Escherichia coli

Nature, 1986 Jul 3-9, 322(6074), 80 - 3
Site-directed mutagenesis of the regulatory light-chain Ca2+/Mg2+ binding site and its role in hybrid myosins; Reinach FC et al.; The regulatory light chains, small polypeptides located on the myosin head, regulate the interaction of myosin with actin in response to either Ca2+ or phosphorylation . The demonstration that the regulatory light chains on scallop myosin can be replaced by light chains from other myosins has allowed us to compare the functional capabilities of different light chains, but has not enabled us to probe the role of features, such as the Ca2+/Mg2+ binding site, that are common to all of them . Here, we describe the use of site-directed mutagenesis to study the function of that site . We synthesized the chicken skeletal myosin light chain in Escherichia coli and constructed mutants with substitutions within the Ca2+/Mg2+ binding site . When the aspartate residues at the first and sixth Ca2+ coordination positions are replaced by uncharged alanines, the light chains have a reduced Ca2+ binding capacity but still bind to scallop myosin with high affinity . Unlike the wild-type skeletal light chain which inhibits myosin interaction with actin, the mutants activate it . Thus, an intact Ca2+/Mg2+ binding site in the N-terminal region of the light chain is essential for regulating the interaction of myosin with actin.

Res Vet Sci, 1986 Jul, 41(1), 63 - 9
Influence of creep feeding and dietary intake after weaning on malabsorption and occurrence of diarrhoea in the newly weaned pig; Hampson DJ et al.; The influence the pattern of creep feeding has on the ability of pigs to absorb xylose after weaning at three weeks old was investigated . All weaned pigs showed a reduction in their ability to absorb xylose one week after weaning, but this was not influenced by the pattern of creep feeding before weaning . Diarrhoea occurred in some animals after weaning, but did not occur in pigs which did not have access to creep food before weaning . This protective effect of withholding creep food was associated with a low dietary intake after weaning with this regimen . Pigs which developed diarrhoea tended to be those which consumed more meal after weaning than their contemporaries . Haemolytic enterotoxigenic Escherichia coli and rotaviruses were present in the faeces of most pigs after weaning, but, in those animals that ate too much and developed diarrhoea, excretion of the E coli continued for approximately twice as long as in animals that remained healthy.

Cancer Res, 1986 Jul, 46(7), 3341 - 7
Mouse peritoneal lymphocytes, a new target for analyzing induction of sister chromatid exchanges on in vivo exposure to a genotoxic agent; Nishi Y et al.; The availability of use of mouse peritoneal lymphocytes as target cells for analyzing sister chromatid exchanges (SCE) upon exposure to a genotoxic drug, cyclophosphamide, was investigated using female ICR mice . Use of these cells overcame the difficulty in use of mouse lymphocyte cultures, recovering sufficient metaphase cells . The greatest advantage of use of peritoneal lymphocytes was that about 1-2 X 10(6) lymphocytes/mouse could easily be recovered from the peritoneal cavity in high purity . Their mitogenic responses were good when Escherichia coli lipopolysaccharide, in combination with 2-mercaptoethanol, was used as mitogens, but they were less when purified phytohemagglutinin was used . In the presence of lipopolysaccharide (60 micrograms/ml) and 2-mercaptoethanol (22-88 microM), the maximum incidence of second division metaphases (greater than 50%) and the highest mitotic index (greater than 4%) were observed 36-40 h after stimulation . Under these conditions, the base-line SCE showed the constant level . The range of intrastrain variations in the base-line SCE was 0.24-0.36/chromosome . The distribution histograms of SCE/chromosome did not fit a single Poisson model, suggesting that these cells are heterogeneous with respect to the base-line SCE . Single s.c . injections 1 h before harvest of doses of 0.75-3.0 mg of cyclophosphamide per kg evoked positive responses, and injections of over 0.375 mg/kg had linear dose-dependent effects . On harvest of cells for up to 192 h after the injection, the maximal induction of SCE attained 1 h after exposure was found to return time dependently to the control level at 192 h . After the initial rapid reduction in the cell number, cellular recovery, measured as the mitotic index and the number of peritoneal exudate cells recovered, returned to the control level within 48 h, without a significant increase thereafter . After maintaining cells under the liquid-holding experiment for various times in vitro following a single exposure to cyclophosphamide for 1 h in vivo, the reduction of their SCE and recovery of their mitotic index were more rapid than those of cells in the time-course experiment . These findings suggest that the association of the recruitment of less- and/or nondamaged cells from their precursors with reduction of the SCE is slight . Repair(s) and, to a lesser extent, selective loss of more damaged cells may be the main factors contributing to the early reduction response of the SCE frequency . The relations of these factors are discussed.

Int J Pediatr Nephrol, 1986 Jul-Sep, 7(3), 141 - 4
Community acquired infections in children on maintenance cyclosporine therapy; Tejani A et al.; Cyclosporine has profound immunosuppressive properties . We reviewed our experience of community acquired infections in 62 children who received the drug from 2 to 36 months . 36 children received the drug for a renal transplant for a mean of 18 months . There were 3 minor and one major episode of infection in this group . The frequency of both minor and major infections in cyclosporine recipients was significantly lower when compared with a group of renal recipients treated with azathioprine . 26 children received cyclosporine for 2 months for resistant nephrotic syndrome . This group had one major and one minor episode of infection . Despite the relative safety of cyclosporine recipients from infectious complications, a more prolonged follow up is necessary.

J Gen Microbiol, 1986 Jul, 132 ( Pt 7), 1859 - 62
Incompatibility between E colicin plasmids; Cooper PC et al.; We have tested the ability of pairs of colicin E plasmids to replicate stably in the same cell line . Although many of the pairs of E colicin plasmids were compatible, plasmids ColE3-CA38, ColE7-K317 and ColE8-J were mutually incompatible, as were ColE5-099, ColE6-CT14 and ColE9-J . Incompatibility between ColE6-CT14 and ColE5-099 or ColE9-J was asymmetrical, whereas incompatibility between the other plasmid pairs was symmetrical.

J Gen Microbiol, 1986 Jul, 132 ( Pt 7), 1843 - 51
Comparative esterase electrophoretic polymorphism of Escherichia coli isolates obtained from animal and human sources; Goullet P et al.; To determine whether enzyme electrophoretic polymorphism in Escherichia coli populations was influenced by environmental background, the mobilities of four electrophoretically variable esterases (A, B, C and I) were examined . The distinction between isolates was established by significant differences in the electrophoretic distribution and the genetic diversity coefficient of individual esterases . Principal components analysis on each population and on all strains revealed three groups of allozymes . The first, characterized by slow electrophoretic mobilities of esterase B, was frequently observed in strains obtained from human extra-intestinal infections and rarely in commensal organisms . The second, characterized by fast mobilities of esterases A and B, was frequently found in animal isolates . The third, characterized by prominence of the most common mobilities of esterases B and A, was recovered in all populations . These results were confirmed by discriminant analysis . Among the 610 strains investigated, 316 electrophoretic types (distinctive combinations of allozymes of the four varieties of esterases) were distinguished, illustrating high esterase polymorphism.

Allergol Immunopathol (Madr), 1986 Jul-Aug, 14(4), 269 - 75
{Polyclonal stimulation activates the basal level of IgM and IgG plaque-forming cells against sheep erythrocytes without inducing a change from IgM to IgG}; Bolos C et al.; In previous works, we have postulated the existence of an immunological equilibrium between persistence of the antigen presence and the B-cell maturation-differentiation; from this hypothesis, it follows that the antigen presence induces the IgM to IgG switch and the final exhaustion of the response which persists if the antigen stimulation persists . On the basis of those results, we have considered of interest to study the direct and indirect polyclonal activation of the background of murine plaque-forming cells (PFC), against sheep erythrocytes . We have investigated this activation by different doses of mitogens, which activate B cells, thus producing various cellular proliferations . The goal was to find out whether polyclonal activation increased the background (as expected) but did not change the IgM/IgG PFC ratio, probably because of a lack of antigenic stimulation . As mitogens, we have used: E . coli lipopolysaccharide (LPS), a known polyclonal activator of B cells which causes a proportional increase in the number of background plaque-forming cells to a given antigen, and pokeweed mitogen (PWM) which, although less frequently used, has been shown to stimulate both T and B lymphocytes . Female Swiss albino mice have been used; 10 mice per group for both control and test groups . The mitogens were administered according to the following doses and protocol: LPS-1 dose of 10, 50 or 100 micrograms; 3 doses of 10, 50, or 100 micrograms (1 dose per week) . PWM-1 dose of 50, 100 or 200 micrograms; 3 doses of 50 or 100 micrograms (1 dose per week).(ABSTRACT TRUNCATED AT 250 WORDS)

Res Vet Sci, 1986 Jul, 41(1), 131 - 2
Experimental Escherichia coli endotoxin-induced sensitisation and abortion in sows; Hussaini SN et al.; Subcutaneous injection of 235 micrograms endotoxin from E coli 08: K87, K88ab into two sows 14 days before the expected farrowing date elicited a toxic shock reaction but the sows recovered and farrowed normally . No reaction was observed in two other sows which had received endotoxin-free 0.9 per cent saline . In a subsequent pregnancy, the same four sows were injected subcutaneously with 23 mg of the same endotoxin preparation . Those which had previously received endotoxin exhibited a severe shock reaction and aborted after 28 and 40 hours, respectively, while the other two sows showed a milder reaction and farrowed normally.

Prikl Biokhim Mikrobiol, 1986 Jul-Aug, 22(4), 507 - 10
{Aspartase activity of Escherichia coli, strain 85, cells grown on glucose-mineral medium with yeast extract}; Shcherbakova VN et al.; The conditions for cultivation of E . coli 85 on a glucose-mineral medium with a yeast extract were optimized . The cells obtained had a high aspartate ammonia-lyase activity . The aspartase activity was determined kinetically both by consumption of the substrate, ammonium fumarate, and by accumulation of the product, aspartic acid.

Mol Biol (Mosk), 1986 Jul-Aug, 20(4), 1024 - 33
{Statistical characteristics in primary structures of functional regions of Escherichia coli genome . II . Non-stationary Markov chains}; Borodovskii MIu et al.; We introduced non-stationary Marcov chains for statistical description of the DNA E . coli structural domains . The values of all needed parameters for those chains was determined by the preliminary statistical processing of a wide set of the E . coli coding regions . It was shown that non-stationary models predict frequencies of occurrences of various combinations of nucleotides within the coding fragments of DNA, better than stationary ones . In particular non-stationary models give good approximation for short and long distance arrangement of nucleotides in the coding regions . The correlation parameters for neighbour codons and for neighbour amino acid residuals in E . coli protein's primary structure was determined from the non-stationary model of the second order . With the aid of the statistical criteria it was found that neighbour residuals in polypeptide chains can't be considered as independent . The new model of the DNA structural domain may be used in computer algorithms for recognition and classification of DNA functional regions.

Mol Biol (Mosk), 1986 Jul-Aug, 20(4), 1014 - 23
{Statistical characteristics in primary structures of functional regions of Escherichia coli genome . I . Frequency characteristics}; Borodovskii MIu et al.; Analysis of the frequencies of occurrence of mono- and dinucleotides in sequenced E . coli DNA fragments was performed . The DNA sequences of total length 135 000 nucleotides were considered . It was demonstrated that the fragments of DNA which have different functional properties also have different parameters of neighbour nucleotides correlation . Moreover, periodical positional dependence of correlation parameters in coding regions was found . The evolution significance of stated observation is discussed, so as the opportunity of using them in the special model of nucleotide's sequences, which is needed for development of the computer recognition algorithms for genomic functional units.

Mol Biol (Mosk), 1986 Jul-Aug, 20(4), 1002 - 7
{Study of the substrate-induced changes in the state of Eco dam methylase using a method of small-angle x-ray scattering}; Tuzikov FV et al.; Interaction of Ecodam methylase (E.C . 2.1.1) with synthetic oligonucleotide substrates of various primary structure was studied by the small angle X-ray scattering method . Complex formation between the enzyme and substrates occurs after addition of double-stranded oligonucleotides to the methylase . In the presence of 1 M NaC1 (when the enzyme is inactive) addition of the synthetic substrates does not result in complex formation . Comparison of the experimental scattering parameters with the calculated ones has been made . The best coincidence of these data is obtained for the model which proposed Ecodam methylase dimer formation in the course of its interaction with the substrates.

J Microsc, 1986 Jul, 143 ( Pt 1), 81 - 8
Developments of new Lowicryl resins for embedding biological specimens at even lower temperatures; Acetarin JD et al.; Two new Lowicryl resins have been developed for embedding biological materials at temperatures down to 210 K (hydrophilic K11M) and to 190 K (hydrophobic HM23) . They have similar properties to Lowicryl K4M and HM20 . The new resins were first tested for low temperature applications by the 'progressive lowering of temperature' procedure and this shows that the low viscosity of K11M and HM23 is favourable for the infiltration of biological specimens . Hardening is achieved through photo-polymerization at these lower temperatures . These properties make K11M and HM23 suitable for cryosubstitution of rapidly frozen material and it is speculated that the preservation of antigenicity may be further improved.

J Antibiot (Tokyo), 1986 Jul, 39(7), 914 - 21
Structural modification of Escherichia coli peptidoglycan induced by bicyclomycin; Pisabarro AG et al.; We have studied the modification of Escherichia coli peptidoglycan induced by bicyclomycin . For this purpose liquid chromatography for peptidoglycan analysis has been used . The main alteration found was an increase of diaminopimelyl-diaminopimelyl bridge containing subunits . Our results show that bicyclomycin impairs the normal breakage of that interpeptidic bond, whose cleavage is needed for the normal remodeling of peptidoglycan and cell growth . Based on the analysis of the possible structure of diaminopimelyl-diaminopimelyl bond and bicyclomycin, we propose a hypothesis on the mechanism of action of bicyclomycin.

Vet Microbiol, 1986 Jul, 12(2), 109 - 18
Septicaemic Escherichia coli and experimental infection of calves; Contrepois M et al.; Three strains of Escherichia coli with a common surface antigen, 31a, capable of adhering to calf enterocytes in vitro were compared to reference strains of septicaemic E . coli (RVC 330 and vir E . coli) . The surface antigen 31a was present in the RVC 330 reference strain . E . coli vir had a surface antigen which was not present in E . coli 31a or E . coli RVC 330 . The RVC 330 and vir reference strains also adhered to calf enterocytes in vitro . Oral infection of calves not receiving colostrum with E . coli 31a was generally followed by septicaemia and death in less than 48 h . Post-mortem examination revealed pneumonia and oedema of the kidneys and gall bladder . Oral infection of calves receiving colostrum had no effect, but intravenous inoculation produced arthritis within 15 days . The comparison of these results with those previously described by other workers did not lead to the identification of pathognomonic characteristics, which could be clearly correlated with properties specific to E . coli 31a . It is suggested that, like ColV and vir, antigen 31a may be a virulence marker for certain strains of bovine septicaemic E . coli . Furthermore, the 31a antigen appears to be carried on a plasmid.

Radiobiologiia, 1986 Jul-Aug, 26(4), 453 - 9
{Thermoinduced radioresistance of Escherichia coli cells and heat shock proteins}; Verbenko VN et al.; Radioresistance of E . coli cells is slightly increased (dose modification factor (DMF) = 1.2) with temperature elevated from 4 degrees to 43 degrees C at the time of gamma-irradiation . However, an appreciable effect of the thermoinduced radioresistance (DMF = 1.7) was observed when the wild-type cells were exposed to gamma-radiation at 15-43 degrees C (but not at 4 degrees C) after 30-min preincubation at 43 degrees C . This effect was absent in htpR mutants, defective in induction of heat shock proteins, and coupled with the decreased post-irradiation DNA degradation in gamma-irradiated htpR+ cells . It is suggested that heat shock proteins are involved in the thermoinduced radioresistance.

Mol Gen Genet, 1986 Jul, 204(1), 126 - 32
The nucleotide sequence of an Escherichia coli chromosomal region containing the genes for ribosomal proteins S6, S18, L9 and an open reading frame; Schnier J et al.; The DNA sequence of a cluster of genes for ribosomal proteins S6 (rpsF), S18 (rpsR) and L9 (rplI), and of their surrounding regions was determined . The order of the genes was established as promoter-rpsF-rpsR-rplI . There is a 315 bp open reading frame that begins seven nucleotides after the end of rpsF and ends immediately before rpsR . Based on the data of insertional mutagenesis experiments with transposon gamma delta, we concluded that these genes probably form an operon . The amino acid sequence deduced from the nucleotide sequence of the genes agrees completely with the published amino acid sequence data for protein S6, but there are discrepancies in the case of proteins S18 and L9 . The C-terminus of protein S6 was deduced to end with two Glu residues, suggesting that the other Glu residues previously found in this protein are added post-translationally as has been predicted (Reeh and Pedersen 1979) . A possible secondary structure in the leader sequence as well as a possible transcriptional terminator after rplI were noticed in the sequence.

Int J Radiat Oncol Biol Phys, 1986 Jul, 12(7), 1207 - 9
DNA damage induced by reductively activated nitroimidazoles--pH effects; Edwards DI et al.; The effect of pH on E . coli DNA damage measured viscometrically and induced by electrolytically reduced metronidazole and misonidazole has been studied, together with the effect on the statistical average number of electrons required for reduction, measured by high-resolution coulometry, and nitrite production measured colorimetrically . In general, nitroimidazole-induced DNA damage is greatest at acid pH and decreased at alkaline pH, but whereas metronidazole exhibits a linear relationship between DNA damage and increased pH, misonidazole shows a plateau between pH 6 and 8 . The electron requirements for complete reduction (n) vary with pH . For misonidazole n increases with an increase in pH both in the absence and presence of DNA with a shallow plateau between pH 6 and 8 . In contrast, for metronidazole, n decreases with increased pH and exhibits breakpoints between pH 6 and 8 . Nitrite (NO2-) production is linear with increased pH for misonidazole but for metronidazole (NO2-) production shows a sudden increase at 7.5 yielding ca . 35% on a molar basis . The results may reflect differences in the relative stability and reactivity of the nitro radical anion.

EMBO J, 1986 Jul, 5(7), 1711 - 7
Timing of initiation of chromosome replication in individual Escherichia coli cells; Skarstad K et al.; The synchrony of initiation of chromosome replication at multiple origins within individual Escherichia coli cells was studied by a novel method . Initiation of replication was inhibited with rifampicin or chloramphenicol and after completion of ongoing rounds of replication the numbers of fully replicated chromosomes in individual cells were measured by flow cytometry . In rapidly growing cultures, with parallel replication of several chromosomes, cells will end up with 2n (n = 1, 2, 3) chromosomes if initiation occurs simultaneously at all origins . A culture with asynchronous initiation may in addition contain cells with irregular numbers (not equal to 2n) of chromosomes . The frequency of cells with irregular numbers of chromosomes is a measure of the degree of asynchrony of initiation . After inhibition of initiation and run-out of replication in rapidly growing B/r A and K-12 cultures, a small fraction of the cells (2-7%) contained 3, 5, 6 or 7 chromosomes . From these measurements it was calculated that initiation at four origins in a single cell occurred within a small fraction, 0.1, of the doubling time (tau) . A dnaA(Ts) mutant strain grown at permissive temperature exhibited a very large fraction of cells with irregular numbers of chromosomes after drug treatment demonstrating virtually random timing of initiation . A similar pattern of chromosome number per cell was found after treatment of a recA strain.

Can J Vet Res, 1986 Jul, 50(3), 438 - 40
Perivascular eosinophilic droplets in swine brain induced by Escherichia coli toxin; Nakamura K et al.; Pigs inoculated intravenously with the culture supernatant and extract of Escherichia coli were investigated histologically . Perivascular eosinophilic droplets and slight vascular degeneration were observed in the medulla oblongata, mid-brain and pons . These droplets were negative for periodic acid-Schiff staining.

Can J Vet Res, 1986 Jul, 50(3), 402 - 9
Relationships between metabolic changes and clinical signs in pregnant sheep given endotoxin; Naylor JM et al.; Groups of four pregnant ewes were allocated to the following feeding and intravenous endotoxin treatments: fed, Escherichia coli endotoxin (50 micrograms/kg X 75), fed, saline, fasted, E . coli endotoxin (50 micrograms/kg X 75) and fasted, saline . Endotoxin administration resulted in depression, fever, hypoglycemia, hypocalcemia and a reduction in nonesterified fatty acid and ketone body concentrations . Depression correlated best with body temperature (r = 0.76), fasted sheep showed smaller increases in body temperature and were less depressed following endotoxin . Three of eight endotoxin treated sheep died, mortality was not related to rectal temperature but was associated with lactic acidosis . Hypoglycemia was not associated with either death or depression . Fed sheep that were unable to stand had lower serum calcium concentrations than standing sheep.

