profile of the three 2-fold axes of symmetry of the face-on orientations, and of both the long and short dimensions of the side-on orientation . The best estimate for the maximum profile of the 2-fold axes of symmetry of the face-on view is 106 +/- 8 A, using glutamine synthetase as an internal size standard . Measurements from micrographs of the isomerase alone, using an external magnification calibration, give the following results: for the maximum profile of the three 2-fold axes of symmetry of the face-on view, 132 +/- 7 A; for the long axis of the side-on view, 136 +/- 10 A; and for the short axis, 105 +/- 6 A . These measurements are consisting with the interpretation of the profiles as representing two different orientations of the L-arabinose isomerase. J Biol Chem, 1978 May 25, 253(10), 3607 - 22 Structural analysis of the tRNA1Tyr gene of Escherichia coli . A 178 base pair sequence that is repeated 3.14 times; Egan J et al.; The distal region of the tRNA1Tyr gene has been sequenced and found to have an unusual structure . It consists of a 178 base pair sequence that is repeated 3.14 times . The first repeat unit commences 19 base pairs before the end of the sequences encoding the mature tRNA, and these 19 base pairs are repeated faithfully at the beginning of each repeat unit . In the last fractional unit the repeated sequence extends only six base pairs beyond this 19 base pair sequence . Sequence information extends for 62 base pairs beyond the 3.14 repeating units, and no resemblance to the repeating sequence, or any other region of the tRNA1Tyr gene, is found . There are only 14 sites at which one of the repeats differs from the others; 11 of these are transitions, and the rest are transversions . The evolutionary implications of the differences are discussed . One of the differences, which occurs in the second repeat unit, corresponds to the location of the in vitro p-dependent transcription termination site . This is discussed along with other implications of the repeated structure. J Biol Chem, 1978 May 25, 253(10), 3415 - 21 Apparent suicidal inactivation of DNA polymerase by adenosine 2',3'-riboepoxide 5'-triphosphate; Abboud MM et al.; Adenosine 2',3'-riboepoxide 5'-triphosphate (epoxyATP) has been found to be a suicidal inactivator of DNA polymerase I from Escherichia coli by the following criteria . Inactivation is complete, is first order in enzyme activity, and shows saturation kinetics with an apparent KD of 30 +/- 10 micron for epoxy ATP . This KD is comparable to the KM of the substrate dATP . The t1/2 for inactivation is 1.3 min . Inactivation requires Mg2+ and the complementary template . The enzyme is protected by dATP but not by an excess of template . Gel filtration of the reaction mixture after inactivation with {3H}epoxy ATP results in the comigration of E . coli DNA polymerase I, the tritium-labeled inactivator, and the DNA template . The stoichiometry of binding approaches 1 mol of {3H}epoxy nucleotide per mol of inactivated enzyme . These results are consistent with the hypothesis that epoxy ATP initially serves as a substrate for the polymerase reaction, elongating the DNA chain by a nucleotidyl unit, and subsequently alkylates an essential base at the primer terminus binding site of the enzyme . Epoxy ATP also inactivates human and viral DNA polymerases but not E . coli RNA polymerase or rabbit muscle pyruvate kinase . Hence epoxy ATP may be a specific suicide reagent for DNA polymerases. J Biol Chem, 1978 May 25, 253(10), 3557 - 62 The deoxyribonuclease induced after infection of KB cells by herpes simplex virus type 1 or type 2 . I . Purification and characterization of the enzyme; Hoffmann PJ et al.; The deoxyribonuclease induced in KB cells by herpes simplex virus (HSV) type 1 and type 2 has been purified . Both enzymes are able to completely degrade single- and double-stranded DNA yielding 5'-monophosphonucleotides as the sole products . A divalent cation, either Mg2+ or Mn2+, is an absolute requirement for catalysis and a reducing agent is necessary for enzyme stability . The maximum rate of reaction is achieved with 5 mM MgCl2 for both HSV-1 and HSV-2 DNase . The optimum concentration for Mn2+ is 0.1 to 0.2 mM and no exonuclease activity is observed when the