Can J Vet Res, 1986 Jul, 50(3), 374 - 9
Time course changes in blood metabolites during endotoxin fever in sheep; Southorn BG et al.; Time course changes in the concentration of plasma amino acids, glucose, insulin, and creatinine were measured in seven mature sheep during fever induced by Escherichia coli (serotype 055:B5) endotoxin . Rectal temperature was increased above that recorded in control animals from 0.75 to 6.25 h postinjection with a maximum rise of 2.3 degrees C . Total amino acid concentrations decreased (P less than 0.05) 4.5 h postinjection and remained depressed (P less than 0.05) until 19 h postinjection . The plasma concentration of each individual amino acid decreased (P less than 0.05) at some point during the experiment with the exception of tryptophan and tyrosine . Glucose concentration decreased (P less than 0.05) and remained depressed until at least 55 h postinjection . Plasma insulin concentration was elevated (P less than 0.05) from 4.5 to 13 h postinjection . Plasma creatinine concentration increased during fever (P less than 0.05) and returned to normal by 31 h postinjection.

Biochemistry, 1986 Jul 1, 25(13), 3852 - 8
Thermodynamic analysis of the lactose repressor-operator DNA interaction; Whitson PA et al.; Kinetic and equilibrium constants for lactose repressor-operator DNA interaction have been examined as a function of salt concentration, size and sequence context of the operator DNA, and temperature . Significant salt effects were observed on kinetic and equilibrium parameters for pLA 322-8, an operator-containing derivative of pBR 322, and pIQ, an operator and pseudooperator-containing derivative of pBR 322 . The association rate constant and equilibrium constant for the 40 base pair operator fragment were also salt dependent . Data for all the DNAs were consistent with a sliding mechanism for repressor-operator association/dissociation {Berg, O . G., & Blomberg, C . (1978) Biophys . Chem . 8, 271-280} . Calculation of the number of ionic interactions based on salt dependence yielded a value of approximately 8 for repressor binding to pIQ and pLA 322-8 vs . approximately 6 for the repressor-40 base pair fragment . These data and the differences in binding parameters for the plasmids vs . the 40 base pair operator are consistent with the formation of an intramolecular ternary complex in the plasmid DNAs . Unusual biphasic temperature dependence was observed in the equilibrium and dissociation rate constants for pLA 322-8, pIQ, and the 40 base pair fragment . These observations coupled with a discontinuity found in the inducer association rate constant as a function of temperature suggest a structural change in the protein . The large positive entropy contributions associated with repressor binding to all the DNAs examined provide the significant driving force for the reaction and are consistent with involvement of ionic and apolar interactions in complex formation.

Biochemistry, 1986 Jul 1, 25(13), 3845 - 52
Dissociation of the lactose repressor-operator DNA complex: effects of size and sequence context of operator-containing DNA; Whitson PA et al.; The dissociation kinetics for repressor-32P-labeled operator DNA have been examined by adding unlabeled operator DNA to trap released repressor or by adding a small volume of concentrated salt solution to shift the Kd of repressor-operator interaction . The dissociation rate constant for pLA 322-8, an operator-containing derivative of pBR 322, was 2.4 X 10(-3) s-1 in 0.15 M KCl . The dissociation rate constant at 0.15 M KCl for both lambda plac and pIQ, each of which contain two pseudooperator sequences, was approximately 6 X 10(-4) s-1 . Elimination of flanking nonspecific DNA sequences by use of a 40 base pair operator-containing DNA fragment yielded a dissociation rate constant of 9.3 X 10(-3) s-1 . The size and salt dependences of the rate constants suggest that dissociation occurs as a multistep process . The data for all the DNAs examined are consistent with a sliding mechanism of facilitated diffusion to/from the operator site . The ability to form a ternary complex of two operators per repressor, determined by stoichiometry measurements, and the diminished dissociation rates in the presence of intramolecular nonspecific and pseudooperator DNA sites suggest the formation of an intramolecular ternary complex . The salt dependence of the dissociation rate constant for pLA 322-8 at high salt concentrations converges with that for a 40 base pair operator . The similarity in dissociation rate constants for pLA 322-8 and a 40 base pair operator fragment under these conditions indicates a common dissociation mechanism from a primary operator site on the repressor.

Biochemistry, 1986 Jul 1, 25(13), 3748 - 51
In vivo function of Escherichia coli pyruvate oxidase specifically requires a functional lipid binding site; Grabau C et al.; The pyruvate oxidase of Escherichia coli is a peripheral membrane flavoprotein that is dramatically activated by lipids . The enzyme strongly binds to phospholipid vesicles in vitro . In vivo, in addition to enzyme activation, binding is thought to be important to provide access of the enzyme to ubiquinone dissolved in the lipid bilayer . It was unclear if both or either of these attributes is needed for enzyme function in vivo . To differentiate between activation and lipid binding, we have constructed, using recombinant DNA techniques, a mutant gene that produces a truncated protein . The truncated protein lacks the last 24 amino acids of the C-terminus of the oxidase (due to introduction of a translation termination codon) and thus is closely analogous to the activated species produced in vitro by limited chymotrypsin cleavage {Recny, M.A., Grabau, C., Cronan, J.E., Jr., & Hager, L.P . (1985) J . Biol . Chem . 260, 14287-14291} . The truncated protein (like the protease-derived species) is fully active in vitro in the absence of lipid, and its activity is not further increased by addition of lipid activators . Moreover, the truncated enzyme fails to bind Triton X-114, a detergent that binds to and activates the wild-type oxidase . Strains producing the truncated protein were devoid of oxidase activity in vivo . This result indicates that binding to membrane lipids is specifically required for function of the oxidase in vivo; activation alone does not suffice.

Ann Rheum Dis, 1986 Jul, 45(7), 566 - 71
Significance of non-pathogenic cross reactive bowel flora in patients with ankylosing spondylitis; McGuigan LE et al.; We have previously shown that antisera raised in rabbits to certain enteric cross reactive strains of bacteria are capable of specifically lysing the peripheral blood lymphocytes of HLA-B27 positive patients with ankylosing spondylitis (B27+ AS+) . We now report that bacteria with cross reactive antigenic determinants are found in the bowel flora of all of 52 B27+ AS+ patients but in only one of 50 HLA-B27 positive normal controls (B27+ AS-) . These organisms are functionally similar to the cross reactive enteric bacteria originally reported . They are not confined to a particular genus or species and their cross reactive serological nature appears to be a property shared by all enteric organisms isolated from B27+ AS+ patients . Organisms with these properties have been shown to persist in the bowel flora of 14 B27+ AS+ patients followed up for more than one year.

Anal Biochem, 1986 Jul, 156(1), 45 - 7
A rapid nonchromatographic assay for aminopropyltransferases; Anton DL; Aminopropyltransferases are key enzymes in the biosynthesis of the polyamines spermidine and spermine . A procedure is described for assaying these enzymes by differential charcoal adsorption of 14C-labeled decarboxylated adenosylmethionine substrate from the labeled polyamine product . This assay is linear with time and enzyme concentration, and is suitable for use with a variety of amine acceptors . This procedure has the advantage, over those previously used, that it is extremely rapid yet very sensitive.

J Appl Physiol, 1986 Jul, 61(1), 185 - 91
Methylprednisolone on circulating eicosanoids and vasomotor tone after endotoxin; Hales CA et al.; Acute pulmonary and systemic vasomotor changes induced by endotoxin in dogs have been related, at least in part, to the production of eicosanoids such as the vasoconstrictor thromboxane and the vasodilator prostacyclin . Steroids in high doses, in vitro, inhibit activation of phospholipase A2 and prevent fatty acid release from cell membranes to enter the arachidonic acid cascade . We, therefore, administered methylprednisolone (40 mg/kg) to dogs to see if eicosanoid production and the ensuing vasomotor changes could be prevented after administration of 150 micrograms/kg of endotoxin . The stable metabolites of thromboxane B2 (TxB2) and 6-ketoprostaglandin F1 alpha (6-keto-PGF1 alpha) were measured by radioimmunoassay . Methylprednisolone by itself did not alter circulating eicosanoids but when given 2.5 h before endotoxin not only failed to inhibit endotoxin-induced eicosanoid production but actually resulted in higher circulating levels of 6-keto-PGF1 alpha (P less than 0.05) compared with animals receiving endotoxin alone . Indomethacin prevented the steroid-enhanced concentrations of 6-keto-PGF1 alpha after endotoxin and prevented the greater fall (P less than 0.05) in systemic blood pressure and systemic vascular resistance with steroid plus endotoxin than occurred with endotoxin alone . Administration of methylprednisolone immediately before endotoxin resulted in enhanced levels (P less than 0.05) of both TxB2 and 6-keto-PGF1 alpha but with a fall in systemic blood pressure and vascular resistance similar to the animals pretreated by 2.5 h . In contrast to the early steroid group in which all of the hypotensive effect was due to eicosanoids, in the latter group steroids had an additional nonspecific effect . Thus, in vivo, high-dose steroids did not prevent endotoxin-induced increases in eicosanoids but actually increased circulating levels of TxB2 and 6-keto-PGF1 alpha with a physiological effect favoring vasodilation.

Am J Pathol, 1986 Jul, 124(1), 1 - 9
Neutropenic responses to intradermal injections of Escherichia coli . Effects on the kinetics of polymorphonuclear leukocyte emigration; Cybulsky MI et al.; Killed Escherichia coli organisms injected intradermally into rabbits induced significant neutropenia and provoked a rapid rise in body temperature . Both the magnitude and the duration of the neutropenia were dose-dependent . After recovery from neutropenia, the rabbits became refractory to its redevelopment when subsequently given an equivalent dose of E coli . The influence of neutropenia and the subsequent refractory period on the rate of polymorphonuclear leukocyte (PMN) emigration into inflammatory sites was examined . Killed E coli organisms (6 X 10(8) per site) were injected into two groups of 20 intradermal sites in each rabbit . The first group (Group F) preceded the second (Group S) by 6 hours . The kinetics of PMN emigration, quantitated with 51Cr-labeled cells, differed in the two groups . In Group S sites an intense PMN influx was measured at 0-4 hours, and subsequently the extent of PMN emigration rapidly declined . In Group F sites a minute PMN influx was detected during the first 4 hours, coinciding with a marked neutropenia . The maximal PMN influx into Group F sites was measured between 6 and 10 hours . Microscopic sections at 4 hours showed a scanty PMN infiltrate and numerous bacteria in the dermis of Group F sites, while extensive phagocytosis of bacteria by PMNs was apparent in Group S sites . By comparing the extent of bacterial phagocytosis in 4-hour-old sites with the magnitudes of PMN emigration between 6 and 10 hours in both groups, we concluded that the phagocytic elimination of killed E coli was not a major mechanism regulating the cessation of local PMN emigration . Instead, we propose that tachyphylaxis or desensitization of sites to inflammatory factors released from E coli is the responsible mechanism.

Proc Natl Acad Sci U S A, 1986 Jul, 83(14), 5101 - 5
Cell cycle-specific replication of Escherichia coli minichromosomes; Leonard AC et al.; The timing of Escherichia coli minichromosome replication in the cell division cycle was examined using an improved procedure for studying plasmid replication frequency . Cultures growing exponentially in glucose/Casamino acids minimal medium were pulse-labeled with {3H}thymidine, and the radioactivity incorporated into plasmid DNA in cells of different ages was analyzed . At the end of the labeling period the bacteria were bound to the surface of a nitrocellulose membrane filter, and the radioactivity in new daughter cells, which eluted continuously from the membrane, was quantitated following agarose gel electrophoresis . The minichromosomes replicated during a discrete interval in the cell division cycle that appeared to coincide with initiation of chromosome replication . In contrast, plasmid pBR322 replicated throughout the division cycle at a rate that increased gradually as a function of cell age . The difference in minichromosome and pBR322 replication was clearly discernible in cells harboring both plasmids . It was also found that the 16 kD gene adjacent to oriC was not a determinant of the timing of minichromosome replication during the division cycle . The results are consistent with the conclusion that minichromosome replication frequency is governed by the same mechanism that controls chromosome replication.

Mutat Res, 1986 Jul, 174(3), 183 - 7
Mutagenic and comutagenic effects of ethionine in Escherichia coli K12; Zgaga Z; A possible mutagenic and comutagenic activity of ethionine, an analog of the amino acid methionine, was investigated in several mutant strains of E . coli K12 . Ethionine was found to act as a weak mutagen only in a mismatch repair deficient mutator strain (mutL) and as a comutagen with 2-aminopurine (2AP) in a wild type E . coli . The latter effect was nor observed in a restriction-deficient strain (r-) nor in a recombination or SOS-deficient recA strain . These effects are interpreted as a consequence of restriction-induced double-strand breaks in hypomethylated E . coli DNA resulting in induction of the SOS mutator effect which generates predominantly mismatch correctable untargeted mutations.

J Clin Microbiol, 1986 Jul, 24(1), 149 - 51
Evaluation and optimization of the DNA filter assay for direct detection of enterotoxigenic Escherichia coli in the presence of stool coliforms; Yam WC et al.; Direct detection of enterotoxigenic Escherichia coli (ETEC) in mixed cultures by DNA filter assay was affected by the presence of other bacteria in the cultures . By shortening incubation to 6 h, bacterial overgrowth was minimized, allowing direct detection of ETEC in stools or mixed cultures initially containing 0.2 to 3% ETEC.

J Bacteriol, 1986 Jul, 167(1), 404 - 6
Genetic and molecular analyses of Escherichia coli N-acetylneuraminate lyase gene; Kawakami B et al.; Two plasmids containing the N-acetylneuraminate lyase (NALase) gene (nanA) of Escherichia coli, pNL1 and pNL4, were constructed . Immunoprecipitation analysis indicated that the 35,000-dalton protein encoded in pNL4 was NALase . The synthesis of NALase in E . coli carrying these plasmids was constitutive.

J Bacteriol, 1986 Jul, 167(1), 383 - 6
Role of leader peptide synthesis in tryptophanase operon expression in Escherichia coli K-12; Stewart V et al.; We used site-directed mutagenesis to replace the Escherichia coli tryptophanase (tna) operon leader peptide start codon with AUC . This change greatly decreased the uninduced rate of tna operon expression, and it also lowered the response to inducer . We conclude that leader peptide synthesis plays an essential role in tna operon expression.

J Bacteriol, 1986 Jul, 167(1), 362 - 7
Anaerobically induced genes of Escherichia coli; Winkelman JW et al.; A collection of anaerobically induced gene fusions were isolated, and representative isolates were characterized with respect to their regulatory properties, phenotypes, and approximate map locations . Four fusion strains that had defects in the anaerobic metabolism of asparagine or aspartate were found . These fusions were all repressed by alternate electron acceptors, ammonia, and glucose but were induced by other sugars . Several other fusion strains which demonstrated no observable phenotype showed diverse regulatory responses . The anaerobically induced fusions were scattered around the Escherichia coli chromosome more or less at random, suggesting that all the isolates examined were in separate genes.

J Bacteriol, 1986 Jul, 167(1), 336 - 45
Cloning, mapping, and sequencing of plasmid R100 traM and finP genes; Fee BE et al.; The fertility control gene finP, the transfer gene traM, and the transfer origin, oriT, of plasmid R100 were isolated on a single 1.2-kilobase EcoRV fragment and were then subcloned as HaeIII fragments . The sequence of the 754-base-pair finP-containing fragment is reported here . In addition to the finP gene, the sequence includes all but two bases of the R100 traM open reading frame and apparently all of the leader mRNA sequence and amino end of the traJ gene of R100 . The sequence contains two open reading frames which encode small proteins on the opposite strand from the traM and traJ genes . It also shows two sets of inverted repeats that have the characteristics of transcription terminators . One set is positioned as if it was the traM terminator, and the other set, which is downstream from the first, sits in the middle of the leader mRNA sequence for traJ . On the bottom strand, this inverted repeat has the structure of a rho-independent terminator . Other less-stable inverted repeats overlap this second terminator in the same way as is seen in attenuation sequences, and the two separate small open reading frames on the bottom strand also totally overlap the stem of the rho-independent terminator, suggesting that their translation would cause shifting of termination to the bottom strand homolog of the putative traM terminator . The finP gene product was not identified, but the gene was mapped to the sequence which contains the traJ gene . It either overlaps traJ or is antisense to it.

J Bacteriol, 1986 Jul, 167(1), 327 - 35
Synthesis of linear plasmid multimers in Escherichia coli K-12; Cohen A et al.; Linear plasmid multimers were identified in extracts of recB21 recC22 strains containing derivatives of the ColE1-type plasmids pACYC184 and pBR322 . A mutation in sbcB increases the proportion of plasmid DNA as linear multimers . A model to explain this is based on proposed roles of RecBC enzyme and SbcB enzyme (DNA exonuclease I) in preventing two types of rolling-circle DNA synthesis . Support for this hypothesis was obtained by derepressing synthesis of an inhibitor of RecBC enzyme and observing a difference in control of linear multimer synthesis and monomer circle replication . Reinitiation of rolling-circle DNA synthesis was proposed to occur by recA+-dependent and recA+-independent recombination events involving linear multimers . The presence of linear plasmid multimers in recB and recC mutants sheds new light on plasmid recombination frequencies in various mutant strains.

J Bacteriol, 1986 Jul, 167(1), 312 - 8
Molecular cloning, DNA sequencing, and enzymatic analyses of two Escherichia coli pyruvate oxidase mutants defective in activation by lipids; Chang YY et al.; Two Escherichia coli pyruvate oxidase (EC 1.2.2.2) mutant genes, poxB3 and poxB4, were cloned on plasmid pBR322 . The poxB3 mutant oxidase which was described previously (Y . Y . Chang and J . E . Cronan, Jr., Proc . Natl . Acad . Sci . USA 81:4348-4352, 1984) was deficient in lipid activation but retained full catalytic activity . The poxB3 mutation was located in the C-terminal half of the gene, and the nucleotide alteration has been determined by DNA sequencing of this part of the gene and by comparing the sequence with that of the wild-type strain (C . Grabau and J . E . Cronan, Jr., submitted for publication) . The poxB3 oxidase mutation is the substitution of a serine residue for Pro-536 . poxB4, another pyruvate oxidase mutant gene, was also deficient in lipid activation . The major difference between the poxB3 and poxB4 oxidase was in the binding of Triton detergents . The poxB4 mutation was also located in the C-terminal half of the gene, and sequence analysis has shown that only one nucleotide base was altered, which resulted in Ala-467 being converted to a threonine residue . The results of the amino acid substitutions in the mutant proteins, leading to the functional alteration of the enzyme, are discussed.

J Bacteriol, 1986 Jul, 167(1), 160 - 7
Effects of signal sequence mutations on the kinetics of alkaline phosphatase export to the periplasm in Escherichia coli; Michaelis S et al.; We isolated a collection of mutants defective in the export of alkaline phosphatase to the periplasm . Two classes of mutants were obtained: one class with lesions unlinked to the phoA gene and a second class harboring linked mutations . Among the former class, one mutant is cold sensitive for growth and may be defective in a component of the Escherichia coli secretory apparatus . Included in the latter class are 47 mutants which are characterized in detail in this report . To facilitate DNA sequence analysis of these mutants, we devised a convenient method that relies on homologous recombination in vivo to transfer phoA mutations from the bacterial chromosome directly onto the genome of a single-stranded M13 phage vector . DNA sequence analysis revealed that our collection of mutants comprises six unique mutations, all of which reside in the phoA signal sequence coding region and lend further support to the notion that the length of the hydrophobic core of the signal sequence is crucial for its function in protein export . Kinetic studies showed that in these mutants, the small fraction of alkaline phosphatase which succeeds in reaching a periplasmic location, despite a defective signal sequence, is translocated across the membrane in a slow, posttranslational fashion.

J Bacteriol, 1986 Jul, 167(1), 130 - 7
Characterization of mutational specificity within the lacI gene for a mutD5 mutator strain of Escherichia coli defective in 3'----5' exonuclease (proofreading) activity; Fowler RG et al.; The mutD (dnaQ) gene of Escherichia coli codes for the epsilon subunit of the DNA polymerase III holoenzyme which is involved in 3'----5' exonuclease proofreading activity . We determined the mutational specificity of the mutator allele, mutD5, in the lacI gene of E . coli . The mutD5 mutation preferentially produces single base substitutions as judged from the enhanced fraction of lacI nonsense mutations and the spectrum of sequenced dominant lacI (lacId) and constitutive lacO (lacOc) mutations which were predominantly (69/71) single nucleotide substitutions . The distribution of amber lacI and sequenced lacId mutations revealed that transitions occur more frequently than transversions . A . T----G . C and G . C----A . T transitions were equally frequent and, with one major exception, evenly distributed among numerous sites . Among the transversions, A . T----T . A events were the most common, A . T----C . G substitutions were rare, and G . C----C . G changes were not detected . Transversions were unequally distributed among a limited number of sites with obvious hotspots . All 11 sequenced transversions had a consensus neighboring sequence of 5'-C-C-(mutated G or A)-C-3' . Although no large deletions or complex mutational events were recovered, sequencing revealed that mutD5 induced single nucleotide deletions within consecutive G X C sequences . An extraordinary A . T----G . C transition hotspot occurred at nucleotide position +6 in the lac operator region; the mutD5 mutation frequency of this single base pair was calculated to be 1.2 X 10(-3).

AJR Am J Roentgenol, 1986 Jul, 147(1), 83 - 6
Intraosseous and intradiscal gas in association with spinal infection: report of three cases; Bielecki DK et al.; The detection of a vacuum phenomenon within the intervertebral disk usually confirms the diagnosis of degenerative disease rather than an infective process . A similar phenomenon in the vertebral body generally indicates ischemic necrosis . However, spinal infection may rarely be accompanied by intradiscal or intraosseous gas so that the latter finding does not entirely exclude the possibility of infection . Three cases are reported to illustrate gas formation in association with vertebral infection.

J Histochem Cytochem, 1986 Jul, 34(7), 909 - 12
Aminoglycoside binding sites in Escherichia coli as revealed by neomycin-gold labeling; Morioka H et al.; A cytochemical technique for demonstration of neomycin binding sites by electron microscopy was developed and applied to Escherichia coli . Neomycin was conjugated chemically with bovine serum albumin (BSA) . Colloidal gold was coated with the conjugated neomycin-BSA . The neomycin-BSA-gold was applied to thin sections of Epon-embedded E . coli and examined . Gold particles were observed on the outer membrane and the cytoplasmic membrane of E . coli . It was probably the ribosomes that were being labeled in the cytoplasm . Different cytochemical controls, including a number of inhibition tests and the use of BSA-gold, proved the specificity of this cytochemical technique and provided the biochemical significance of the observations.

J Clin Immunol, 1986 Jul, 6(4), 284 - 91
Secretory antibodies in IgA-deficient and immunosuppressed individuals; Mellander L et al.; Total levels of IgM and secretory IgM as well as specific antibodies to poliovirus type I antigen, Escherichia coli O antigens, and beta-lactoglobulin were measured in unstimulated and stimulated saliva as well as nasal secretion using an enzyme-linked immunosorbent assay (ELISA) . The levels of these antibodies in IgA-deficient adults with and without frequent respiratory infections and children under immunosuppressive therapy for malignant disease were compared to those in normal adults and infants 1-7 months of age . The IgA-deficient adults had significantly higher IgM levels (P less than 0.002) than the normal adults as well as higher levels of IgM antibodies to poliovirus type I (P less than 0.05) and E . coli O antigen (P less than 0.002) . There was a less pronounced IgM anti-beta-lactoglobulin compensation . Secretory component (SC)-carrying antibodies against all three antigens were lower than in normal adults . The infants studied had levels of IgM in secretions close to those of the normal adults and significantly lower than those of the IgA-deficient adults (P less than 0.001) but with a higher proportion of SC-carrying IgM . The increase in total IgM and specific bacterial and viral IgM antibodies in saliva above that of the normal adults was significant (P less than 0.001-0.005) for those IgA-deficient individuals without, but not for those with, frequent infections . There was, however, no significant difference between the levels in the two groups of IgA-deficient adults.(ABSTRACT TRUNCATED AT 250 WORDS)

Crit Care Clin, 1986 Jul, 2(3), 405 - 28
Pathology of the adult respiratory distress syndrome; Meyrick B; Despite the wide range of insults that can lead to the development of ARDS, a common sequence of pathologic changes can be identified in the lung . These changes can be divided into three phases: the acute, or exudative, phase (up to 6 days), in which hyaline membranes are a characteristic feature; the subacute, or proliferative, phase (4 to 10 days), in which metaplasia of the alveolar lining cells and early evidence of fibrosis are seen; and the chronic phase (8 days and on), when organizing fibrosis is a major finding . Structural changes of chronic pulmonary hypertension are also found in the patients with ARDS of longer duration . The mechanism by which these pulmonary changes occur is unknown . Studies of experimental models of ARDS may offer the best opportunity to elucidate the mechanisms . For example, a single infusion of E . coli endotoxin into sheep mimics the pathophysiologic changes of ARDS, offering a model for study of the initial insult on the lung . In addition, animals exposed to high concentrations of oxygen also show morphologic changes similar to those seen in patients with ARDS . Whether the hyperoxia is responsible for such changes, or whether it potentiates the injury induced by some other insult, is not certain.

Mol Gen Genet, 1986 Jul, 204(1), 180 - 4
Autoregulation of the nar operon encoding nitrate reductase in Escherichia coli; Bonnefoy V et al.; Nitrate reductase is demonstrated to exert an autogenous control on its own synthesis . This effect requires the participation of the molybdenum cofactor . Use of strains in which the control region of the nar operon is mutated reveals two loci in this region: one, affected in strain LCB94, is common to both autoregulation and induction by nitrate while the other, mutated in strain LCB188, is specific for the induction by nitrate . It is proposed that the autogenous control prevents the unnecessary accumulation of the nitrate reductase subunits in the cytoplasm.

EMBO J, 1986 Jul, 5(7), 1667 - 73
Heat shock and ecdysterone activation of the Drosophila melanogaster hsp23 gene; a sequence element implied in developmental regulation; Mestril R et al.; The regulation of the Drosophila melanogaster hsp23 gene by heat shock and ecdysterone has been analysed by measuring activities of hsp--Escherichia coli beta-galactosidase hybrid genes in transfected hormone-sensitive D . melanogaster cells . Mutation analysis identified multiple, distinct promoter elements . A sequence element, which also occurs in the promoters of several other developmentally regulated Drosophila genes, is present in regions of the hsp23 promoter that are essential for its ecdysterone, but not its heat-regulated activity; this element may represent a binding site for an ecdysterone--receptor complex . Mutant promoters that can be activated only by heat shock or by hormone have been constructed . Thus the two types of regulation of the hsp23 gene can function independently of each other.

Can J Microbiol, 1986 Jul, 32(7), 594 - 601
Physicochemical roles of soluble metal cations in the outer membrane of Escherichia coli K-12; Ferris FG et al.; Atomic absorption spectroscopy of isolated native and EDTA-modified (lipopolysaccharide-depleted) outer membrane revealed trace amounts of potassium, manganese, and iron (1.0-7.0 nmol/mg dry weight outer membrane) . Sodium, magnesium, and calcium were approximately one order of magnitude more plentiful, but EDTA-modified outer membrane was deficient in calcium . When metal-binding assays were conducted to find the binding capacity of native and EDTA-modified outer membrane, potassium bound poorly compared with sodium . However, there was no difference in the binding of these ions between the OM preparations . In contrast, reduced amounts of magnesium, calcium, manganese, and iron III bound to the EDTA-modified OM . Partitioning of intact cells in a biphasic dextran-polyethyleneglycol system indicated that the reduced lipopolysaccharide content of the EDTA-modified outer membrane increased the hydrophobicity of the cell surface . Exposure of control and EDTA-treated cells to divalent metal salt solutions before phase partitioning also increased cell surface hydrophobicity . Freeze-etching showed that sodium ions had no effect on the membrane fractures observed in control cells, but with EDTA-treated cells, this cation increased the occurrence of small outer membrane fractures (plateaus) which are characteristic of EDTA treatment . Both magnesium and manganese increased the frequency of outer membrane cleavage in control cells, whereas calcium did not . In contrast, all three divalent metallic ions increased the frequency and extent of cleavage in the outer membrane of EDTA-treated cells.

Biochimie, 1986 Jul-Aug, 68(7-8), 935 - 40
Vitamin-D dependent 9 kDa calcium-binding protein gene: cDNA cloning, mRNA distribution and regulation; Thomasset M et al.; Cholecalciferol (calcitriol) the active hormonal form of vitamin D induces the synthesis of at least two intracellular calcium-binding proteins (Ka = 10(6) M-1), the cholecalcins (CaBP) in mammals . We used the synthesis of these proteins to study the genomic steroid-like action of vitamin D . The 9 kDa CaBP is mainly concentrated in the duodenum while 28 kDa CaBP is located in the kidney and cerebellum . Complementary DNA copies of rat intestinal 9 kDa CaBP mRNA were cloned in E . coli . The deduced amino acid sequence for 9 kDa CaBP contains two 'EF hand' domains corresponding to calcium-binding sites I and II . The homology observed suggests, after comparison with the structures of other intracellular CaBPs, that rat 9 kDa CaBP mRNA contains the remains of an untranslated calcium-binding site III-like structure seen in 28 kDa CaBP from kidney and cerebellum of rat . Northern blots showed that the cDNA sequence hybridizes to a homogeneous 500-600 nucleotide mRNA species from rat duodenum . Larger mRNA species encoding 28 kDa CaBP were undetectable in rat kidney and cerebellum even under low stringency conditions . These findings demonstrate that there is no cross-hybridization between 9 kDa and 28 kDa CaBP mRNAs, and Southern analysis indicates that there are distinct genes coding for each rat cholecalcin . The cDNA probe was used to analyze the specific 9 kDa CaBP gene expression along the intestine of growing rats and during gestation and fetal development.(ABSTRACT TRUNCATED AT 250 WORDS)

J Bacteriol, 1986 Jul, 167(1), 350 - 5
Characterization of iucA and iucC genes of the aerobactin system of plasmid ColV-K30 in Escherichia coli; de Lorenzo V et al.; A cloned 8.3-kilobase-pair DNA fragment carrying all the genes (iucABCD iutA) of the aerobactin iron transport system of plasmid pColV-K30 was subjected to in vitro mutagenesis to afford mutant genes iucA, iucC, and iucA iucC . Complementation analyses and identification of aerobactin precursors accumulated by Escherichia coli cells harboring the different constructions allowed assignment of the iucA and iucC genes to discrete steps in biosynthesis of the siderophore from N epsilon-acetyl-N epsilon-hydroxylysine and citrate . Plasmid pVLN10, a derivative carrying a DNA fragment complementing an iucC mutation, expressed in a minicell system a single 62,000-dalton protein as the product of this gene.

J Bacteriol, 1986 Jul, 167(1), 201 - 4
Diverse effects of the MalE-LacZ hybrid protein on Escherichia coli cell physiology; Ito K et al.; The hybrid protein between the periplasmic maltose-binding protein and the cytoplasmic beta-galactosidase (the MalE-LacZ hybrid protein) was previously shown to block the export of envelope proteins when synthesized in large amounts . Now we show that the hybrid protein exerts another major effect on the cell, that is, induction of the heat shock proteins . This latter effect was dependent on the htpR gene product but independent of the function of the signal sequence on the hybrid protein . On the other hand, the previously reported induction of the SecA protein by the hybrid protein was independent of htpR and may be caused by the reduced protein export ability of the cell . The functional htpR gene is essential for viability of the cell in which the basal level of the hybrid protein is synthesized, whereas in the absence of the hybrid protein htpR is dispensable at low temperature . These results indicate that the hybrid protein somehow generates a signal or stress that is similar to what the cell experiences at elevated temperatures.

J Bacteriol, 1986 Jul, 167(1), 101 - 9
Mutations specifically affecting ligand interaction of the Trg chemosensory transducer; Park C et al.; The Trg transducer mediates chemotactic response to galactose and ribose by interacting, respectively, with sugar-occupied galactose- and ribose-binding proteins . Adaptation is linked to methylation of specific glutamyl residues of the Trg protein . This study characterized two trg mutations that affect interaction with binding protein ligands but do not affect methylation or adaptation . The mutant phenotypes indicated that the steady-state activity of methyl-accepting sites is independent of ligand-binding activity . The mutation trg-8 changed arginine 85 to histidine, and trg-19 changed glycine 151 to aspartate . The locations of the mutational changes provided direct evidence for functioning of the amino-terminal domain of Trg in ligand recognition . Cross-inhibition of tactic sensitivity by the two Trg-linked attractants implies competition for a common site on Trg . However, the single amino acid substitution caused by trg-19 greatly reduced the response to galactose but left unperturbed the response to ribose . Thus Trg must recognize the two sugar-binding proteins at nonidentical sites, and the complementary sites on the respective binding proteins should differ . trg-8 mutants were substantially defective in the response to both galactose and ribose . An increase in cellular content of Trg-8 protein improved the response to galactose but not to ribose . It appears that Trg-8 protein is defective in the generation of the putative conformational change induced by ligand interaction . The asymmetry of the mutational defect implies that functional separation of interaction sites could persist beyond the initial stage of ligand binding.

Blood, 1986 Jul, 68(1), 46 - 57
Effects of purified bacterially synthesized murine multi-CSF (IL-3) on hematopoiesis in normal adult mice; Metcalf D et al.; Normal adult C57BL, BALB/c, and C3H/HeJ mice were injected intraperitoneally three times daily for up to 6 days with 102,000 U (200 ng) per injection of purified, bacterially synthesized, Multipotential colony-stimulating factor (CSF) (Interleukin-3) (rMulti-CSF) and compared with control mice injected with serum/saline with or without added endotoxin (1 ng/mL) . Mice injected with rMulti-CSF exhibited tenfold rises in blood eosinophil and twofold to threefold rises in neutrophil and monocyte levels . The spleens from mice injected with rMulti-CSF showed a 50% increase in weight, elevated levels of maturing granulocytes, eosinophils, nucleated erythroid cells and megakaryocytes, and up to 100-fold rises in mast cells . Progenitor cell frequencies in the spleen were elevated sixfold to 18-fold . No significant changes were observed in the marrow . Sixfold to 15-fold rises were observed in peritoneal cell populations of mice injected with rMulti-CSF with evidence of increased peritoneal macrophage phagocytic activity . Livers of C57BL mice, but not of the other strains, exhibited increased numbers of infiltrating hematopoietic cells whereas rises in mast cell numbers were observed in the mesenteric lymph node, skin, and gut in BALB/c and C3H/HeJ mice . Endotoxin was excluded as being responsible for the observed changes except possibly those involving peritoneal macrophage phagocytic activity . The results indicate that the injection of normal mice with rMulti-CSF significantly stimulates the same types of hematopoietic populations as are stimulated in vitro by Multi-CSF and indicate that this and other CSFs should be useful in stimulating hematopoietic repopulation and functional activity in vivo.

J Immunol, 1986 Jul 1, 137(1), 357 - 61
Immunogenicity of the repetitive and nonrepetitive peptide regions of the divergent CS protein of Plasmodium knowlesi; Sharma S et al.; The circumsporozoite (CS) protein of the Nuri strain of the simian malarial parasite Plasmodium knowlesi was expressed as a fusion protein in E . coli . This fusion protein cross-reacted with the polyclonal monkey sera raised against irradiated sporozoites of another strain (H strain) of P . knowlesi . The antibody against the repeat units of the H strain CS protein was affinity purified from the polyclonal sera by using synthetic repeat peptides . The affinity-purified antibody did not cross-react with the Nuri CS fusion protein . The immunogenicity of different regions of the CS protein was additionally studied by using several synthetic peptides . All but the most COOH-terminal peptide showed cross-reactivity with the polyclonal sera . Because the repeat regions of the CS protein of the two strains are diverse, whereas the non-repetitive regions are immunogenic and conserved, the latter may be better suited for a potential vaccine.

Mol Cell Biol, 1986 Jul, 6(7), 2695 - 703
Termination-reinitiation occurs in the translation of mammalian cell mRNAs; Peabody DS et al.; Many examples of internal translation initiation in eucaryotes have accumulated in recent years . In many cases terminators of upstream reading frames precede the internal initiation site, suggesting that translational reinitiation may be a mechanism for initiation at internal AUGs . To test this idea, a series of recombinants was constructed in the mammalian expression vector pSV2 . Each contained a dicistronic transcription unit comprising the coding sequence for mouse dihydrofolate reductase (DHFR) followed by the gene for xanthine-guanine phosphoribosyl transferase (XGPRT) from Escherichia coli . Various versions of this pSV2dhfr-gpt recombinant plasmid altered the location at which the DHFR reading frame was terminated relative to the XGPRT initiation codon and demonstrated that this is a critical factor for the expression of XGPRT activity in transfected Cos-1 cells . Thus, when the DHFR frame terminated upstream or a very short distance downstream of the XGPRT initiator AUG, substantial levels of XGPRT activity were observed . When the DHFR frame terminated 50 nucleotides beyond the XGPRT initiator, activity was reduced about twofold . However, when the DHFR and XGPRT sequences were fused in-frame so that ribosomes which initiated at the DHFR AUG did not terminate until they encountered the XGPRT terminator, production of XGPRT activity was abolished . This dependence of internal translation initiation on the position of terminators of the upstream reading frame is consistent with the hypothesis that mammalian ribosomes are capable of translational reinitiation.

Mol Biol (Mosk), 1986 Jul-Aug, 20(4), 1008 - 13
{Expression of aminoglycoside phosphotransferase gene is under the control of recA promoter}; Kiselev VI et al.; A novel expression vector using the 300 bp promotor-operator fragment of the recA gene of Escherichia coli has been constructed . The strength of the recA promotor was examined by assaying aminoglycoside phosphotransferase (APT) activity expressed from APT gene placed downstream of the promotor . We have observed, that some plasmids, containing N-portion of recA gene caused a large increase in radiosensitivity of host bacteria cells.

Biochem Cell Biol, 1986 Jul, 64(7), 638 - 46
Purification and characterization of catalase HPII from Escherichia coli K12; Loewen PC et al.; Catalase (hydroperoxidase II or HPII) of Escherichia coli K12 has been purified using a protocol that also allows the purification of the second catalase HPI in large amounts . The purified HPII was found to have equal amounts of two subunits with molecular weights of 90,000 and 92,000 . Only a single 92,000 subunit was present in the immunoprecipitate created when HPII antiserum was added directly to a crude extract, suggesting that proteolysis was responsible for the smaller subunit . The apparent native molecular weight was determined to be 532,000, suggesting a hexamer structure for the enzyme, an unusual structure for a catalase . HPII was very stable, remaining maximally active over the pH range 4-11 and retaining activity even in a solution of 0.1% sodium dodecyl sulfate and 7 M urea . The heme cofactor associated with HPII was also unusual for a catalase, in resembling heme d (a2) both spectrally and in terms of solubility . On the basis of heme-associated iron, six heme groups were associated with each molecule of enzyme or one per subunit.

Vet Q, 1986 Jul, 8(3), 195 - 203
The effects of ACTH, prednisolone and Escherichia coli endotoxin on some clinical haematological and blood biochemical parameters in dwarf goats; van Miert AS et al.; ACTH (microgram kg-1 i.v.) and prednisolone (1 microgram-1 i.v.) caused a moderate but statistically significant inhibition of rumen contractions, whereas no effects on heart rate and body temperature were observed . Both hormones induced hyperglycaemia and leucocytosis, characterised by moderate lymphopenia and a profound increase in the number of circulating neutrophils . A significant decrease in plasma iron and increase in plasma zinc concentrations were observed . After 3 daily i.m . injections of ACTH (10 micrograms-1 day-1) decreases were seen in both serum Alkaline phosphatase (ALP) activity and plasma trace metal concentrations; heart rate was significantly higher . Intraveneous injection of E . coli endotoxin (0.1 microgram kg-1) caused shivering, fever, inhibition of rumen contractions, changes in heart rate, lymphopenia, neutropenia followed by neutrophilic leucocytosis, hypoferraemia, hypozincaemia, hypoglycaemia and a decline in serum ALP activity . ACTH, given i.m . for 3 days, reduced the febrile responses to E . coli endotoxin, modified the changes in heart rate, intensified the inhibition of rumen contractions, and induced a more marked decrease in the number of circulating neutrophils . ACTH pretreatment did not affect the endotoxin-induced decrease in blood glucose concentrations nor the drop in plasma zinc and iron values . These results suggest that glucocorticosteroids are not primarily involved in the fall in plasma iron and zinc concentrations during E . coli endotoxin-induced fever, the effects of endotoxin released glucocorticosteroids on white blood cells and blood glucose are masked by some other effect(s) of endotoxin, and in dwarf goats, ACTH has antipyretic properties without influencing normal body temperature . This effect is probably not dependent on adrenal cortical activity.

Mol Gen Genet, 1986 Jul, 204(1), 82 - 4
Role of the RecF gene product in UV mutagenesis of lambda phage; Wood RD et al.; E . coli recF mutants have a greatly reduced capacity for Weigle mutagenesis of ultraviolet light-irradiated lambda phage . A recF 332::Tn3 mutation was introduced into an E . coli recA441 lexA51 strain which constitutively expresses SOS functions . Weigle mutagenesis of phage lambda could occur in the resulting strain in the absence of host cell irradiation, and was increased when the recA441 (tif) allele was activated by increased temperature and excess adenine . The inability of recF strains to support Weigle mutagenesis can therefore be ascribed to a defect in expression of SOS functions after irradiation.

Mol Gen Genet, 1986 Jul, 204(1), 70 - 4
Mistranslation during phenylalanine starvation; Parker J et al.; Starvation for phenylalanine led to leucine misincorporation frequencies of 0.1 and 0.6 at UUC codons in the argI transcript of Escherichia coli, but no detectable misincorporation at a UUU codon . Under similar starvation conditions the relative synthesis of full sized MS2 coat protein, encoded by the RNA virus or a DNA copy, is greatly reduced, preventing analysis of the protein . This reduction in amount is unaffected by a rpsL mutation.