concentration of Mn2+ is greater than 1 mM . The rate of reaction at the optimal Mg2+ concentration is 3- to 5-fold greater than that at the optimal Mn2+ concentration . In the presence of Mg2+, the enzymes are inhibited upon the addition of Mn2+, Ca2+, and Zn2+ . The enzymatic reaction is also inhibited by spermine and spermidine, but not by putrescine . Crude and purified HSV-1 and HSV-2 DNase can degrade both HSV-1 and HSV-2 DNA, but native HSV-1 DNA is hydrolyzed at only 22% of the rate and HSV-2 DNA at only 32% of the rate of Escherichia coli DNA . Although HSV-1 and HSV-2 DNase were similar, minor differences were observed in most other properties such as pH optimum, inhibition by high ionic strength, activation energy, and sedimentation coefficient . However, the enzymes differ immunologically. Biochim Biophys Acta, 1978 May 24, 534(1), 89 - 98 Effect of temperature on fluorescence and circular dichroism of Escherichia coli dihydrofolate reductase and its complexes; Kitchell BB et al.; Dihydrofolate reductase and its complexes have been studied by fluorescence and circular dichroism . NADPH, trimethoprim, pyrimethamine, or Methotrexate binding causes small changes in the enzyme far ultraviolet CD which possibly arise from alterations in polypeptide backbone of the enzyme; however, their effects on enzyme far ultraviolet CD are also explained as the result of ligand interactions with enzyme aromatic groups . In ternary complexes of the enzyme, fluorescence properties of bound NADPH are surprisingly sensitive to the type of inhibitor bound nearby . The effect of temperature on the enzyme and its complexes is clearly shown by changes in enzyme fluorescence and CD . At temperatures near 45 degrees C, the enzyme undergoes an irreversible denaturation, as shown by major alterations in enzyme far ultraviolet CD and by an increased rate of fluorescence quenching . Binary complexes with NADPH or Methotrexate stabilize the enzyme towards this heat denaturation, whereas bound trimethoprim and pyrimethamine do not . Ternary complexes with NADPH and any of the ligands are more stable than the enzyme itself toward heat denaturation . Fluorescence-temperature and fluorescence polarization studies show that near 30 degrees C the enzyme undergoes a reversible transition that is modified by NADPH or methotrexate. Biochim Biophys Acta, 1978 May 23, 518(3), 530 - 4 Pairing properties of the methylester of 5-carboxymethyl uridine in the wobble position of yeast tRNA3Arg; Weissenbach J et al.; At optimum magnesium concentration (10 mM) both yeast tRNA1Arg and tRNA3Arg are able to bind to poly (A,G) and A-G-A in presence of Escherichia coli robisomes . With A-G-G only tRNA1Arg ginds, wherea tRNA3Arg (anticodon mcm5 U-C-U) is not bound . This result means that the methylcarboxymethyl substituant in position 5 of U prevents its wobble with G. Eur J Biochem, 1978 May 16, 86(2), 603 - 6 Evidence for synthesis of alkaline phosphatase on membrane-bound polysomes in Escherichia coli; Varenne S et al.; Polysomes containing nascent chains of alkaline phosphatase have been isolated from a membrane-bound polysome preparation . Indirect immunoprecipitation using conformation-specific antibodies has been employed . This technique provides a good enrichment of these polyribosomes since routinely no more than than 10--15% of non-specific immunoprecipitation was observed . The yield of the procedure is generally 40% but can be increased if higher non-specific immunoprecipitation is tolerated . Antibodies, previously described, directed against uncoiled or folded monomers of alkaline phosphatase can be used as primary antibody to recognize the nascent chains contained in membrane-bound polysomes which suggests that these chains are partially folded. Eur J Biochem, 1978 May 16, 86(2), 325 - 34 Complete nucleotide sequence of the simian-virus 40 Hind-G fragment and localisation of the carboxyl terminus of the VP1 protein; van Heuverswyn H et al.; The restriction fragment Hind-G represents 7.0% of the simian virus 40 (SV40) genome . The information present in fragment Hind-G is expressed as part of the major, late 16-S messenger RNA . The complete nucleotide sequence of the fragment Hind-G has now been determined by application of the procedure of