Mol Gen Genet, 1986 Jul, 204(1), 17 - 23
Feedback regulation of RNA polymerase subunit synthesis after the conditional overproduction of RNA polymerase in Escherichia coli; Bedwell DM et al.; The beta and beta' subunits of RNA polymerase are thought to be controlled by a translational feedback mechanism regulated by the concentration of RNA polymerase holoenzyme . To study this regulation in vivo, an inducible RNA polymerase overproduction system was developed . This system utilizes plasmids from two incompatibility groups that carry RNA polymerase subunit genes under lac promoter/operator control . When the structural genes encoding the components of core RNA polymerase (alpha, beta and beta') or holoenzyme (alpha, beta, beta' and sigma 70) are present on the plasmids, induction of the lac promoter results in a two fold increase in the concentration of functional RNA polymerase . The induction of RNA polymerase overproduction is characterized by an initial large burst of beta beta' synthesis followed by a gradual decrease as the concentration of RNA polymerase increases . Overproduction of RNA polymerase in a strain carrying an electrophoretic mobility mutation in the rpoB gene results in the specific repression of beta beta' synthesis off the chromosome . These results indicate that RNA polymerase feedback regulation controls beta beta' synthesis in vivo.

Mol Gen Genet, 1986 Jul, 204(1), 148 - 52
Sequence of the promoter and 5' coding region of pepN, and the amino-terminus of peptidase N from Escherichia coli K-12; McCaman MT et al.; The pepN gene of Escherichia coli K-12 has been cloned onto a multi-copy plasmid and shown to encode a polypeptide which co-migrates with purified peptidase N . Transformed strains have been shown to contain up to a one hundred fold increase in the amount of peptidase N . We isolated the peptidase N protein and determined the sequence of its first 15 amino acids . By restriction mapping, we identified and subcloned the 5' region of the pepN gene and then determined its nucleotide sequence . Comparison of the actual amino acid sequence with that predicted from the extended open reading frame found in the DNA sequence indicated that peptidase N is not synthesized as a pre-protein precursor . The presumed region preceding the open reading frame contained nucleotide sequence having homology to the procaryotic promoter consensus sequences for the -35 and the -10 regions and the ribosome binding site.

Mol Gen Genet, 1986 Jul, 204(1), 120 - 5
Lytic activity localized to membrane-spanning region of phi X174 E protein; Buckley KJ et al.; Lytic activity of the phi X174 E (lysis) protein had previously been localized to the amino terminal 51 amino acids (a.a.) of the molecule (Blasi and Lubitz 1985) . This E gene lytic activity has here been further localized to the amino terminal 29 a.a., a region of the protein which is thought to just span the cell membrane (Young and Young 1982) . phi X174 E gene fusions to both the lacZ gene and the chloramphenicol acetyl transferase (CAT) gene resulted in fusion proteins with lytic activity . Fusion to a third protein, trpE, did not result in lytic activity . These results support a model of oligomerization of the phi X174 E protein for lytic activity since both beta-galactosidase and CAT exist as tetramers in their native state . A difference in the composition of the charged amino acids at the cytoplasmic boundary between the various fusion proteins could also account for these results, since these amino acids may play a role in proper anchoring of the E protein in the cell membrane . In a spontaneous E gene mutant, which introduces a proline residue at position 9 of the E protein, lytic activity of the E protein was decreased, but not abolished . The presence of the helix-breaking proline at this position may interfere with insertion of the lysis protein into the cell membrane, leading to the decreased functional activity of the protein.

Mol Gen Genet, 1986 Jul, 204(1), 115 - 9
Regions associated with the stable maintenance of plasmid pSC101 and its tetracycline resistance; Makino S et al.; Two regions tentatively called unsA and unsR were identified on pSC101 . One, unsA, corresponds to less than 650 bp of the N-terminal in the tetracycline resistance structural gene and seems to inhibit stable maintenance of pSC101 . The other, unsR, is defined within the 1 kb XhoI-EcoRI region located upstream of the tetracycline resistance structural gene and is a regulatory gene clearly distinct from tetR (Unger et al . 1984); it serves as a suppressor of the unsA function.

Mol Gen Genet, 1986 Jul, 204(1), 1 - 7
Nucleotide sequence of the lig gene and primary structure of DNA ligase of Escherichia coli; Ishino Y et al.; The DNA ligase of Escherichia coli catalyses the NAD-dependent formation of phosphodiester linkages between 5'-phosphoryl and 3'-hydroxyl groups in DNA . It is essential for DNA replication and repair of damaged DNA strands . We determined the nucleotide sequence of the lig gene of Escherichia coli coding for DNA ligase and flanking regions . The coding frame of the gene was confirmed by the amino acid composition and the amino- and carboxyl-terminal amino acid sequences of the purified ligase . The ligase consists of 671 amino acid residues with a molecular weight of 73,690.

Genetika, 1986 Jul, 22(7), 1073 - 80
{Isolation and characteristics of compound transposons Tn1000::Tn5}; Egorov IA et al.; Two types of compound transposons were derived . In the first case, transposon Tn5 is inserted into the gene responsible for Tn1000 transposase synthesis . In the other, Tn5 is inserted into the region near the left end of Tn1000, where no functionally significant genes were found . It is known that translocation of the compound transposons does not depend on their size and takes place with the efficiency close to that characteristic of the intact Tn1000 . Insertion of Tn5 into the gene coding for Tn1000 transposase results in sharp decrease in the efficiency of Tn1000 translocations . This effect, however, may be eliminated by introduction into the cell of the intact Tn1000.

Genetika, 1986 Jul, 22(7), 1061 - 6
{Restriction mapping of recombinant plasmids carrying the genes for arginine biosynthesis in Escherichia coli K-12}; Nersisian AA et al.; ArgA and argECBH genes of Escherichia coli K-12 were cloned on the pBR322 vector . Restriction maps of the recombinant plasmids were constructed . Deletion mutants of these recombinant plasmids, retaining the functional argA and argE genes, were obtained using different restriction enzymes . All of the recombinant derivatives have the replication properties of the pBR322 vector.

Can J Microbiol, 1986 Jul, 32(7), 591 - 3
Restriction endonuclease activities in the legionellae; Chen GC et al.; Fifteen legionellae isolates were studied for the presence of restriction endonucleases . At least three different specificities were found in nine of these isolates . One particular activity was present in two groups of isolates isolated from widely separated geographic areas.

Biochimie, 1986 Jul-Aug, 68(7-8), 1001 - 7
Cloning of yeast lysyl- and phenylalanyl-tRNA synthetase genes; Mirande M et al.; Cloning of yeast lysyl- and phenylalanyl-tRNA synthetase genes was accomplished by probing a lambda gt11 recombinant DNA expression library with antibodies directed against the purified enzymes . Several DNA clones encoding either the alpha or the beta subunit of phenylalanyl-tRNA synthetase were isolated . In each case, the inserted DNA was oriented in the same direction with respect to the lambda gt11 lacZ transcription unit, giving rise to the expression of hybrid proteins . The corresponding DNA fragments constitute suitable hybridization probes for the isolation of complete nucleotide sequences encoding the alpha and beta subunits of the enzyme . Recombinant DNA lambda gt11 clones encoding lysyl-tRNA synthetase were also selected . One of these contained yeast DNA inserted with the opposite orientation with respect to lacZ . The lysogen corresponding to that recombinant DNA phage produced an active, native lysyl-tRNA synthetase . The 3.6 kbp DNA insert contained all the information necessary for the expression of yeast lysyl-tRNA synthetase in E . coli.

Plasmid, 1986 Jul, 16(1), 52 - 62
A common plasmid of Chlamydia trachomatis; Palmer L et al.; A 7.4-kb plasmid is a common and perhaps essential component of the Chlamydia trachomatis genome . This plasmid occurs as 10 copies per chlamydial chromosomal equivalent . It is unable to replicate in Escherichia coli . Complete plasmid genomes from eight serovars of C . trachomatis have been isolated in E . coli as cloned sequences ligated to pBR322 . Restriction enzyme cleavage site mapping indicates that these plasmids are closely related . Homologous plasmid sequences have also been detected by DNA hybridization in all of the 200 clinically isolated strains of C . trachomatis which have been examined . DNA sequences homologous to the C . trachomatis plasmid were not found in eucaryotic DNA nor in a plasmid of similar size isolated from C . psittaci . C . trachomatis plasmid genes are expressed in vivo and the plasmid encoded gene products may play a role in the intracellular growth of this organism . Plasmid encoded genes were also expressed from the cloned C . trachomatis plasmid in E . coli minicells and using an E . coli S-30 in vitro transcription translation extract.

Plasmid, 1986 Jul, 16(1), 30 - 6
Location and cloning of the ultraviolet-sensitizing function from the chromosomally associated IncJ group plasmid, R391; Pembroke JT et al.; The IncJ plasmid R391, which specifies a uv-sensitizing function, has been shown to be associated with chromosomal DNA . Deletions originating from Tn10 insertion into the kanamycin-resistance determinant of plasmid R391 gave rise to uv-resistant derivatives . This apparent linkage between the kanamycin-resistance determinant and the uv-sensitizing gene(s) was used to clone the uv-sensitizing function from plasmid R391 into pUR222 . A recombinant plasmid containing both functions (KanR and Uvs+) was obtained . The uv-sensitizing function was mapped to a 4-kb EcoRI fragment.

Plasmid, 1986 Jul, 16(1), 15 - 29
Evidence for the involvement of the incC locus of broad host range plasmid RK2 in plasmid maintenance; Thomas CM; Plasmid pRK2501 is a deletion derivative of broad host range plasmid RK2 and encodes two trfB-regulated operons: the trfA operon which codes for both kilD, which interferes with plasmid maintenance if unregulated, and trfA whose protein product(s) is essential for replication from oriVRK2; and part of the trfB operon, containing both trfB/korA/korD, whose product negatively regulates transcription of both trfA and trfB operons, and incC, the product of which interferes in trans with inheritance of RK2 and certain of its derivatives . Plasmid pRK2501ts3 is a derivative with a point mutation in trfB, rendering plasmid maintenance temperature sensitive . Transcriptional fusions of the trfB operon and the galK gene demonstrate that this mutation derepresses trfB operon transcription at both 30 and 42 degrees C . The trfA operon is also derepressed by this mutation . Since the trfB gene product appears to be defective at both permissive and non-permissive temperatures the temperature sensitivity of pRK2501ts3 must be due to a secondary effect . In fact, it appears to arise from the inhibitory behavior of derepressed incC at the non-permissive temperature since a major class of "revertant" of pRK2501ts3 contains deletions inactivating incC and a reconstruction experiment demonstrates that such a deletion is sufficient for "reversion." Maxicell experiments show that at the non-permissive temperature the trfA operon polypeptide products are produced at much lower levels, an effect partly reversed by a deletion affecting incC . It is proposed that incC normally plays a role in maintenance of IncP plasmids by modulation of trfA operon expression.

Genetics, 1986 Jul, 113(3), 483 - 97
Transitory derepression and the maintenance of conjugative plasmids; Lundquist PD et al.; It has been proposed that bacterial plasmids cannot be maintained by infectious transfer alone and that their persistence requires positive selection for plasmid-borne genes . To test this hypothesis, the population dynamics of two laboratory and five naturally occurring conjugative plasmids were examined in chemostat cultures of E . coli K-12 . Both laboratory plasmids and three of the five wild plasmids failed to increase in frequency when introduced at low frequencies . However, two of the naturally occurring plasmids rapidly increased in frequency, and bacteria carrying them achieved dominance in the absence of selection for known plasmid-borne genes . Three hypotheses for the invasion and persistence of these two plasmids were examined . It is concluded that although these two extrachromosomal genetic elements are repressed for conjugative pili synthesis, as a consequence of high rates of transfer during periods of transitory derepression in newly formed transconjugants, they become established and are maintained by infectious transfer alone . The implications of these observations to the theory of plasmid maintenance and the evolution of repressible conjugative pili synthesis are discussed.

Proc Natl Acad Sci U S A, 1986 Jul, 83(14), 5076 - 80
Mutations of the ras gene product p21 that abolish guanine nucleotide binding; Clanton DJ et al.; We have constructed several point mutations affecting the GTP-binding site of p21, the ras-encoded protein . Both lysine (116K) and tyrosine (116Y) mutations of asparagine-116, which, by analogy with the crystal structure of elongation factor Tu (EF-Tu), has critical interactions with the guanine base, abolish GTP binding and transforming activities of p21 . These activities are retained by proteins with a mutation at position 117 or 118 . Both 116K and 116Y mutant p21s, when overproduced in Escherichia coli, are apparently devoid of GTP-binding and autokinase activities . Similarly, the mutant DNAs do not transform NIH 3T3 cells in a focus-forming assay . By cotransfection with pSV-neo, cell clones resistant to the neomycin analog G418 have been isolated . Cells transfected with 116K or 116Y mutant DNA are contact inhibited . In contrast to competent clones, the defective mutants have no detectable phosphorylated p21 . The present results suggest that the basic structure of the GTP-binding site is conserved between p21 and EF-Tu and that this binding site is crucial for ras gene function.

Proc Natl Acad Sci U S A, 1986 Jul, 83(14), 5057 - 61
Escherichia coli mutS-encoded protein binds to mismatched DNA base pairs; Su SS et al.; The Escherichia coli mutS gene product is involved in mismatch correction in this organism . We have purified a biologically active form of the 97,000 Mr protein to near homogeneity from an overproducing strain . Enzymatic and chemical protection ("footprinting") experiments have demonstrated that mutS-encoded protein specifically binds to DNA regions containing a single base-pair mismatch . The protein displayed variable affinity for the limited set of mismatches tested (G-T greater than G-A approximately equal to A-C greater than T-C).

Proc Natl Acad Sci U S A, 1986 Jul, 83(13), 4695 - 9
Inquiries into the structure-function relationship of ribonuclease T1 using chemically synthesized coding sequences; Ikehara M et al.; The genes for ribonuclease T1 and its site-specific mutants were chemically synthesized and introduced to Escherichia coli . All enzymes were fusion products produced by joining the synthetic gene at specific restriction sites to the synthetic gene for human growth hormone in a plasmid containing the E . coli trp promoter . The fusion protein from this plasmid contained 66% of the amino-terminal sequences of the human growth hormone, which were recognizable immunologically . RNase T1 or its mutants were cleaved from the fusion protein with cyanogen bromide . The synthetic RNase T1 endowed with the revised wild-type triad Gly-Ser-Pro, residues 71-73, was fully functional, readily hydrolyzing pGpC bonds, whereas a mutant enzyme having the originally reported, erroneous triad Pro-Gly-Ser was totally inactive . Various amino acid substitutions were also introduced to the guanosine recognition region comprised of residues 42-45, Tyr-Asn-Asn-Tyr . Substitution of either of the tyrosine residues noted above with phenylalanine had no dramatic effect on the enzyme's function . Replacement of asparagine-43 with arginine or alanine also caused only a small change in the hydrolyzing activity--a mutant enzyme maintained greater than 50% of the wild-type activity . In sharp contrast, when aspartic acid or alanine was substituted for asparagine-44, the activity was dramatically reduced to a few percent of the wild-type activity.

Mutat Res, 1986 Jul, 166(1), 89 - 98
Microinjection of Escherichia coli UvrA, B, C and D proteins into fibroblasts of xeroderma pigmentosum complementation groups A and C does not result in restoration of UV-induced unscheduled DNA synthesis; Zwetsloot JC et al.; The UV-induced unscheduled DNA synthesis (UDS) in cultured human fibroblasts of repair-deficient xeroderma pigmentosum complementation groups A and C was assayed after injection of identical activities of either Uvr excinuclease (UvrA, B, C and D) from Escherichia coli or endonuclease V from phage T4 . Under conditions where the T4 enzyme was able to induce repair synthesis in both XP complementation groups in agreement with earlier observations (de Jonge et al., 1985), no effect of the UvrABCD excinuclease could be observed either when the enzymatic complex was injected into the cytoplasm, or when it was delivered directly into the nucleus . In addition, no effect of the E . coli excinuclease was found on the repair ability of normal repair-proficient human fibroblasts . We conclude that the UvrABCD excinuclease may not work on DNA lesions in human chromatin.

Mutat Res, 1986 Jul, 166(1), 17 - 22
Role of the radB gene in postreplication repair in UV-irradiated Escherichia coli uvrB; Sargentini NJ et al.; In UV-irradiated Escherichia coli, the radB101 mutation sensitized uvrB recF cells 4-fold and uvrB recB cells 1.2-fold, but did not sensitize uvrB recB recF cells . The radB mutation had very little effect (1.2-fold or less) on the repair of UV radiation-induced DNA daughter-strand gaps in uvrB cells, but it did cause about a 3-fold deficiency in the repair of the DNA double-strand breaks that arise in association with nonrepaired daughter-strand gaps in UV-irradiated uvrB recF cells . Thus, the radB gene does not appear to be involved in the recF-dependent or recF recB-independent processes for the repair of DNA daughter-strand gaps, but is involved in the recB-dependent postreplication repair of DNA double-strand breaks.

J Nucl Med, 1986 Jul, 27(7), 1147 - 9
Advantage of indium-111 leukocytes over ultrasound in imaging an infected renal cyst; Fortner A et al.; Indium-111-labeled leukocyte scanning is a highly sensitive and specific method of detecting abscesses . This report describes a patient with polycystic kidneys and a single infected cyst . Ultrasound could not determine which cyst was infected, but the infected cyst could be localized by {111In}leukocyte imaging in conjunction with a {99mTc}DMSA renal scan . The two radionuclide studies were used to identify an infected renal cyst and direct ultrasound guided aspiration.

J Gen Virol, 1986 Jul, 67 ( Pt 7), 1427 - 34
Infectious bronchitis immunity: its study in chickens experimentally infected with mixtures of infectious bronchitis virus and Escherichia coli; Cook JK et al.; The live infectious bronchitis (IB) vaccine, H120, protected chickens against intranasal challenge with a mixture of Escherichia coli strains (E . coli Pool) and IB virus (IBV) strains of the same (Massachusetts) serotype as H120; it usually also protected against challenge with the E . coli Pool and IBV strains of other serological types . When these challenge strains were themselves used as vaccines they usually protected against challenge with a mixture of the E . coli Pool and an IBV strain of the Massachusetts serotype (VF69-149) or an IBV strain not of the Massachusetts serotype (HVI-116) . Poor protection, when observed, was most common in those experiments involving a minority of the IBV strains that had been incriminated in recent outbreaks of disease in vaccinated flocks of chickens . Much lower concentrations of IBV strain VF69-149 and E . coli O18 were found in the nose, trachea and spleen of H120-vaccinated chickens killed at different times after they were given a mixture of these organisms than were found in these sites in similarly challenged unvaccinated chickens . Some protection against challenge with IBV and the E . coli Pool was also observed in chickens vaccinated with an inactivated IBV strain; it was much less effective than that obtained following vaccination with the corresponding live IBV strain.

J Clin Invest, 1986 Jul, 78(1), 177 - 84
Effects of the putative neutrophil-generated toxin, hypochlorous acid, on membrane permeability and transport systems of Escherichia coli; Albrich JM et al.; Titrimetric addition of hypochlorous acid (HOCl) or chloramine (NH2Cl) to suspensions of Escherichia coli decreases their ability to accumulate 14C-labeled glutamine, proline, thiomethylgalactoside, and leucine in a manner that approximately coincides with loss of cell viability; quantitative differences in cellular response are observed with the two oxidants . Inhibition of beta-galactosidase activity in E . coli ML-35, a strain lacking functional lactose permease, is complex and also depends upon the identity of the oxidant . Membrane proton conductivities and glycerol permeabilities are unchanged by addition of HOCl or NH2Cl in excess of that required for inactivation . The combined results are interpreted to indicate that the locus of HOCl attack is the cell envelope, that HOCl inactivation does not occur by loss of membrane structural integrity, that loss of transport function can be identified with either selective oxidative inhibition of the transport proteins or loss of cellular metabolic energy, and that different mechanisms of inactivation may exist for HOCl and NH2Cl.

J Clin Microbiol, 1986 Jul, 24(1), 16 - 20
The vector homology problem in diagnostic nucleic acid hybridization of clinical specimens; Ambinder RF et al.; Nucleic acid hybridization techniques using cloned probes are finding application in assays of clinical specimens in research and diagnostic laboratories . The probes that we and others have used are recombinant plasmids composed of viral inserts and bacterial plasmid vectors such as pBR322 . We suspected that there was material homologous to pBR322 present in many clinical samples . because hybridization occurred in samples which lacked evidence of virus by other techniques . If the presence of this vector-homologous material was unrecognized, hybridization in the test sample might erroneously be interpreted as indicating the presence of viral sequences . In this paper we demonstrate specific hybridization of labeled pBR322 DNA with DNA from various clinical samples . Evidence is presented that nonspecific probe trapping could not account for this phenomenon . In mixing experiments, it is shown that contamination of clinical samples with bacteria would explain such a result . Approaches tested to circumvent this problem included the use of isolated insert probes, alternate cloning vectors, and cold competitor pBR322 DNA in prehybridization and hybridization mixes . None proved entirely satisfactory . We therefore emphasize that it is essential that all hybridization detection systems use a control probe of the vector alone in order to demonstrate the absence of material with vector homology in the specimen tested.

J Bacteriol, 1986 Jul, 167(1), 82 - 8
Cloning and expression of the Escherichia coli glgC gene from a mutant containing an ADPglucose pyrophosphorylase with altered allosteric properties; Leung P et al.; A mutant strain of Escherichia coli K-12, designated 618, accumulates glycogen at a faster rate than wild-type strain 356 . The mutation affects the ADPglucose pyrophosphorylase regulatory properties (N . Creuzat-Sigal, M . Latil-Damotte, J . Cattaneo, and J . Puig, p . 647-680, in R . Piras and H . G . Pontis, ed., Biochemistry of the Glycocide Linkage, 1972) . The enzyme is less dependent on the activator, fructose 1,6 bis-phosphate for activity and is less sensitive to inhibition by the inhibitor, 5'-AMP . The structural gene, glgC, for this allosteric mutant enzyme was cloned into the bacterial plasmid pBR322 by inserting the chromosomal DNA at the PstI site . The glycogen biosynthetic genes were selected by cotransformation of the neighboring asd gene into an E . coli mutant also defective in branching enzyme (glgB) activity . Two recombinant plasmids, pEBL1 and pEBL3, that had PstI chromosomal DNA inserts containing glgC and glgB were isolated . Branching enzyme and ADPglucose pyrophosphorylase activities were increased 240- and 40-fold, respectively, in the asd glgB mutant, E . coli K-12 6281 . The E . coli K-12 618 mutant glgC gene product was characterized after transformation of an E . coli B ADPglucose pyrophosphorylase mutant with the recombinant plasmid pEBL3 . The kinetic properties of the cloned ADPglucose pyrophosphorylase were similar to those of the E . coli K-12 618 enzyme . The inserted DNA in pEBL1 was arranged in opposite orientation to that in pEBL3.

J Bacteriol, 1986 Jul, 167(1), 393 - 5
Regulation of glycerol kinase by enzyme IIIGlc of the phosphoenolpyruvate:carbohydrate phosphotransferase system; de Boer M et al.; Wild-type glycerol kinase of Escherichia coli is inhibited by both nonphosphorylated enzyme IIIGlc of the phosphoenolpyruvate:carbohydrate phosphotransferase system and fructose 1,6-diphosphate . Mutant glycerol kinase, resistant to inhibition by fructose 1,6-diphosphate, was much less sensitive to inhibition by enzyme IIIGlc . The difference between the wild-type and mutant enzymes was even greater when inhibition was measured in the presence of both enzyme IIIGlc and fructose 1,6-diphosphate . The binding of enzyme IIIGlc to glycerol kinase required the presence of the substrate glycerol.

EMBO J, 1986 Jul, 5(7), 1645 - 51
Adenovirus-2 early region IA protein synthesized in Escherichia coli extracts indirectly associates with DNA; Ko JL et al.; The interaction of adenovirus-2 (Ad2) early region IA (EIA) protein (encoded by the 13S mRNA) with DNA was examined using EIA protein synthesized in Escherichia coli extracts directed by a plasmid containing the cloned EIA gene . Without any purification, this protein when chromatographed over calf thymus DNA immobilized on cellulose, showed at least two types of salt-sensitive activities after associating with equal efficiency to both single- and double-stranded DNA; however, a putative C-terminal proteolytic fragment of the EIA protein (identified by immunoprecipitation with anti-serum specific to the EIA carboxy-terminus) showed 10-fold greater affinity to double- versus single-stranded DNA . When examined with Ad2 DNA, the EIA protein had a retention that was at least 2-fold higher compared to calf thymus DNA, suggesting some substrate specificity . It was also found that a 1.0 M salt concentration was required for the elution of the EIA protein from pBR322 DNA containing cloned regulatory sequences of adenovirus early regions II and III . This suggests that the strength of the protein interaction depends on the target DNA sequence . Finally, addition of uninfected HeLa cell extract to bacterial extracts containing EIA-like protein potentiated the association of the protein to double-stranded calf thymus DNA up to 7-fold . These data support the hypothesis that the EIA protein interacts with target DNA, presumably mediated by co-factor(s) in an indirect fashion.

Eur J Biochem, 1986 Jul 1, 158(1), 43 - 9
Biochemical comparison of the Neurospora crassa wild type and the temperature-sensitive and leucine-auxotroph mutant leu-5 . Purification of the cytoplasmic and mitochondrial leucyl-tRNA synthetases and comparison of the enzymatic activities and the degradation patterns; Kunugi S et al.; The cytoplasmic leucyl-tRNA synthetases of Neurospora crassa wild type (grown at 37 degrees C) and mutant (grown at 28 degrees C) were purified approximately 1770-fold and 1440-fold respectively . Additional enzyme preparations were carried out with mutant cells grown for 24 h at 28 degrees C and transferred then to 37 degrees C for 10-70 h of growth . The mitochondrial leucyl-tRNA synthetase of the wild type was purified approximately 722-fold . The mitochondrial mutant enzyme was found only in traces . The cytoplasmic leucyl-tRNA synthetase from the mutant (grown at 37 degrees C) in vivo is subject of a proteolytic degradation . This leads to an increased pyrophosphate exchange, without altering aminoacylation . Proteolysis in vitro by trypsin or subtilisin of isolated cytoplasmic wild-type and mutant leucyl-tRNA synthetases, however, did not establish and difference in the degradation products and in their catalytic properties . Comparing the cytoplasmic wild-type and mutant enzymes (grown at 28 degrees C) via steady-state kinetics did not show significant differences between these synthetases either . The rate-determining step appears to be after the transfer of the aminoacyl group to the tRNA, e.g . a conformational change or the release of the product . Besides leucine only isoleucine is activated by the enzymes with a discrimination of approximately 1:600; however, no Ile-tRNALeu is released . Similarly these enzymes, when tested with eight ATP analogs, cannot be distinguished . For both enzymes six ATP analogs are neither substrates nor inhibitors . Two analogs are substrates with identical kinetic parameters . The mitochondrial wild-type leucyl-tRNA synthetase is different from the cytoplasmic enzyme, as particularly exhibited by aminoacylating Escherichia coli tRNALeu but not N . crassa cytoplasmic tRNALeu . The presence of traces of the analogous mitochondrial mutant enzyme could be demonstrated . Therefore, the difference between wild-type and mutant leu-5 does not rest in the catalytic properties of the cytoplasmic leucyl-tRNA synthetases . Differences in other properties of these enzymes are not excluded . In contrast the activity of the mitochondrial leucyl-tRNA synthetase of the mutant is approximately 1% of that of the wild-type enzyme.

Proc Natl Acad Sci U S A, 1986 Jul, 83(13), 4650 - 4
Nucleotide sequence and expression of the selenocysteine-containing polypeptide of formate dehydrogenase (formate-hydrogen-lyase-linked) from Escherichia coli; Zinoni F et al.; The gene (fdhF) coding for the selenopolypeptide of the benzylviologen-linked formate dehydrogenase of Escherichia coli was cloned and its nucleotide sequence was determined . The fdhF gene contains, within an open reading frame coding for a protein of 715 amino acids (calculated molecular weight, 79,087), an opal (UGA) nonsense codon in amino acid position 140 . Existence of this nonsense codon was confirmed by physical recloning and resequencing . Internal and terminal deletion clones and lacZ fusions of different N-terminal parts of fdhF were constructed and analyzed for selenium incorporation . Selenylated truncated polypeptide chains or beta-galactosidase fusion proteins were synthesized when the deletion clones or gene fusions, respectively, contained the fdhF gene fragment coding for the selenopolypeptide sequence from amino acid residue 129 to amino acid residue 268 . Translation of the lacZ part of the fusions required the presence of selenium in the medium when the N-terminal fdhF part contained the UGA codon and was independent of the presence of selenium when a more upstream part of fdhF was fused to lacZ . The results are consistent with a co-translational selenocysteine incorporation mechanism.

Proc Natl Acad Sci U S A, 1986 Jul, 83(13), 4599 - 603
Replication of UV-irradiated single-stranded DNA by DNA polymerase III holoenzyme of Escherichia coli: evidence for bypass of pyrimidine photodimers; Livneh Z; Replication of UV-irradiated circular single-stranded phage M13 DNA by Escherichia coli RNA polymerase (EC 2.7.7.6) and DNA polymerase III holoenzyme (EC 2.7.7.7) in the presence of single-stranded DNA binding protein yielded full-length as well as partially replicated products . A similar result was obtained with phage G4 DNA primed with E . coli DNA primase, and phage phi X174 DNA primed with a synthetic oligonucleotide . The fraction of full-length DNA was several orders of magnitude higher than predicted if pyrimidine photodimers were to constitute absolute blocks to DNA replication . Recent models have suggested that pyrimidine photodimers are absolute blocks to DNA replication and that SOS-induced proteins are required to allow their bypass . Our results demonstrate that, under in vitro replication conditions, E . coli DNA polymerase III holoenzyme can insert nucleotides opposite pyrimidine dimers to a significant extent, even in the absence of SOS-induced proteins.

Mol Gen Genet, 1986 Jul, 204(1), 8 - 16
The tobacco mitochondrial ATPase subunit 9 gene is closely linked to an open reading frame for a ribosomal protein; Bland MM et al.; A transcribed segment of mitochondrial DNA (mtDNA) from Nicotiana tabacum contains the F0-ATPase subunit 9 gene, an open reading frame with homology to the E . coli small subunit ribosomal protein S13 and an open reading frame with homology to a portion of the mammalian "URF 1" protein, recently shown to be a component of the NADH:ubiquinone reductase complex (NADH:Q 1) . The transcriptional patterns of the tobacco ATPase 9 gene and S13-like open reading frame share eight RNA species indicating the two sequences are part of the same transcriptional unit . A maize mtDNA fragment contains the S13 homologous sequence and the NADH:Q 1 homologous sequence in an orientation similar to tobacco . The S13-like sequence is present as a single copy in maize and tobacco, as two copies in wheat, and is absent in pea and bean . We discuss the distribution and orientation of the S13-like and "URF 1"-like sequences and the possibility that they are active genes.

Mol Gen Genet, 1986 Jul, 204(1), 75 - 81
Organization and expression of genes involved in the biosynthesis of 987P fimbriae; de Graaf FK et al.; A chromosomal DNA segment encoding the biosynthesis of 987P fimbriae was isolated by cosmid-cloning and subsequent subcloning into pBR322 . The 12 kb DNA segment expressed five polypeptides with apparent molecular weights of 81,000, 39,000, 28,500, 20,500, and 16,500, respectively . The location of the corresponding genes was determined by insertional mutagenesis using Tn5 . The 20.5 K polypeptide was identified as the 987P fimbrial subunit by its reaction with specific anti-987P antibodies . The 81, 39, and 28.5 K polypeptides appeared to be accessory proteins involved in 987P production.

J Clin Lab Immunol, 1986 Jul, 20(3), 143 - 5
The effects of fimbriate and non-fimbriate strains of Escherichia coli on the oxidative reactions of neutrophils from a variety of species; Beswick PH et al.; 5 fimbriate strains and 5 non-fimbriate strains of Escherichia coli were tested for their ability to enhance the cyanide insensitive oxygen consumption of human, bovine and ovine blood neutrophils in the absence of serum opsonins . Neutrophils from all 3 animal species showed significantly increased oxygen consumption when challenged with fimbriate strains . Only ovine neutrophils were stimulated by non-fimbriate strains although to a lesser extent than by fimbriate strains . In the presence of serum the stimulation of ovine neutrophils was the same whether fimbriate or non-fimbriate strains were used.

J Hosp Infect, 1986 Jul, 8(1), 47 - 56
Susceptibility of porin- and lipopolysaccharide-deficient strains of Escherichia coli to some antiseptics and disinfectants; Russell AD et al.; The sensitivities of some outer membrane protein (Omp) and/or lipopolysaccharide (LPS)-defective mutants of Escherichia coli to chlorhexidine and some quaternary ammonium compounds (QACs) were examined . Wild-type strains, with no defect in Omp or LPS were the most resistant to QACs, whereas LPS-deficient strains were the most sensitive . As expected, QAC resistance could not be related to the presence or absence of any specific porin(s) . Chlorhexidine was highly active against all strains, inhibitory concentrations lying within the narrow range of 1 and 2 mg l-1.

Arch Biochem Biophys, 1986 Jul, 248(1), 116 - 20
N,N'-dicyclohexylcarbodiimide and 4-chloro-7-nitrobenzofurazan bind to different beta subunits of the F1 ATPase of Escherichia coli; Stan-Lotter H et al.; The fluorescent thiol reagent 2-(4'-iodoacetamidoanilino)naphthalene-6-sulfonic acid (IAANS) labels the gamma, delta, and one of the three beta subunits of the F1 ATPase from Escherichia coli (ECF1) . This is the same beta subunit which incorporates 4-chloro-7-nitrobenzofurazan (Nbf) {H . Stan-Lotter and P . D . Bragg (1986) Eur . J . Biochem . 154, 321-327} . After inactivation of ECF1 with N,N'-dicyclohexylcarbodiimide (DCCD), IAANS labels in addition to the beta, gamma, and delta subunits also the alpha subunit . This suggests a conformational change of ECF1 upon binding of DCCD . The beta subunit which incorporates DCCD does does not bind IAANS . Likewise, IAANS-modified ECF1 does not incorporate DCCD into the same beta subunit . It is concluded that DCCD and Nbf bind to different beta subunits . Since neither of these reagents binds to that beta subunit which can be crosslinked to to the epsilon subunit by 1-ethyl-3-{3-(dimethylamino)propyl}carbodiimide, these data show that there is a difference in the chemical reactivity of each of the three beta subunits of ECF1, despite their identical primary structures . This suggests that there is an asymmetry in the F1 molecule.

Proc Natl Acad Sci U S A, 1986 Jul, 83(13), 4631 - 5
Cloning and sequencing of the pertussis toxin genes: operon structure and gene duplication; Nicosia A et al.; Pertussis toxin, a protein composed of five different subunits (S1, S2, S3, S4, and S5), is the major virulence factor of Bordetella pertussis . We have cloned and sequenced a DNA fragment of 4.7 kilobases that contains the genes coding for the five subunits . The genes are clustered within 3.2 kilobases in the following order: S1, S2, S4, S5, and S3 . A sequence closely resembling Escherichia coli promoters is found only before the S1 gene, and a possible termination signal is present at the end of the S3 gene, which suggests that the pertussis toxin genes are organized in a single operon . A possible Shine-Dalgarno sequence is present before the S1 gene but not before the other four genes that 8-12 nucleotides upstream from the ATG codon show a new consensus sequence, 5'TCC(T)GG3', possibly involved in the regulation of translation . We have also found sequence homology between the S2 and S3 genes and their protein products indicating that gene duplication played a major role in the evolution of pertussis toxin.

J Bacteriol, 1986 Jul, 167(1), 407 - 10
Functional relationship among the gene clusters encoding F7(1), F7(2), F9, and F11 fimbriae of human uropathogenic Escherichia coli; Van Die I et al.; The P fimbrial gene clusters encoding the serologically different F7(1), F7(2), F9, and F11 fimbriae were compared functionally . The results show that these gene clusters are closely related.

J Bacteriol, 1986 Jul, 167(1), 389 - 92
Roles of H+-ATPase and proton motive force in ATP-dependent protein translocation in vitro; Chen LL et al.; Membrane vesicles from an Escherichia coli mutant with a deletion of the uncBC operon required ATP to translocate proteins, thus ruling out an essential role of F1F0-H+-ATPase in ATP-dependent protein translocation . Moreover, proteins could be translocated in the absence of proton motive force . At suboptimal ATP concentrations, D-lactate stimulated protein translocation, indicating that proton motive force, although insufficient to support translocation, could facilitate the process.

Vopr Med Khim, 1986 Jul-Aug, 32(4), 47 - 51
{Effective complexes of the antileukemic enzyme L-asparaginase with dextran sulfate}; Karsakevich AS et al.; Enzymatic and antileukemic effects of the complexes of L-asparaginase from E . coli and biologically active polymer dextran sulfate were studied to increase their therapeutic properties . The complex was characterized by more distinct substrate specificity, by an increase in the stability during storage, in thermostability as well as in the resistance to proteolysis . The increased antileukemic activity of the complex was observed in experimental lymphoid leukemia L5178y in mice . Use of the complex of L-asparaginase and dextran sulfate enabled to decrease distinctly the therapeutic dose of the enzyme.

Biochem Cell Biol, 1986 Jul, 64(7), 681 - 91
The anthranilate aggregate of Escherichia coli: kinetics of inhibition by tryptophan of phosphoribosyltransferase; Gonzalez JE et al.; The kinetic mechanism of the phosphoribosyltransferase reaction is shown to be rapid equilibrium random bi bi with an enzyme-anthranilate-pyrophosphate abortive complex . We present a rate equation that not only predicts the observed kinetic patterns but also accommodates the fact that feedback inhibition is partial, even though tryptophan (Ki = 0.5 microM) and phosphoribosylpyrophosphate (Km = 50 microM) are competitive . Neither ligand completely abolishes the effect of the other . Instead, the binding of one ligand leads to a mutual elevation in the dissociation constant of the opposing ligand by a factor of two to three . Tryptophan inhibition is noncompetitive with respect to anthranilate (Km = 0.58 microM) and does not diminish the rate of interconversion of ternary complexes . Tryptophan cooperativity, with respect to the inhibition of phosphoribosyltransferase, conforms to the concerted Monod-Wyman-Changeux formulation (kinetic Hill coefficient = 2), whereas tryptophan as an inhibitor of anthranilate synthase more closely conforms to a Koshland model of sequential cooperativity with a kinetic Hill coefficient of 1.4 . The aggregate contains only one class of tryptophan sites . Thus the first tryptophan molecule bound to the aggregate maximally inhibits both phosphoribosyltransferase active centers and one of the two anthranilate synthase catalytic sites . The remaining anthranilate synthase subunit thereupon is converted into a form with less (but not zero) affinity for chorismate and a greater affinity for a second molecule of tryptophan.

Ann Inst Pasteur Immunol, 1986 Jul-Aug, 137D(1), 63 - 78
Immunogenicity of Plasmodium falciparum antigenic determinants produced by Escherichia coli recombinant clones; Mercereau-Puijalon O et al.; The immunogenicity of two parasite antigens produced by Escherichia coli as proteins fused to beta-galactosidase was investigated in three animal species: mice, rabbits and squirrel monkeys . 2L protein carries 71 amino acids of a parasite antigen and 11.1 protein carries 23 repeats of a 9-amino-acid repetitive unit . The humoral response was studied using indirect immunofluorescence and immunoprecipitation . The results indicate that immunization of mice, rabbits and squirrel monkeys using SDS-denatured 2L fusion protein induced antibodies able to bind to parasite antigen 2L in the IFA or in the immunoprecipitation assays . Immunization using the native fusion protein did not induce antibodies able to immunoprecipitate the 2L parasite antigen . The same observation was made for the animals immunized with 11.1 recombinant protein . In this case, the antibody response was also measured by ELISA using synthetic dimers of the repeat as antigen . In mice and rabbits, high titres of anti-11.1 antibodies were found by ELISA . However, when the antigen produced by the parasite itself was used to evaluate the response, low titres were found . This indicates that the animals produced high levels of antibodies to a structure which is not exposed in the parasite . In squirrel monkeys, the same observation was made, but the overall levels of the response to 11.1 antigen were considerably lower than those observed in mice or rabbits.

Biochemistry, 1986 Jul 1, 25(13), 3916 - 25
NMR evidence for dynamic secondary structure in helices II and III of the RNA of Escherichia coli; Leontis NB et al.; A new ribonuclease A (RNase A) resistant fragment of the 5S ribonucleic acid (RNA) from Escherichia coli has been isolated and characterized . This fragment comprises helix III and most of helix II of the parent molecule, a part of the 5S RNA molecule for which several energetically equivalent secondary structures have been proposed {De Wachter, R., Chen, M.-W., & Vandenberghe, A . (1984) Eur . J . Biochem . 143, 175-182} . The imino proton spectrum of this fragment has been studied by nuclear magnetic resonance methods at 500 MHz . The data obtained are readily rationalized in terms of one of the structures proposed for this region of 5S RNA . They also suggest that upon heating, this structure is replaced by a second, different one, consistent with the view that the helix II-helix III region of 5S RNA is able to switch between alternative structures . Among the products of the nucleolytic digestion of 5S RNA is a species whose sequence indicates that RNase A can ligate RNA as well as hydrolyze it.