Maxam and Gilbert {Proc . Natl Acad . Sci . U.S.A . (1977) 74, 560-564} . It contains 369 nucleotide base pairs . On the basis of the termination code words in the strand with the same polarity as the late mRNA, two illegitimate reading frames can be defined . Therefore the third, open frame must code for the carboxyl terminal part of the VP1 protein . It terminates within fragment Hind-G with a TGA signal . This stop codon is followed by a non-translated region of the mRNA of about 83 nucleotides . The latter contains the sequence A-A-U-A-A-A, common to all other eukaryotic mRNA molecules so far studied . The Hind-G fragment also contains sequences which presumably play a role in the synthesis, processing and/or expression of early mRNA; these aspects are discussed in the following paper. Eur J Biochem, 1978 May 16, 86(2), 317 - 24 Nucleotide sequence of the restriction fragment Hind-F-EcoRI1 of simian-virus-40 DNA (part of the VP1 gene); Contreras R et al.; The nucleotide sequence of the simian virus 40 (SV40) genome region between the cleavage sites for restriction endonucleases EcoRI (map position 0) and HindII (map position 0.05) has been determined mainly by the partial chemical DNA degradation procedure of Maxam and Gilbert . This fragment represents 5.3% of the genome of SV40 and is located in the late region, internally in the VP1 gene . The message strand shows only one open reading frame for translation into protein, which connects to the one for the preceding fragment . On this basis part of the amino acid sequence of the VP1 protein is presented. Biochemistry, 1978 May 16, 17(10), 1922 - 8 Ferric enterobactin transport system in Escherichia coli K-12 . Extraction, assay, and specificity of the outer membrane receptor; Hollifield WC Jr et al.; An outer membrane preparation from cells of Escherichia coli K-12 grown in low iron medium was found to retain ferric enterobactin binding activity following solubilization in a Tris-HCl, Na2EDTA buffer containing Triton X-100 . Activity was measured by means of a DEAE-cellulose column which separated free and receptor bound ferric enterobactin . The binding activity was greatly reduced in preparations obtained from cells grown in iron rich media or from cells of a colicin B resistant mutant grown in either high or low iron media . Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis enabled correlation of this lack of activity to a single band missing in the outer membrane profile of the colicin B mutant . Evidence was obtained for in vitro competition between ferric enterobactin and colicin B for the extracted receptor . The binding specificity of the extracted receptor was examined by competition between ferric enterobactin and several iron chelates including a carbocyclic analogue of enterobactin, cis-1,5,9-tris(2,3-dihydroxybenzamido)cyclododecane . The ferric form of the latter compound supported growth of siderophore auxotrophs, apparently without hydrolysis to dihydroxybenzoic acid and resynthesis into enterobactin . These data may require revision of the accepted mechanism of enterobactin mediated iron utilization. J Mol Evol, 1978 May 12, 11(1), 47 - 56 Relationship between gene function and gene location in Escherichia coli; Riley M et al.; Genes of Escherichia coli were grouped according to the "biochemical relatedness" of the enzymes they specifiy, using two schemes to determine relatedness: similarity of reaction or similarity of reactants . The tendency of biochemically related genes as so defined to lie approximately 90 degrees or 180 degrees from one another on the circular genetic map was analyzed statistically . Of the classes analyzed, only the genes for the enzymes of glucose catabolism showed a significant departure from random distribution in this respect . The glucose catabolism genes showed a pronounced tendency to lie either 90 degrees of 180 degrees from one another (P = ca . 10(-9)), and, furthermore, most of these genes were found to lie in only four gene clusters on the E . coli genome . The significance of this observation is discussed in relation to evolutionary mechanisms and to mechanisms of gene expression. Biochim Biophys Acta, 1978 May 11, 524(1), 37 - 44 Affinity chromatography studies with the pyruvate dehydrogenase complex of wild-type Escherichia coli; Visser J et al.; 1 . The lifetime of thiamine pyrophosphate-Sepharose 2B affinity matrices synthesized according to Matsuura et al . (Matsuura, A., Iwashina, A . and Nose, Y . (1973) Biochem . Biophys . Res . Commun . 