Anal Biochem, 1986 Jul, 156(1), 136 - 9
Requirement for cysteine in the color silver staining of proteins in polyacrylamide gels; Chuba PJ et al.; To determine whether cysteine residues have a contribution to the mechanism of color silver staining, we silver stained sodium dodecylsulfate polyacrylamide gel electrophoresis separations of proteins which have few or no cysteines . Proteins without cysteine stained negatively (yellow against a yellow background) with silver . Proteins with one or more cysteines stained orange, red, brown, or green/gray depending on the mole percentage of cysteine and whether they contained covalently attached lipids . The colors could not be correlated with the mole percentages of cysteine of these proteins indicating that some components other than cysteine affect the staining color of cysteine-containing proteins . Silver staining of amino acids, sugars, nucleotide bases, or lipopolysaccharide dot-blotted onto nitrocellulose paper implicated adenine, lipids, the basic amino acids, and glutamine, but not sugars or other amino acids in silver/protein complexes.

Arch Biochem Biophys, 1986 Jul, 248(1), 53 - 61
H+/ATP stoichiometry of proton pumps from Neurospora crassa and Escherichia coli; Perlin DS et al.; A kinetic method has been used to measure the apparent stoichiometry of H+ ions translocated per ATP split by membrane-bound {H+}-ATPases . In this method, membrane vesicles are suspended in well-buffered medium, ATP is added, and a fluorescent probe of delta pH (acridine orange) is used to detect the formation of a steady-state pH gradient . At the steady state, it is assumed that proton pumping in one direction is exactly balanced by the leak of protons in the opposite direction . The pump is then rapidly turned off by the addition of an appropriate inhibitor, and the initial rate of relaxation of delta pH is used to infer the pump rate . This rate is divided by the rate of ATP hydrolysis, measured under the same condition, to give the apparent H+/ATP stoichiometry . The method has been applied to two different {H+}-ATPases, the plasma-membrane ATPase of Neurospora (a Mr = 100,000 integral membrane protein) and the ATPase of Escherichia coli (which belongs to the F0F1 group) . The Neurospora ATPase displayed an apparent stoichiometry close to 1 H+/ATP (0.82-1.23), in agreement with previous estimates from electrophysiological measurements on whole cells . In contrast, the E . coli ATPase yielded an apparent stoichiometry close to 2 H+/ATP (1.90), consistent with several published values obtained by both kinetic and thermodynamic methods for bacterial, mitochondrial, and chloroplast ATPases.

Proc Natl Acad Sci U S A, 1986 Jul, 83(14), 5062 - 6
tRNA anticodon replacement experiments show that ribosomal frameshifting can be caused by doublet decoding; Bruce AG et al.; The expression of certain normal genes requires a specific ribosomal frameshift event because the mRNA has the coding information for one protein in two different reading frames . One of several possible mechanisms for this involves recognition of a nontriplet codon by a noncognate tRNA . The AGUC-decoding Escherichia coli tRNASer3 reads a GCA alanine codon to cause a -1 frameshift . Replacement of the anticodon of tRNAPhe with the anticodon of tRNASer3 allows the constructed tRNA to cause this frameshifting . By altering the anticodon loop nucleotides at positions 33-36 in the constructed tRNAPhe molecules, the tRNA was found to recognize a 2-base codon . Instead of the usual anticodon, positions 34-36, the nucleotides in positions 34 and 35 form essential base pairs with the first two positions of the alanine codon . The uridine in position 36 is also required but not for base pairing.

Proc Natl Acad Sci U S A, 1986 Jul, 83(14), 5000 - 4
Mechanism for the autogenous control of the crp operon: transcriptional inhibition by a divergent RNA transcript; Okamoto K et al.; Expression of the crp gene is negatively autoregulated by the complex of cyclic AMP and its receptor protein (cAMP-CRP) . We find a second promoter in this region that is strongly activated in vitro and in vivo by cAMP-CRP . Transcription from this promoter is initiated 3 nucleotides upstream and on the opposite strand from the start of crp mRNA . The addition of the purified 5' segment of the divergent RNA specifically inhibits crp transcription in vitro . cAMP-CRP does not block crp expression if the new promoter is altered so that divergent RNA cannot be made . The initial nucleotides of the divergent RNA are complementary to 10 of the first 11 nucleotides of the crp mRNA . Since the next 11 nucleotides of crp mRNA are A + U-rich, and RNA hybrid between the divergent RNA and the 5' end of crp mRNA could produce a structure similar to a rho-independent terminator, leading to inhibition of crp transcription.

Mutat Res, 1986 Jul, 166(1), 9 - 16
5-Azacytidine: survival and induction of the SOS response in Escherichia coli K-12; Barbe J et al.; Survival and induction of the SOS system by 5-azacytidine, an analog of cytidine, were studied in Escherichia coli K-12 . This compound did not produce any effect on the viability of dcm and dam dcm mutants . Furthermore, recA430 and lexA1 strains (both mutations interfere with LexA repressor cleavage but not recombination proficiency) were more resistant than the wild-type strain of E . coli K-12 . In contrast, recBC and recA13 mutants were more sensitive to 5-azacytidine than the wild type . Transient exposure of E . coli to 5-azacytidine for 60 min induced both recA-dependent inhibition of cell division and induction of lambda prophage in Dcm+ strains but not in Dcm- mutants . Expression of both functions was dependent on recBC exonuclease . On the other hand, 5-azacytidine was unable to trigger the induction of umuCD and mucB genes and no amplification of RecA protein synthesis in either Dcm+ or Dcm- strains was observed . These last results are in agreement with previously reported data suggesting that there is a discrimination in the expression of the several SOS functions and that some SOS genes may be induced without amplification of RecA protein synthesis.

J Bacteriol, 1986 Jul, 167(1), 319 - 26
Replication of plasmid RK2 in vitro by a DNA-membrane complex: evidence for initiation of replication and its coupling to transcription and translation; Kornacki JA et al.; The following results with an in vitro replication system utilizing a plasmid RK2 DNA-membrane complex indicate that the essential trfA-encoded replication protein of RK2 is present and active in the complex . (i) A complex extracted from a conditional replication mutant of RK2, which contains a temperature-sensitive mutation in trfA, displayed extensive DNA synthesis at the permissive temperature but little activity at the restrictive temperature . A control wild-type RK2 complex showed no inhibition of DNA synthesis at the restrictive temperature . (ii) Analysis of plasmid-encoded proteins revealed that the trfA-specified replication protein and other proteins which may be involved in the replication and maintenance of RK2 are located physically in the complex . Semiconservative plasmid DNA replication by the DNA-membrane complex was indicated by density shift experiments; DNA synthesized in the presence of a heavy-density precursor banded primarily in a heavier-density area of a neutral CsCl density gradient and consisted mostly of heavy- and light-density single-stranded DNA as determined by alkaline CsCl density gradient centrifugation . Plasmid RK2 DNA replication by the DNA-membrane complex appears to be coupled to transcription and translation as indicated by the following results: the inhibitory effects of chloramphenicol on both DNA and protein synthesis by the complex; the stimulation of replication by components normally required for protein synthesis (tRNA and all the common amino acids); the synthesis of RNA and protein by the complex; and the synthesis of specific RK2-encoded proteins.

J Bacteriol, 1986 Jul, 167(1), 25 - 9
Escherichia coli grpE gene codes for heat shock protein B25.3, essential for both lambda DNA replication at all temperatures and host growth at high temperature; Ang D et al.; We have identified the grpE gene product as the B25.3 heat shock protein of Escherichia coli on the following evidence: (i) a protein similar in size and isoelectric point to B25.3 was induced after infection of UV-irradiated bacteria by lambda grpE+ transducing phage, (ii) mutant phage lambda grpE40, isolated by its inability to propagate on grpE280 bacteria, failed to induce the synthesis of the B25.3 protein, and (iii) lambda grpE+ revertants, derived from phage grpE40 as able to propagate on grpE280 bacteria, simultaneously recovered the ability to induce synthesis of the B25.3 protein . In addition, we show that E . coli bacteria carrying the grpE280 mutation are temperature sensitive for bacterial growth at 43.5 degrees C . Through transductional analysis and temperature reversion experiments, it was demonstrated that the grpE280 mutation is responsible for both the inability of lambda to replicate at any temperature tested and the lack of colony formation at high temperature . At the nonpermissive temperature the rates of synthesis of DNA and RNA were reduced in grpE280 bacteria.

Mol Cell Endocrinol, 1986 Jul, 46(2), 131 - 5
Purification of pituitary and biosynthetic human growth hormone using monoclonal antibody immunoadsorbent; Jonsdottir I et al.; This report describes the purification of human growth hormone from crude pituitary extract and lysate of recombinant E . coli by an immunoadsorbent purification with monoclonal antibody coupled to solid phase . By a single-immunoaffinity chromatography step pure hGH can be obtained from either origin as revealed by SDS-PAGE followed by silver staining or immunoblotting . An additional ion-exchange chromatography step results in homogeneous 22 kDa hGH preparations . Furthermore, this method may be used for isolation of a pituitary hGH variant which has higher binding affinity for this monoclonal antibody than the major 22 kDa form . This study clearly illustrates the potential of monoclonal antibody immunoadsorbents for purification of different molecular forms of hGH.

Carcinogenesis, 1986 Jul, 7(7), 1231 - 4
DNA repair pathway in alkylated human cells: apurinic/apyrimidinic intermediate resolved by Escherichia coli endonuclease IV-coupled alkaline elution; Moran MF et al.; Apurinic/apyrimidinic (AP) sites were measured in HeLa cells by digestion of cellular DNA with Escherichia coli endonuclease IV, an AP-specific endonuclease, prior to alkaline elution . The absence of non-specific endonuclease activity allowed endonuclease IV-sensitive AP sites to be detected with the sensitivity of conventional alkaline elution . Cells that were alkylated with dimethyl sulfate contained AP sites that were repaired along with DNA single-strand breaks during a post-alkylation recovery period . These results show that DNA alkylation products are repaired, at least in part, via an AP intermediate suggesting a repair pathway initiated by DNA glycosylases followed by DNA incision by AP endonuclease.

Biochem Biophys Res Commun, 1986 Jun 30, 137(3), 964 - 9
Catalytic function of a tyrosyl residue in tryptophanase; Kakizono T et al.; Tryptophanase has an essential tyrosyl residue/active site which can be modified by tetranitromethane . Pyridoxal 5'-phosphate can prevent this modification efficiently, whereas pyridoxal 5'-phosphate N-oxide cannot, indicating that the free pyridinium N is required for the interaction of the coenzyme with the tyrosyl residue, probably via a hydrogen bond . The weakened binding of the coenzyme to the modified enzyme was confirmed on gel filtration, the modified enzyme being dissociated from the coenzyme seven-fold faster than the native enzyme . Furthermore, absorption spectral analyses demonstrated that the modified enzyme can catalyze the transaldimination step, but fails to abstract the alpha-H of substrates . The tyrosyl residue, therefore, not only participates in coenzyme binding, but also contributes to alpha-H labilization.

J Chromatogr, 1986 Jun 25, 360(2), 385 - 95
Reversed-phase high-performance liquid chromatography peptide mapping of bovine somatotropin; Hartman PA et al.; Reversed-phase high-performance liquid chromatography peptide mapping techniques have been used to examine the primary structure of bovine somatotropin (bSt) isolated from Escherichia coli modified by recombinant DNA techniques (rbSt) and from bovine pituitary (pbSt) . Peptide fragments arising from tryptic digestion of bSt were separated on Baker wide pore C4 or C8 columns with linear gradients (acetonitrile-water with 0.1% trifluoroacetic acid) . Major peaks eluted in a consistent order, but significant day-to-day variation in retention times was observed . Isolated peptide fragments were analyzed via acid hydrolysis followed by formation and separation of the phenylthiocarbamyl amino acids . These correspond to expected tryptic fragments.

J Biol Chem, 1986 Jun 25, 261(18), 8230 - 6
The complete amino acid sequence of potato alpha-glucan phosphorylase; Nakano K et al.; The complete amino acid sequence of potato alpha-glucan phosphorylase has been determined . The monomer contains 916 amino acids with a molecular weight of 103,916 . About one-fourth of the amino-terminal threonine is blocked by an acetyl group . Sequence comparison among phosphorylases from potato tuber, rabbit muscle, and Escherichia coli reveals the presence of a characteristic 78-residue insertion in the middle of the polypeptide chain of the potato enzyme . Except for the large inserted portion, 51 and 40% of the amino acids in the potato enzyme are identical with the rabbit muscle and E . coli enzymes, respectively . The regions relevant to the regulation of activity are completely different among the three enzymes, whereas those involved in the catalytic reaction are well conserved . The potato enzyme sequence is consistent with the tertiary structure of the rabbit muscle enzyme . The 78-residue insertion is located at the junction of the amino- and carboxyl-terminal domains on the molecular surface near the glycogen storage site . This insertion could account for the substrate discrimination of the potato enzyme . The molecular evolution of phosphorylase is discussed based on the presence of the large insertion of the potato enzyme.

Nucleic Acids Res, 1986 Jun 25, 14(12), 5067 - 80
The gene II proteins of the filamentous phages IKe and Ff (M13, fd and f1) are not functionally interchangeable during viral strand replication; Peeters BP et al.; Gene II protein is the only phage-encoded protein required for the double-stranded DNA replication of the distantly related filamentous phages IKe and Ff (M13, fd and f1) . Complementation studies have demonstrated that, despite a significant degree of homology between the nucleotide sequences of the gene II's of IKe and Ff and the core's (domains A) of their viral strand replication origins, the biological functions of the gene II proteins are not interchangeable . The specificity of these proteins is not determined by the nucleotide sequence (domain B) which is required for efficient initiation of viral strand replication of Ff . In fact, our data indicate that a sequence with a similar function as domain B in Ff does not form part of the viral strand replication origin of IKe.

Nucleic Acids Res, 1986 Jun 25, 14(12), 4865 - 79
The involvement of host replication proteins and of specific origin sequences in the in vitro replication of miniplasmid R1 DNA; Ortega S et al.; The in vitro replication of R1 miniplasmid promoted by purified preparations of the plasmid encoded RepA protein in cell extracts of E . coli is resistant to rifampicin and can be completely inhibited by antibodies against DnaG, the primase of the cell, as well as by antibodies against proteins DnaB and SSB . R1 replication is abolished in extracts deficient in the DnaA protein . This deficiency is efficiently complemented by purified preparations of the DnaA protein . The in vitro replication of plasmid R1 is also abolished in DnaC deficient extracts and by a 10 bp deletion (nucleotides 1463-1472) within the minimal origin region . These data indicate the requirement of the DnaA, DnaB, DnaC, DnaG and SSB replication proteins of the host, as well as of specific oriR1 sequences for the RepA dependent replication of plasmid R1 . The implications of these results for the initiation of R1 replication are discussed.

J Biol Chem, 1986 Jun 25, 261(18), 8081 - 4
Two-electron reduced mercuric reductase binds Hg(II) to the active site dithiol but does not catalyze Hg(II) reduction; Miller SM et al.; Mercuric reductase contains FAD and a redox-active disulfide which is reduced to a thiol/thiolate pair in two-electron reduced enzyme (EH2) (Fox, B . and Walsh, C.T . (1982) J . Biol . Chem . 257, 2498-2503) . A charge transfer interaction between the thiolate and oxidized FAD gives EH2 a characteristic absorption spectrum, very similar to that found with other flavoprotein disulfide oxidoreductases . We have examined the reaction of EH2 with HgCl2 (+/- mercaptoethanol) in stopped-flow kinetic and static titration experiments . In the absence of mercaptoethanol, reaction of EH2 with HgCl2 yields a final spectrum which is indistinguishable from that of oxidized enzyme . The nature of the final species was examined by titration of enzyme thiols with 5,5'-dithiobis-2,2'-nitrobenzoic acid under denaturing conditions in the presence of NaI to displace any Hg(II) bound to enzyme thiols . These studies demonstrate that EH2 tightly complexes Hg(II) with its active site thiols, but is incapable of reducing Hg(II) to Hg0 . For the latter reaction to occur, additional reducing equivalents are required . In catalysis, the enzyme must first be reduced to EH2 after which it cycles between EH2 and EH2 X NADPH forms . This is in contrast to other flavoprotein disulfide oxidoreductases which cycle between Eox and EH2 forms in catalysis (Williams, C . H., Jr . (1976) in The Enzymes (Boyer, P . D., ed) 3rd Ed., Vol . 13, pp . 89-173, Academic Press, New York) . With mercuric reductase, exogenous thiols are required for catalytic reduction of Hg(II) to Hg0 . We have shown that this is due to prevention or reversal of formation of an abortive complex of Hg(II) with the thiol/thiolate pair of EH2.

Nucleic Acids Res, 1986 Jun 25, 14(12), 5013 - 8
Specificity of ionizing radiation-induced mutagenesis in the lac region of single-stranded phage M13 mp10 DNA; Ayaki H et al.; M13 mp10 single-stranded phage DNA was irradiated with 60 Co gamma-rays, and transfected into Escherichia coli . One hundred and sixteen mutant clones having lesions in the lac insert were selected, and mutational sites were examined by DNA sequence analysis . Fourteen out of the 15 nucleotide changes thus detected were base substitutions, and the rest was a base addition . Transitions and transversions were almost equal in number . Mutational events were observed at cytosine residues more frequently than at other residues, and the predominant base change was a C ---- T transition . Possible roles in gamma-ray-induced mutagenesis played by the misincorporation of dAMP owing to radiolytic derivatives of cytosine residues and/or formation of apurinic/apyrimidinic sites are discussed.

J Biol Chem, 1986 Jun 25, 261(18), 8534 - 9
Role of polycationic C-terminal portion in the structure and activity of recombinant human interferon-gamma; Arakawa T et al.; Purified recombinant human interferon-gamma, produced in Escherichia coli, was digested with trypsin under mild conditions, resulting in a preparation containing approximately 90% of a Mr = 15,800 protein and 10% of a 14,400 protein . The Mr = 15,800 protein has an intact N terminus and the Mr = 14,400 protein lacks 14 N-terminal residues . Both proteins lack C terminus of approximately 13 residues . This preparation containing the Mr = 15,800 and 14,400 proteins was identical with the intact protein with respect to conformation and dimerization, as analyzed by circular dichroism and gel filtration . However, the antiviral activity of this preparation was 1000-fold lower than that of the intact molecule . Since the majority of this preparation is the Mr = 15,800 protein, these results suggest that the C terminus does not affect the protein conformation and self-association, but greatly alters antiviral activity.

J Biol Chem, 1986 Jun 25, 261(18), 8528 - 33
Purification and characterization of guanine phosphoribosyltransferase from Giardia lamblia; Aldritt SM et al.; Giardia lamblia, a flagellated parasitic protozoan and the causative agent of giardiasis, lacks de novo purine biosynthesis and exists on salvage of adenine and guanine by adenine phosphoribosyltransferase and guanine phosphoribosyltransferase . Guanine phosphoribosyltransferase from G . lamblia crude extracts has been purified to apparent homogeneity by Sephacryl S-200 gel filtration followed by C-8-GMP-agarose and 2',3'-GMP-agarose affinity chromatography, resulting in an overall recovery of 77% and a purification of 83,000-fold . The molecular weight of the native enzyme as estimated by gel filtration and isokinetic sucrose gradients was found to be 58,000-63,000, with a subunit molecular weight of approximately 29,000, as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis . Mono P chromatofocusing chromatography gives rise to a major activity peak eluting from the column at a pH of 6.75 and two minor activity peaks at pH of 5.3 and 5.2 . Hypoxanthine and xanthine can be recognized by the enzyme as substrates but at Km values 20 times higher than that observed with guanine . G . lamblia guanine phosphoribosyltransferase is immunologically distinct from human hypoxanthine-guanine phosphoribosyltransferase and Escherichia coli xanthine-guanine phosphoribosyltransferase, and G . lamblia DNA fragments are incapable of hybridizing with mouse neuroblastoma hypoxanthine-guanine phosphoribosyltransferase DNA or E . coli xanthine phosphoribosyltransferase DNA under relatively relaxed conditions . All evidence presented suggests that G . lamblia guanine phosphoribosyltransferase may be qualified as a potential target for antigiardiasis chemotherapy.

Nucleic Acids Res, 1986 Jun 25, 14(12), 5001 - 12
Genomic structure of the large RNA segment of infectious bursal disease virus; Hudson PJ et al.; The larger RNA segment of infectious bursal disease virus (IBDV: Australian strain 002-73) has been characterized by cDNA cloning and nucleotide sequence analysis . We believe IBDV is the first birnavirus to be sequenced and so have confirmed the coding region by N-terminal amino acid sequence analysis of intact viral proteins and several tryptic peptide fragments . The large RNA segment encodes in order the 37-kDa, 28-kDa and 32-kDa proteins within a continuous open reading frame and the primary translation product appears to be subsequently processed into the mature viral proteins . The large protein precursor is still processed into the 32-kDa host protective immunogen when expressed as a fusion protein in E . coli . These results are in marked contrast to the predictions from in vitro translation data that birnavirus genomes are expressed as polycistronic templates . We can now propose that birnaviruses, in particular IBDV, possess monocistronic segments and that the precursor is proteolytically processed in vivo . The sequence data presented for the 32-kDa host protective immunogen may provide the basic information needed for the production of an effective subunit vaccine against this commercially important virus.