51, 241-246) has been improved . The matrix interacts with bacterial pyruvate dehydrogenase complexes . 2 . The synthesis of a stable thiochrome-Sepharose 2B matrix is described . 3 . Both matrices bind the pyruvate dehydrogenase complex of Escherichia coli in a 50 mM phosphate buffer, pH 7.0 . Elution is possibly by an increase in ionic strength but not by the cofactor or metal-cofactor complexes . 4 . The presence of Mg2+, reduces the capacity of the affinity matrices but leads to higher specificity for the multienzyme complex . 5 . The pyruvate dehydrogenase complex of E . coli has been successfully purified by combining a classical purification step with these affinity chromatography systems . The method is less suitable for large scale operation. J Biol Chem, 1978 May 10, 253(9), 2898 - 901 Reconstitution of the ribonucleotide reductase enzyme from Ehrlich tumor cells; Cory JG et al.; Ribonucleotide reductase from Ehrlich tumor cells was separated by chromatography on blue dextran/Sepharose into two protein fractions (Tris and Dye fractions) . Neither fraction alone had reductase activity, but when combined, constituted an active enzyme system . Heat treatment of either fraction resulted in an inactive combination . The approximate molecular size of the active component of the Tris and Dye fractions was determined to be 5.7 S and 6.5 S, respectively, compared to 9 S for the intact enzyme . The Tris fraction was inactivated by hydroxylamine while the dye fraction was inactivated by pyridoxal phosphate/BH4-treatment . The inactivation of the Dye fraction was prevented by ATP . These data would indicate that the Tris and Dye fractions were comparable in function to the B2 and B1 proteins, respectively, of the Escherichia coli ribonucleotide reductase. J Biol Chem, 1978 May 10, 253(9), 3313 - 9 A multienzyme system for priming the replication of phiX174 viral DNA; McMacken R et al.; Synthesis of the oligonucleotides that prime replication of phiX174 single-stranded DNA employs complex protein machinery of the host cell which is probably used by the cell to replicate its own chromosome . Primer synthesis depends on at least five proteins (DNA binding protein, dnaB and dnaC proteins, protein i, and protein n) and ATP to form a replication intermediate and another protein, primase (dnaG protein), to assemble the oligonucleotide by template transcription . The data in this paper show that ribo- and deoxyribonucleoside triphosphates can serve as substrates and form hybrid primers when present together . Both RNA and DNA primers were initiated with ATP . At least three of the four base-pairing nucleoside triphosphates were required for the transcription that generates effective primers . Over 90% of the RNA and DNA transcripts were extended into complementary strands by DNA polymerase III holoenzyme . At optimal triphosphate concentrations, the rate and extent of primer formation were greater from ribonucleoside triphosphates than from deoxyribonucleoside triphosphates . Uncoupled from DNA replication, the length of RNA primers was 14 to 50 residues, the DNA primers 4 to 20 residues . The fingerprint pattern of an RNase digest of RNA primers has a complexity suggestive of transcription from many sites on the phiX174 template . The multienzyme priming system is highly specific for phiX174 DNA as template. J Biol Chem, 1978 May 10, 253(9), 2990 - 9 A DNase for apurinic/apyrimidinic sites associated with exonuclease III of Hemophilus influenzae; Clements JE et al.; An endonuclease purified from Hemophilus influenzae made single strand breaks in DNA containing apurinic or apyrimidinic sites but had no detectable endonuclease activity on untreated native DNA . The new 5'-termini created at the cleavage sites were base-free deoxyribose 5-phosphate residues . The enzyme preparation also catalyzed the exonucleolytic release of 5'-mononucleotides from bihelical DNA and the hydrolysis of DNA 3'-terminal phosphomonoesters . The phosphatase-exonuclease activity was indistinguishable from that reported by Gunther and Goodgal (J . Biol . Chem . (1970) 245, 5341-5349) and resembled that of exonuclease III of Escherichia coli . The endonucleolytic and exonucleolytic activities could not be separated by electrophoresis, sedimentation, or gel filtration, and they were also affected simultaneously by mutation . The enzymatic activities appear to be functions of a single monomeric protein (M(r) = 30,000). Biochim Biophys Acta, 1978 May 10, 502(2), 345 - 53 Specific labelling of the (Ca2+ + Mg2+)-ATPase of Escherichia coli with 8-azido-ATP and 4-chloro-7-nitrobenzofurazan; Verheijen JH et al.; 1 . 