J Biol Chem, 1986 Jun 25, 261(18), 8096 - 9
Differential translation of the genes encoding the proton-translocating ATPase of Escherichia coli; Klionsky DJ et al.; Translation of the gene for the b subunit of the Escherichia coli proton-translocating ATPase has been examined . Oligonucleotide-directed site-specific mutagenesis was used to mutate certain nucleotides in the intergenic region between uncE (c) and uncF (b) . One of the changes was predicted to lower the stability of a proposed stem structure which blocked the ribosome binding site of the uncF mRNA segment . The result of the mutation is a nearly 3-fold increase in the rate of synthesis of the b polypeptide . Another mutation was introduced which changed the initiation codon for uncF from GUG to AUG . This change resulted in an approximately 2-fold increase in the synthesis rate of the b polypeptide . These results suggest that secondary structure in the mRNA and the use of a less efficient initiation codon play a role in restricting translation initiation of the uncF mRNA segment . These mechanisms may, in part, explain how the polypeptides of the ATPase complex are synthesized in approximately the same relative amounts as they appear in the assembled complex.

Nucleic Acids Res, 1986 Jun 25, 14(12), 4803 - 21
Cross-linking of initiation factor IF3 to Escherichia coli 30S ribosomal subunit by trans-diamminedichloroplatinum(II): characterization of two cross-linking sites in 16S rRNA; a possible way of functioning for IF3; Ehresmann C et al.; The initiation factor IF3 is platinated with trans-diamminedichloroplatinum(II) and cross-linked to Escherichia coli 30S ribosomal subunit . Two cross-linking sites are unambiguously identified on the 16S rRNA: a major one, in the region 819-859 in the central domain, and a minor one, in the region 1506-1529 in the 3'-terminal domain . Specific features of these sequences together with their particular location within the 30S subunit lead us to postulate a role for IF3, that conciliates topographical and functional observations made so far.

Biochim Biophys Acta, 1986 Jun 23, 871(3), 257 - 67
The catalytic active site of thioredoxin: conformation and homology with bovine pancreatic trypsin inhibitor; Lunn CA et al.; Rabbit polyclonal antibody was raised to a chemically synthesized nonapeptide (Trp-Ala-Glu-Trp-Cys-Gly-Pro-Cys-Lys) corresponding to the active-site sequence of Escherichia coli thioredoxin . The antiserum efficiently inhibited thioredoxin activity in the standard thioredoxin reductase/NADPH coupled assay . This inhibition was blocked by preincubation of the antiserum with the nonapeptide . Tight association of the E . coli thioredoxin to the active-site antibody required SDS denaturation . These results suggest that thioredoxin reductase (NADPH: oxidized-thioredoxin oxidoreductase, EC 1.6.4.5) alters the conformation of thioredoxin sufficiently to permit binding to the antibody . The antiserum bound to plant and liver thioredoxins . Bovine pancreatic trypsin inhibitor, whose active site (Gly-Pro-Cys-Lys) is homologous to that of thioredoxin, also competes for the active-site antibody . This result led to experiments showing that thioredoxin can inhibit the digestion of cytochrome c by trypsin . The ability of thioredoxin to act as a trypsin inhibitor analogue provides a rationale for thioredoxin's resistance to digestion by trypsin.

FEBS Lett, 1986 Jun 23, 202(1), 45 - 8
Phospholipase A2 activity in T-lymphocytes; Goppelt-Struebe M et al.; Phospholipase A2 activity was shown indirectly in T-lymphocytes from rat thymus and a permanent T-cell line by the liberation of arachidonic acid from phospholipids . In addition, phospholipase A2 activity was measured directly with two different substrates, phosphatidylcholine and labelled E . coli.

J Mol Biol, 1986 Jun 20, 189(4), 711 - 4
Co-operativity value of DNA RecA protein interaction . Influence of the protein quaternary structure on the binding analysis; Takahashi M et al.; We show that an erroneous estimation of the quaternary structure of free protein distorts the quantitative analysis of its interaction with DNA, affecting especially the co-operativity value found . This could explain the discrepancy reported for the co-operativity value of the RecA-DNA interaction . The large cluster observed by electron microscopy indicates a very high co-operativity, whereas analysis of the binding isotherm indicates a moderate one, on the assumption of monomer . But if RecA is a large oligomer, the latter analysis would give a much higher co-operativity value and the former a smaller one, and they would be in accordance . Our sedimentation and light-scattering experiments suggest an oligomerization of about 30-mer or more, and support this explanation.

J Mol Biol, 1986 Jun 20, 189(4), 701 - 7
Ultrastructure of native lipoprotein from Escherichia coli envelopes; Manstein DJ et al.; The free form of the major lipoprotein from Escherichia coli cells envelopes has been purified to homogeneity by gentle extraction procedures and conventional chromatographic separations in a non-ionic detergent . The morphology of paracrystals obtained from homogeneous protein was investigated by low-dose electron microscopy . Electron diffraction of the paracrystals was consistent with alpha-helices arranged perpendicularly to the main cross-band with a periodicity of 20 nm.

J Mol Biol, 1986 Jun 20, 189(4), 653 - 62
Mechanism of ribosomal translocation . Translocation limits the rate of Escherichia coli elongation factor G-promoted GTP hydrolysis; Robertson JM et al.; The pre-steady-state kinetics of GTP hydrolysis catalysed by elongation factor G and ribosomes from Escherichia coli has been investigated by the method of quenched-flow . The GTPase activities either uncoupled from or coupled to the ribosomal translocation process were characterized under various experimental conditions . A burst of GTP hydrolysis, with a kapp value greater than 30 s-1 (20 degrees C) was observed with poly(U)-programmed vacant ribosomes, either in the presence or absence of fusidic acid . The burst was followed by a slow GTP turnover reaction, which disappears in the presence of fusidic acid . E . coli tRNAPhe, but not N-acetylphenylalanyl-tRNAPhe (N-AcPhe-tRNAPhe), stimulates the GTPase when bound in the P site . If the A site of poly(U)-programmed ribosomes, carrying tRNAPhe in the P site, is occupied by N-AcPhe-tRNAPhe, the burst of Pi discharge is replaced by a slow GTP hydrolysis . Since, under these conditions, N-AcPhe-tRNAPhe is translocated from the A to the P site, this GTP hydrolysis very probably represents a GTPase coupled to the translocation reaction.

Biochim Biophys Acta, 1986 Jun 20, 867(3), 107 - 13
Alteration of the exonuclease activities of DNA polymerase I by captan; Freeman-Wittig MJ et al.; DNA polymerase I is a multifaceted enzyme with one polymerizing and two exonuclease activities . Captan was previously shown to be an inhibitor of this enzyme's polymerizing activity and this report measures the effects of captan on the two exonuclease activities . When the holoenzyme was tested, captan enhanced the degradation of poly(dA-dT), T7 DNA and, to a significantly lesser extent, heat-denatured DNA . However, when the effects of captan were tested as a function of substrate concentration, the stimulatory influence was measured only at high substrate concentrations . At low concentrations of DNA, captan was inhibitory . Inhibition and enhancement each showed an ED50 of the same value (approx . 100 microM) . By assaying the two exonuclease activities separately it was shown that the differential effect on the holoenzyme by captan was the result of a combined inhibition of the 3'----5' exonuclease and enhancement of the 5'----3' exonuclease . Klenow fragment with poly(dA-dT) as substrate was used to assay for 3'----5' exonuclease activity . Captan inhibited this exonuclease and the inhibition could be prevented by the addition of greater concentrations of substrate . Holoenzyme and poly(rA)-poly(dT) were used to assay for 5'----3' exonucleolysis, which was enhanced at higher concentrations of substrate in the presence of captan.

Cell, 1986 Jun 20, 45(6), 879 - 84
Resolution of linear minichromosomes with hairpin ends from circular plasmids containing vaccinia virus concatemer junctions; Merchlinsky M et al.; The junctions, separating unit-length genomes in intracellular concatemeric forms of vaccinia virus DNA, are duplex copies of the hairpin loops that form the ends of mature DNA molecules present in infectious virus particles . Circular E . coli plasmids with palindromic junction fragments were replicated in vaccinia virus-infected cells and resolved into linear minichromosomes with vector DNA in the center and vaccinia virus DNA hairpins at the two ends . Resolution did not occur when the concatemer joint was less than 250 bp or when plasmids were transfected into uninfected cells, indicating requirements for a specific DNA structure and viral trans-acting factors . These studies indicate that concatemers can serve as replicative intermediates and account for the generation of flip-flop sequence variation of the hairpins at the ends of the mature vaccinia virus genome.

J Mol Biol, 1986 Jun 20, 189(4), 643 - 52
Evidence for an intermediate in DNA synthesis involving pyrophosphate exchange . A possible role in fidelity; Lecomte P et al.; The incorporation of exogenous deoxyribonucleotide monophates (dNMP) was measured under conditions of ongoing DNA synthesis, providing arguments for the existence of a {DNAn X dNMP X PPi} intermediate in the nucleotide incorporation step of DNA synthesis: (formula; see text) . The existence of such an intermediate is suggested by an apparent exchange of both dNMP and pyrophosphate (PPi) moieties of the deoxyribonucleotide triphosphate (dNTP) substrate with exogenous molecules . Such exchange and the incorporation of exogenous dNMP into DNA, strictly require ongoing DNA synthesis, suggesting that the energy for exchange reactions is provided by the cleavage of dNTP substrate . We propose that nucleotide selection during ongoing DNA synthesis results largely from the different relative rates of forward (beta) and backward (-alpha) reactions involving the {DNAn X dNMP X PPi} intermediate: the forward (incorporation) reaction is expected to predominate for the correct nucleotide, whereas the backward (abortive) reaction is expected to predominate for incorrect nucleotides.

Cell, 1986 Jun 20, 45(6), 801 - 15
Genetic evidence that Tn10 transposes by a nonreplicative mechanism; Bender J et al.; We present genetic evidence that the tetracycline resistance element Tn10 transposes by a nonreplicative mechanism . Heteroduplex Tn10 elements containing three single base pair mismatches were constructed on lambda phage genomes and allowed to transpose from lambda into the bacterial chromosome . Analysis of TetR colonies resulting from such transpositions suggests that information from both strands of the transposing Tn10 element is transmitted faithfully to its transposition product . The simplest interpretation of these results is that the transposing element is excised from the donor molecule and inserted into the target molecule without being replicated . A mismatch 70 base pairs from one end of the transposon is preserved, suggesting that there is little or no replication, even at the termini of the element, during transposition in vivo.

J Mol Biol, 1986 Jun 20, 189(4), 633 - 41
Expression, purification and operator binding of the transposon Tn1721-encoded Tet repressor; Klock G et al.; The regulation of expression of the Tn1721-encoded tetracycline-resistance determinant is described at the molecular level . The transcriptional control element consists of overlapping divergent promoters, which are negatively regulated by two operators with nearly identical sequence . The mRNA for the regulatory gene tetR is translated without a ribosome-binding site . This result is confirmed by S1 nuclease mapping and RNA sequencing of the tetR mRNA . The start nucleotide for transcription of this mRNA is the adenosine residue of the sequence 5'-AUG . Determination of the N-terminal amino acid sequence of the purified Tet repressor proves that this AUG is the initiation codon for translation . The Tet repressor protein is further used to map the two tet operators by DNase I footprinting . Tight contacts of the protein to the N-7 positions of two guanosine residues in each operator are determined from methylation protection experiments with dimethylsulfate . The differential regulation and positive control of transcription of the tetR gene that is possible with this arrangement of promoters and operators is discussed.

Biochim Biophys Acta, 1986 Jun 20, 867(3), 81 - 8
Plasmid genes increase membrane permeability in Escherichia coli; Ono T et al.; The membrane permeability to o-nitrophenyl beta-D-galactoside is increased in the presence of rifampicin in Escherichia coli cells carrying srnB+ or pnd+ plasmids, but not in the cells carrying srnB- or pnd- mutant plasmids . The same permeability alteration was also observed at 42 degrees C when a rpoC4- mutant strain was used as a host strain in the absence of rifampicin . These results and the blockage of the effects by action of chloramphenicol suggest that the increase of permeability to o-nitrophenyl galactoside was caused by the expression of srnB+ or pnd+ gene, respectively . srnB+ gene expression leads to massive RNA degradation, probably through the activation of the rna+ gene product . In an rna- strain carrying the srnB+ plasmid, the extent of RNA degradation was reduced, whereas the permeability to o-nitrophenyl galactoside was increased to the same level as in the rna+ strain . Also, the increase in permeability to o-nitrophenyl galactoside was observed at 30 degrees C, although high-temperature incubation (42 degrees C) was necessary for the induction of RNA degradation . These results suggest that the alteration in permeability is a more direct effect of the expression of srnB+ or pnd+ gene and that the RNA degradation is a secondary phenomenon caused by the alteration in the membrane.

Biochemistry, 1986 Jun 17, 25(12), 3690 - 5
Magnesium ion dependent equilibria, kinetics, and thermodynamic parameters of Artemia ribosome dissociation and subunit association; Goss DJ et al.; The influence of magnesium ion concentration on the equilibrium and kinetics of Artemia ribosome dissociation and subunit association has been studied by laser light scattering . Ribosomal aggregation was found to be reduced by addition of 0.1-0.05 mM spermidine and KCl concentrations of 100 mM . The ribosomes were found to be stable at low {Mg2+}, and the curves obtained for ribosome-subunit equilibrium were independent of the direction and origin of the magnesium ion titration . Thermodynamic parameters were obtained from the temperature-dependent equilibria and have been compared to those of wheat germ and Escherichia coli type A ribosomes . The entropy term calculated for the association of 40S and 60S subunits is small, and the reaction is exothermic . The entropy term is negative, favoring subunit dissociation, and contributes less to the free energy than the enthalpy term . Rate constants for ribosome dissociation and subunit association have been determined . The reaction curves gave no evidence for sequential processes and were homogeneous.

Biochemistry, 1986 Jun 17, 25(12), 3682 - 90
Binding of Escherichia coli protein synthesis initiation factor IF1 to 30S ribosomal subunits measured by fluorescence polarization; Zucker FH et al.; The interaction of initiation factor IF1 with 30S ribosomal subunits was measured quantitatively by fluorescence polarization . Purified IF1 was treated with 2-iminothiolane and N-{{(iodoacetyl)-amino}ethyl}-5-naphthylamine-1-sulfonic acid in order to prepare a covalent fluorescent derivative without eliminating positive charges on the protein required for biochemical activity . The fluorescent-labeled IF1 binds to 30S subunits and promotes the formation of N-formylmethionyl-tRNA complexes with 70S ribosomes . Analyses of mixtures of fluorescent-labeled IF1 and 30S ribosomal subunits with an SLM 4800 spectrofluorometer showed little change in fluorescence spectra or lifetimes upon binding, but a difference in polarization between free and bound forms is measurable . Bound to free ratios were calculated from polarization data and used in Scatchard plots to determine equilibrium binding constants and number of binding sites per ribosomal subunit . Competition between derivatized and nonderivatized forms of IF1 was quantified, and association constants for the native factor were determined: (5 +/- 1) X 10(5) M-1 with IF1 alone; (3.6 +/- 0.4) X 10(7) M-1 with IF3; (1.1 +/- 0.2) X 10(8) M-1 with IF2; (2.5 +/- 0.5) X 10(8) M-1 with both IF2 and IF3 . In all cases, 0.9-1.1 binding sites per 30S subunit were detected . Divalent cations have little effect on affinities, whereas increasing monovalent cations inhibit binding . On the basis of the association constants, we predict that greater than 90% of native 30S subunits are complexed with all three initiation factors in intact bacterial cells.

Biochemistry, 1986 Jun 17, 25(12), 3570 - 5
Heterogeneity of lipid A: structural determination by 13C and 31P NMR of lipid A fractions from lipopolysaccharide of Escherichia coli 0111; Baltzer LH et al.; Purified lipid A from Escherichia coli 0111 was fractionated by thin-layer chromatography, and seven major bands were studied by 13C and 31P NMR . All lipid A fractions except one had fatty acids, 3-hydroxytetradecanoic acid, 3-(acyloxy)tetradecanoic acid, and phosphate groups bonded to the diglucosamine backbone . The remaining fraction was shown to be phosphatidylethanolamine . The number of substituents found showed that in all fractions all sites available for C-acylation (C-3, C-4, and C-3') and N-acylation (C-2 and C-2') carried acylic substituents . The number, ranging from four to six, and type of ester-bound carboxylic acid residues as well as the number of phosphate groups differed among the fractions . The three fastest moving bands all had three unsubstituted hydroxy fatty acids and one phosphate group (C-4'), while the slower moving bands had four hydroxy fatty acids and two phosphate groups . Unsubstituted 3-hydroxytetradecanoic acid residues were amide-bound to the disaccharide in all but one of the fractions . In summary, the heterogeneity of E . coli 0111 lipid A is found to be a consequence of a variation of the number and composition of carboxylic acid residues and of varying phosphate content.

Biochemistry, 1986 Jun 17, 25(12), 3666 - 72
1H NMR (500 MHz) identification of aromatic residues of gene 32 protein involved in DNA binding by use of protein containing perdeuterated aromatic residues and by site-directed mutagenesis; Prigodich RV et al.; Preparation of gene 32 protein containing perdeuterated tyrosyl and phenylalanyl residues has allowed the resolution of separate 1H NMR signals for the Tyr and Phe residues of the protein by NMR difference spectra . Upfield shifts in the chemical shifts of a number of aromatic protons previously observed to accompany deoxyoligonucleotide complex formation with gene 32 protein {Prigodich, R . V., Casas-Finet, J., Williams, K . R., Konigsberg, W., & Coleman, J . E . (1984) Biochemistry 23, 522-529} can be assigned to five Tyr and two Phe residues that must form part of the DNA binding domain . Site-directed mutation of Tyr-115 to Ser-115 results in the disappearance of a set of 2,6 and 3,5 tyrosyl protons that are among those moved upfield by oligonucleotide complex formation . These findings suggest that the amino acid sequence from Tyr-73 to Tyr-115 which contains six of the eight Tyr residues of the protein forms part of the DNA binding surface.

Biochem J, 1986 Jun 15, 236(3), 643 - 9
Overproduction of the cyclic AMP receptor protein of Escherichia coli and expression of the engineered C-terminal DNA-binding domain; Gronenborn AM et al.; Overproduction of the cyclic AMP receptor protein (CRP) from Escherichia coli, up to 25% of the soluble cell protein, has been achieved in an inducible host-vector system under transcriptional control of the lambda promoter PL . This system is ideally suited for large scale production and purification of CRP . In addition, a structural gene for the DNA-binding domain of CRP has been constructed . To this end the nucleotide sequence coding for the C-terminus was fused to the sequence coding for the first 10 N-terminal amino acids and cloned into suitable vectors . Good expression was achieved using the lambda PL promoter . The gene product, beta CRP, is recognized by anti-CRP antibodies.

J Biol Chem, 1986 Jun 15, 261(17), 8070 - 5
Translocation of nascent non-signal sequence protein in heated Escherichia coli; Yatvin MB et al.; Exposure of Escherichia coli to heat resulted in 1) selective inhibition of protein synthesis, 2) synthesis of heat shock proteins, and 3) altered subcellular distribution of newly synthesized proteins . Either 5 min or 1 h at 48 degrees C increases outer membrane proteins of Coomassie Blue-stained gels . After 1 h, there was a loss of stained proteins from the soluble fraction . Much greater changes in the distribution of radiolabeled (newly synthesized) proteins were observed, with marked increases in the number of outer membrane protein species and a corresponding loss of soluble fraction proteins . Three major species of radiolabeled proteins from heat-treated cells remain in the soluble fraction; these proteins have apparent Mr 56,000, 69,200, and 79,400 . Cells were labeled with L-{35S} methionine at either 37 or 48 degrees C and chased with non-radiolabeled methionine before a temperature shift to either 48 or 37 degrees C, respectively . Only proteins synthesized at elevated temperature participated in translocation . It is suggested that heat disordering of membrane lipids promotes interlipidic connections between the inner and outer membrane providing pathways for protein movement to the outer membrane and may be the mechanism whereby a cell quickly responds to environmental temperature stress . The response does not require but may trigger synthesis of mRNA.

J Biol Chem, 1986 Jun 15, 261(17), 7995 - 6
Crystallization and x-ray diffraction studies of a phosphate-binding protein involved in active transport in Escherichia coli; Kubena BD et al.; We have obtained single crystals of a phosphate-binding protein (Mr = 34,400) that serves as initial receptor in osmotic shock-sensitive active transport in Escherichia coli . The crystals, suitable for high resolution crystallographic analysis, belong to the space group P2(1)2(1)2(1) . The unit cell has dimensions of a = 41.97, b = 64.66, and c = 124.6 A and contains four protein molecules . Including this phosphate-binding protein, there are now a total of six different binding protein structures currently under investigation in our laboratory, the others being those specific for L-arabinose, D-galactose, D-maltose, sulfate, or leucine/isoleucine/valine.