8-Azido-ATP is a substrate for Escherichia coli (Ca2+ + Mg2+)-ATPase (E . coli F1) . 2 . Illumination of E . coli F1 in the presence of 8-azido-ATP causes inhibition of ATPase activity . The presence of ATP during illumination prevents inhibition . 3 . 8-Azido-ATP and 4-chloro-7-nitrobenzofurazan (NbfCl) bind predominantly to the alpha subunit of the enzyme, but also significantly to the beta subunit . 4 . The alpha subunit of E . coli F1 seems to have some properties that in other F1-ATPases are associated with the beta subunit. J Biol Chem, 1978 May 10, 253(9), 3298 - 304 ATP utilization by rep protein in the catalytic separation of DNA strands at a replicating fork; Kornberg A et al.; Hydrolysis of ATP by rep protein proceeds in the presence of a single-stranded region of DNA 4 residues long, but the true effector for rep ATPase appears to be a replicating fork rather than a random coil . At or near a fork in duplex DNA, rep ATPase action is different from what it is on DNA lacking secondary structure (single-stranded): (i) Km for ATP is lower, (ii) specificity is for ATP and dATP with no action on other nucleoside triphosphates, (iii) sensitivity to certain ATP analogs is reduced, (iv) presence of a DNA-nicking enzyme (e.g . cistron A protein induced by phiX174) is required, and (v) Escherichia coli DNA binding protein facilitates rather than inhibits . During the separation of strands accompanying replication, 2 molecules of nucleoside triphosphate (ATP or dATP) are hydrolyzed for every nucleotide polymerized . Utilization of ATP by rep protein may provide energy for catalytic strand separation at a fork in advance of replication. J Biol Chem, 1978 May 10, 253(9), 3292 - 7 Purification of the rep protein of Escherichia coli . An ATPase which separates duplex DNA strands in advance of replication; Scott JF et al.; The product of the rep gene of Escherichia coli catalytically separates phiX174 duplex DNA strands in advance of their replication, utilizing ATP in the process (Scott, J . F., Eisenberg, S., Bertsch, L . L., and Kornberg, A . (1977) Proc . Natl . Acad . Sci . U . S . A . 74, 193-197) . The enzyme has now been purified to near-homogeneity . Relatively large quantities were obtained from ColE1-plasmid-containing cells in which the enzyme level was 7 to 10 times above wild type . The assay for rep protein was based on its essential role, with phage-induced cistron A protein, in enzymatic synthesis of phage phiX174 (+) strands, using duplex circular DNA as template . The protein exhibits a molecular weight of 65,000 under denaturing and reducing conditions . The turnover number of the enzyme is approximately 6800 ATP molecules/min in strand separation as measured by extent of replication, or in an uncoupled reaction using single-stranded DNA effector. J Biol Chem, 1978 May 10, 253(9), 3123 - 8 The epsilon subunit of Escherichia coli coupling factor 1 is required for its binding to the cytoplasmic membrane; Sternweis PC; The coupling factor, F1-ATPase of Escherichia coli (ECF1) contains five different subunits, alpha, beta, gamma, delta, and epsilon . Properties of delta-deficient ECF1 have previously been described . F1-ATPase containing only the alpha, beta, and gamma subunits was prepared from E . coli by passage of delta-deficient ECF1 through an affinity column containing immobilized antibodies to the epsilon subunit . The delta, epsilon-deficient enzyme has normal ATPase activity but cannot bind to ECF1-depleted membrane vesicles . Both the delta and epsilon subunits are required for the binding of delta, epsilon-deficient ECF1 to membranes and the restoration of oxidative phosphorylation . Either delta or epsilon will bind to the deficient enzyme to form a four-subunit complex . Neither four-subunit enzyme binds to depleted membranes . The epsilon subunit, does, however, slightly improve the binding affinity between delta and delta-deficient enzyme suggesting a possible interaction between the two subunits . Neither subunit binds to trypsin-treated ECF1, which contains only the alpha and beta subunits . A role for gamma in the binding of epsilon to F1 is suggested . epsilon does not bind to ECF1-depleted membranes . Therefore, the in vitro reconstitution of depleted membranes requires an initial complex formation between epsilon and the rest of ECF1 prior to membrane attachment . Reconstitution experiments indicate that only one epsilon is required per functional ECF1 molecule. Biochim Biophys Acta, 1978 May 4, 509(1), 148 - 58 Use of salicylic acid to measure the apparent intracellular pH in the Ehrlich ascites-tumor cell and Escherichia coli; Garcia-Sancho J et al.; The distribution of salicylic acid between the intracellular and extracellular phases has been used to estimate the intracellular pH in the Ehrlich cell and Escherichia coli . The validity of the method was established by: (i) comparison of the results obtained with salicylic acid with those obtained with 5,5-dimethyloxazolidine-2,4-dione; (ii) by following changes of the apparent intracellular pH under circumstances in which such changes are predictable, e.g., the addition of weak acids or proton conductors to the incubation medium during incubation at acidic pH; (iii) by comparison of the apparent intracellular pH changes with the uptake of H+ by the cells estimated from the changes of the medium pH . Optimal results are obtained with this indicator when the extracellular pH is below 5.5, because in this case the indicator is to a sufficient extent in its penetrating form, so that its movement can reflect intracellular pH changes occurring in less than 30 s . When the intracellular pH falls below 5.2 measurable binding of salicylic acid to the intracellular material of the Ehrlich cell takes place, but above this pH no binding has been found . The Ehrlich cell and cells of Escherichia coli behaved similarly under various experimental circumstances tested, but striking difference were found in the inherent permeability of the membrane to H+ and in the changes in this parameter by lowering the temperature to 2 degrees C. Biochim Biophys Acta, 1978 May 3, 540(2), 301 - 12 The use of mutants in the study of structure-function relationships in cloacin DF13; Gaastra W et al.; A bacteriocin from cells with a mutant Clo DF13 plasmid (cloacin clp03 . immunity protein complex) and a bacteriocin from cells containing the recombinant plasmic Clo DF13 :: Tn901 (cloacin pJN82) have been isolated . Both bacteriocins like wild-type cloacin DF13, are still able to inhibit in vitro protein synthesis, but their in vivo killing activity is absent . Comparison of some physicochemical characteristics of the cloacin clp03 . immunity protein complex and wild-type cloacin complex showed no significant differences . From a comparison of the binding capacity to specific receptors on sensitive cells, the translocation through the cell wall, and the interaction with cytoplasmic membranes, it could be concluded that the cloacin clp03 complex is hampered in its translocation from the outer membrane receptor site to the cytoplasmic membrane, resulting in the observed lack in killing activity . Cloacin pJN82 is shortened at the C-terminal of the molecule by approximately ten amino acid residues . Together with its loss of in vivo killing activity it has lost its capacity to bind immunity protein . Since the immunity protein probably not only provides cloacin-producing cells with "immunity" but is also involved in the translocation of the bacteriocin to the interior of sensitive cells, the absence of this protein is probably the reason for the lack of killing activity of cloacin pJN82 . The implications of these findings for the topography of the cloacin molecule as suggested by de Graaf et al . (de Graaf, F.K., Stukart, M.J., Boogerd, F.C . and Metselaar, K . (1978) Biochemistry, in press) are discussed. Biochim Biophys Acta, 1978 May 3, 540(2), 190 - 6