J Immunol, 1986 Jun 15, 136(12), 4561 - 4
Cloning, sequence, and expression of bovine interferon-gamma; Cerretti DP et al.; Bovine interferon-gamma (IFN-gamma) sequences have been isolated by screening a cDNA library with a human IFN-gamma cDNA probe . The cDNA library was constructed from RNA isolated from concanavalin A-stimulated bovine lymph node cells . The open reading frame predicts that the bovine IFN-gamma precursor is composed of 166 amino acids with a predicted m.w . of 19,393 . Alignment of the amino acid sequence with human IFN-gamma indicates that mature bovine IFN-gamma is composed of 143 amino acids with a predicted m.w . of 16,858 . It has an amino acid homology of 63% with human IFN-gamma, and 47% with murine IFN-gamma . Biologically active bovine IFN-gamma was synthesized in an Escherichia coli expression system.

J Biol Chem, 1986 Jun 15, 261(17), 7659 - 62
An analysis of the structure of the product of the rbsA gene of Escherichia coli K12; Buckel SD et al.; The predicted amino acid sequence of rbsA, a gene from the high affinity ribose transport operon (rbs) of Escherichia coli K12, is homologous to the products of hisP, malK, and pstB, components of the histidine, maltose, and phosphate high affinity transport operons . The recent finding by Hobson et al . (Hobson, A . C., Weatherwax, R., and Ames, G.F.-L . (1984) Proc . Natl . Acad . Sci . U.S.A . 81, 7333-7337) that the hisP and malK products bind ATP suggests that these four gene products may be involved in coupling the energy from ATP to drive the active transport in their respective transport systems . Each gene product contains a sequence of glycine and basic residues which are characteristic of an ATP-binding site (Walker, J.E., Saraste, M., Runswick, M.J., and Gay, N.J . (1982) EMBO J . 1, 945-951) . Interestingly the N- and C-terminal halves of rbsA are also homologous, suggesting that a primordial gene duplication and subsequent fusion of the products occurred.

J Biol Chem, 1986 Jun 15, 261(17), 7797 - 806
Coupling between the sodium and proton gradients in respiring Escherichia coli cells measured by 23Na and 31P nuclear magnetic resonance; Castle AM et al.; The relationship between the steady-state sodium gradient (delta pNa) and the protonmotive force developed by endogenously respiring Escherichia coli cells has been studied quantitatively, using 23Na NMR for measurement of intracellular and extracellular sodium concentrations, 31P NMR for measurement of intracellular and extracellular pH, and tetraphenylphosphonium distribution for measurement of membrane potential . At constant protonmotive force, the sodium concentration gradient was independent of extracellular concentrations over the measured range of 4-285 mM, indicating that intracellular sodium concentration is not regulated . The magnitude of delta pNa was measured as a function of the composition and magnitude of the protonmotive force . At external pH values below 7.2, delta pNa was parallel to delta pH but showed no simple relationship to the membrane potential; above pH 7.2 the parallel relationship began to diverge, with delta pH continuing to decrease but delta pNa starting to level off or increase . Although plots of delta pNa versus delta pH had slopes of close to 1, the value of delta pNa consistently exceeded that of delta pH by approximately 0.4 units, indicating a partially electrogenic character to the putative H+/Na+ antiport . The apparent stoichiometry was 1.13 +/- 0.01 at external pH below 7.2 . The possible significance of this nonintegral stoichiometry is discussed according to a model in which two distinct integral stoichiometries (possibly 1H+/1Na+ and 2H+/1Na+) are available with some relative probability; the model predicts futile cycling of sodium ions and a dissipative proton current . In the course of this study, we discovered that the magnitude of the pH gradient developed by the cells was osmolarity-dependent, yielding steady-state intracellular pH values that varied from 7.1 at 100 mosm to 7.7 at 800 mosm.

J Biol Chem, 1986 Jun 15, 261(17), 7652 - 8
The nucleotide sequences of the rbsD, rbsA, and rbsC genes of Escherichia coli K12; Bell AW et al.; The nucleotide sequences of rbsD, rbsA, and rbsC have been determined . These genes encode components of the high affinity ribose transport system in Escherichia coli, and together with the sequences of rbsB (Groarke, J.M., Mahoney, W.C., Hope, J.N., Furlong, C.E., Robb, F.T., Zalkin, H., and Hermodson, M.A . (1983) J . Biol . Chem . 258, 12952-12956) and rbsK (Hope, J.N., Bell, A.W., Hermodson, M.A., and Groarke, J.M . (1986) J . Biol . Chem . 261, 7663-7668), they complete the nucleotide sequence of the first five genes of the rbs operon . Nuclease S1 mapping places the transcriptional start site for the operon 29 base pairs upstream from the most likely translational start site for rbsD . The open reading frames of rbsD, rbsA, and rbsC encode proteins of 139, 501, and 321 amino acid residues, respectively . The character of the proteins varies widely, from very hydrophilic for the rbsA product to exceedingly hydrophobic for the rbsC product . The intercistronic spaces between the three genes are very short, with the stop codons of the upstream genes overlapping the ribosome-binding sites of the downstream genes . This may imply translational control of expression of these genes, the products of which presumably form a membrane-bound transport complex.

J Immunol, 1986 Jun 15, 136(12), 4509 - 14
Interleukin 1 alpha and interleukin 1 beta bind to the same receptor on T cells; Kilian PL et al.; Pure, E . coli-derived recombinant murine interleukin 1 alpha (IL 1 alpha) was labeled with 125I and used for receptor binding studies . The 125I-IL 1 binds to murine EL-4 thymoma cells in a specific and saturable manner . Scatchard plot analysis for binding studies carried out at 4 degrees C reveals a single type of high affinity binding site with an apparent dissociation constant of approximately 2.6 X 10(-10) M and the presence of approximately 1200 binding sites per cell . The rate of association of the 125I-IL 1 with EL-4 cells is slow, requiring more than 3 h to reach apparent steady state at 4 degrees C . Cell-bound 125I-IL 1 cannot be dissociated from EL-4 cells upon removal of unbound 125I-IL 1 and incubation of the cells at 4 degrees C in the presence or absence of unlabeled IL 1 . Unlabeled recombinant murine IL 1 competes for 125I-IL 1 binding in a dose-dependent manner, whereas interferon-alpha A, interleukin 2 (IL 2), epidermal growth factor, and nerve growth factor have no effect . The 125I-IL 1 binding site is sensitive to trypsin, suggesting that it is localized on the cell surface . We have also examined the ability of purified recombinant human IL 1 alpha and IL 1 beta to compete for binding of the radiolabeled murine IL 1 to its receptor and to stimulate IL 2 production by EL-4 cells . Previous reports have shown that human IL 1 alpha is approximately 60% homologous in amino acid sequence with murine IL 1, but that human IL 1 beta is only about 25% homologous with either murine IL 1 or human IL 1 alpha . Despite these marked differences, however, we report here that both human IL 1 proteins are able to recognize the same binding site as mouse IL 1 . In addition, murine as well as both human IL 1 proteins stimulate IL 2 production by EL-4 cells.

Biochem Biophys Res Commun, 1986 Jun 13, 137(2), 649 - 56
Identification of cDNA clones for ligninase from Phanerochaete chrysosporium using synthetic oligonucleotide probes; Zhang YZ et al.; Four cDNA clones for ligninase were isolated from the cDNA library (constructed into the PstI site of E . coli vector pUC9) representing 6 day-old lignin degrading culture of Phanerochaete chrysosporium by the use of three synthetic oligonucleotide probes corresponding to partial amino acid sequences of tryptic peptides of the ligninase . Each of the three probes, 14.1, 14.2 and 25, represents a mixture of 32 12- or 14-base long oligonucleotides . Three cDNA clones hybridized with probe 14.1 but not with probe 25 or 14.2, but one cDNA clone hybridized with all of the three probes . Differential hybridization studies showed that these clones are unique to 6-day poly(A) RNA, but not to 2-day poly(A) RNA.

Biochim Biophys Acta, 1986 Jun 13, 858(1), 217 - 20
Contamination of commercial preparations of xanthine oxidase by a Ca2+-dependent phospholipase A2; Gamache DA et al.; Using {1-14C}oleate-labelled autoclaved Escherichia coli as substrate, we demonstrate that many, but not all, commercial preparations of xanthine oxidase contain phospholipase A2 activity as a contaminant . Phospholipase A2 activity (64.3-545.6 nmol phospholipid hydrolyzed per min per mg protein) was optimal in the neutral to alkaline pH range, was Ca2+-dependent, and was unaffected by the addition of xanthine . Phospholipase A2 activity was totally inhibited by 1.0 mM EDTA while radical production by xanthine plus xanthine oxidase was unaffected by EDTA . Even chromatographically purified xanthine oxidase (Sigma Grade III) contained substantial phospholipase A2 activity (64.3 nmol/min per mg) . Since the preparation of xanthine oxidase employs proteolytic digestion of milk or buttermilk by pancreatin, an extract of pancreas which is an organ rich in phospholipase A2 activity, we speculate that the contaminant phospholipase A2 is introduced by this treatment . Because xanthine oxidase is used extensively to study free radical-induced cell injury and membrane phospholipid alterations, the presence of a potent extracellular phospholipase A2 may have influenced previously published reports and such studies in the future should be interpreted with care.

Biochem Biophys Res Commun, 1986 Jun 13, 137(2), 847 - 54
Production and characterization of monoclonal antibodies against recombinant human tumor necrosis factor/cachectin; Liang CM et al.; Tumor necrosis factor is a monokine, which causes cytolysis of many transformed cells . In this study we have found that in addition to cytotoxicity recombinant Escherichia coli-derived human tumor necrosis factor, like cachectin, inhibited the lipoprotein lipase of 3T3-L1 preadipocytes . Both effects were inhibited by monoclonal anti-tumor necrosis factor antibodies . Monoclonal antibodies against recombinant human tumor necrosis factor were produced by fusing splenocytes of immune mice with P3X63Ag8 653 myeloma cells . The monoclonal antibodies, namely BG 2-4, were of IgG2a, IgG, and IgG2a subclasses . These monoclonal antibodies neutralized the cytotoxicity of natural and recombinant human tumor necrosis factor but not that of rabbit or mouse tumor necrosis factor . They also neutralized the cachectin activity of human tumor necrosis factor in the 3T3-L1 embryonic cell assay . These results indicate that the functional structure(s) of human tumor necrosis factor responsible for the cytotoxicity and cachectin activities are likely to be closely related.

Biochem Biophys Res Commun, 1986 Jun 13, 137(2), 884 - 91
Characterization of monoclonal antibodies directed against pyruvate oxidase from Escherichia coli: modulation of antibody-induced inhibition by enzyme conformation; Barassi CA et al.; Monoclonal antibodies have been prepared against pyruvate oxidase, a flavoprotein dehydrogenase isolated from Escherichia coli . Six monoclonals were obtained, but only one was found to bind to the native form of the enzyme . This monoclonal, 1I1, was a potent inhibitor . Although this antibody inhibited the unactivated and lipid-activated forms of the enzyme, it had much less of an inhibitory effect on the protease-activated form of the enzyme, although the antibody still bound to this form . Hence, the coupling between antibody binding and the conformation at the active site can itself be modulated by the conformation of the protein.

Biochim Biophys Acta, 1986 Jun 13, 858(1), 67 - 82
Characterisation in vivo of the reactive thiol groups of the lactose permease from Escherichia coli and a mutant; exposure, reactivity and the effects of substrate binding; Page MG et al.; The reactivity and accessibility of the reactive thiol groups of the native lactose permease and a mutant have been studied in a number of circumstances and with a number of reagents, in particular using the specific thiol-disulphide exchange reaction . Seven different reactive states of the thiol in the native protein have been characterised by their different second-order rate constants . Interconversion between these states is dependent on the magnitude of the protonmotive force, pH and substrate binding . In the absence of galactoside, reactivity is controlled by an ionisation with apparent pKa 9.3 . This pKa is not affected by the protonmotive force, but it is lowered in the presence of external galactoside . The conformation adopted by the permease when in equilibrium with saturating galactoside appears to be different from that of the intermediate that accumulates during net turnover . In the former state, the reactivity of the thiol group is depressed, whereas in the latter state it is enhanced . The thiol group of the native protein is buried in a hydrophobic environment that has a dielectric constant considerably lower than that of water . The environment is not greatly perturbed by changes in the magnitude of the protonmotive force, but it is affected by the binding of galactoside . In a strain which carries the YUN mutation (Wilson, T.H . and Kusch, M . (1972) Biochim . Biophys . Acta 255, 786-797), two reactive thiols were characterised . The more reactive of the two is more exposed than the thiol group of the native molecule and is in an environment that has a dielectric constant close to that of water . The less reactive thiol appears to be more deeply buried than that of the native protein . Thus the mutation appears to produce a conformation change in the central portion of the polypeptide chain that results in greater exposure of the reactive thiol to the aqueous environment.

Biochem Biophys Res Commun, 1986 Jun 13, 137(2), 795 - 800
Modulation of the relaxing activity of Escherichia coli topoisomerase I by single-stranded DNA binding proteins; Srivenugopal KS et al.; Removal of negative superhelical turns in ColE1 plasmid DNA by Escherichia coli topoisomerase I was markedly enhanced by the presence of single-stranded DNA binding protein from E . coli . A lack of species specificity makes unlikely the possibility of physical association between topoisomerase I and single-stranded DNA binding proteins . Stabilization of single-stranded regions in supercoiled DNA by single-stranded DNA binding protein would appear to be the basis of the enhancement of topoisomerase activity.

Nucleic Acids Res, 1986 Jun 11, 14(11), 4453 - 69
The trfB region of broad host range plasmid RK2: the nucleotide sequence reveals incC and key regulatory gene trfB/korA/korD as overlapping genes; Thomas CM et al.; We report the nucleotide sequence of the trfB region of broad host range plasmid RK2 . This region encodes the following loci: trfB, identical to korA and korD, which encodes a key transcriptional repressor of certain RK2 operons; incC, which appears to be involved in plasmid maintenance, possibley through post-transcriptional regulation of trfA product levels; the start of korB, which encodes a second transcriptional repressor of operons involved in stable inheritance of RK2 . These loci are expressed as part of the trfB operon . In combination with deletion analysis, transcriptional and translation fusions and 'maxicell' analysis of polypeptides, the DNA sequence allows a number of conclusions to be drawn . First, the korB ORF start codon overlaps the incC ORF stop codon, suggesting the possibility of translational coupling between these two genes . Second, the trfB ORF lies entirely within the first third of the incC ORF using a different phase . Third, the incC ORF appears to contain a second transcriptional start whose function appears to be coupled to translation of the trfB ORF . Analysis of codon usage in the region of overlap between incC and trfB suggests that the incC gene may have evolved before the trfB gene . Determination of the DNA sequence of a mutant in which the product of trfB is rendered defective for transcriptional repression reveals an amino acid alteration within a region of this polypeptide which exhibits homology to the alpha helix-turn-alpha helix motif characteristic of many DNA binding proteins, and which is probably responsible for recognition of the trfB operator by this protein.

Nucleic Acids Res, 1986 Jun 11, 14(11), 4437 - 51
Complete nucleotide sequence of the Escherichia coli recC gene and of the thyA-recC intergenic region; Finch PW et al.; The nucleotide sequence of a 6,000 bp region of the E . coli chromosome that includes the 3' end of the coding region for the thyA gene and the entire recC gene has been determined . The proposed coding region for the RecC protein is 3369 nucleotides long, which would encode a polypeptide consisting of 1122 amino acids with a calculated molecular mass of 129 kDa . Mung bean nuclease mapping of a recC specific transcript produced in vivo indicates that transcription of recC is initiated 80 bp upstream of the translational start point . A weak promoter sequence situated 5' to the transcription initiation point has been identified . In the 1953 bp thyA-recC intergenic region there are three open reading frames that would code for polypeptides of molecular mass 30 kDa, 13.5 kDa and 12 kDa, respectively . Although the first and third of these open reading frames are preceded by possible ribosome binding sites, no obvious promoter sequences could be identified . Moreover, transcripts for these reading frames could not be detected.

Nucleic Acids Res, 1986 Jun 11, 14(11), 4393 - 400
The nucleotide sequence of the gene for gamma-glutamylcysteine synthetase of Escherichia coli; Watanabe K et al.; The nucleotide sequence of the gsh I gene for gamma-glutamylcysteine synthetase(GSH I) of Escherichia coli B has been determined . The gsh I structural gene contains 1557 bases in length and predicted a ploypeptide of 518 amino acids with a calculated molecular weight of 58,251 . The value is in good agreement with that obtained from gel filtration and SDS/PAGE of GSH I . The initiation codon 5 bp downstream of putative Shine-Dalgarno sequence was an unusual TTG, which encoded methionine . For transcription, two sets of consensus promoter signals(-10 and -35 regions) overlapping each other were identified . The terminator signal shows the favored stem-loop structure with an adequate free energy delta G = -22.80 kcal/mol.

Nucleic Acids Res, 1986 Jun 11, 14(11), 4543 - 56
Binding of the EcoRII methylase to azacytosine-containing DNA; Friedman S; Binding of DNA(cytosine-5)methyltransferases to azacytosine containing DNA is stimulated by the presence of S-adenosyl-methionine or its analogs sinefungin or S-adenosyl-L-homocysteine . Methylation of the DNA is therefore not necessary for binding to occur . There is no relationship between the affinity of the analog for the EcoRII enzyme and its ability to stimulate binding . The DNA-enzyme complex partially dissociates on incubation in 0.1% sodium dodecyl sulfate and 0.5 M ammonium acetate . Some of this DNA could again form a tight complex with enzyme, indicating that DNA-enzyme complex formation is reversible . Binding occurs when the second cytosine in the sequence CCAGG is substituted by azacytosine . This is the cytosine that would normally be methylated by the enzyme . The binding is therefore due to specific interaction of the methylase with azacytosine at the site it would normally methylate.

FEBS Lett, 1986 Jun 9, 201(2), 267 - 70
Organization of citric acid cycle enzymes into a multienzyme cluster; Barnes SJ et al.; The possibility that some of the enzymes of the citric acid cycle may be loosely associated into a multienzyme cluster has been investigated using extracts prepared by gentle disruption of cells . Gel filtration and sucrose density gradient centrifugation have shown that five sequential enzymes of the cycle specifically associate into a cluster: fumarase, malate dehydrogenase, citrate synthase, aconitase and isocitrate dehydrogenase . Ultrasonication destroys the abilities of the enzymes to associate . The cluster could catalyse the sequence of reactions leading from fumarate to oxoglutarate and has been found in extracts of several bacterial species as well as rat liver mitochondria.

Science, 1986 Jun 6, 232(4755), 1258 - 64
Pertussis toxin gene: nucleotide sequence and genetic organization; Locht C et al.; The current pertussis vaccines, although efficacious, in some instances produce undesirable side effects . Molecular engineering of pertussis toxin, the major protective antigen, could provide a safer, new generation of vaccines against whooping cough . As a first critical step in the development of such a vaccine, the complete nucleotide sequence of the pertussis toxin gene was determined and the amino acid sequences of the individual subunits were deduced . All five subunits are coded by closely linked cistrons . A promoter-like structure was found in the 5'-flanking region, suggesting that the toxin is expressed through a polycistronic messenger RNA . The order of the cistrons is S1, S2, S4, S5, and S3 . All subunits contain signal peptides of variable length . The calculated molecular weights of the mature subunits are 26,024 for S1, 21,924 for S2, 21,873 for S3, 12,058 for S4, and 11,013 for S5 . Subunits S2 and S3 share 70% amino acid homology and 75% nucleotide homology . Subunit S1 contains two regions of eight amino acids homologous to analogous regions in the A subunit of both cholera and Escherichia coli heat labile toxins.

Cell, 1986 Jun 6, 45(5), 761 - 70
A second virus-encoded proteinase involved in proteolytic processing of poliovirus polyprotein; Toyoda H et al.; The poliovirus polyprotein is cleaved at three different amino acid pairs . Viral polypeptide 3C is responsible for processing at the most common pair (glutamineglycine) . We have found that a cDNA fragment encoding parts of the capsid protein region (P1) and the nonstructural protein region (P2), and including the P1-P2 processing site (tyrosine-glycine), can be expressed in E . coli . The translation product was correctly processed . Disruption of the coding sequence of 2A, a nonstructural polypeptide mapping carboxy-terminal to the tyrosine-glycine cleavage site, by linker mutagenesis or deletion, prevented processing . Deletion of the adjacent polypeptide 2B had no such effect . Antibodies against 2A specifically inhibited processing at the 3C'-3D' processing site (tyrosine-glycine) in vitro . We conclude that poliovirus encodes the second proteinase 2A, which processes the polyprotein at tyrosine-glycine cleavage sites.

Cell, 1986 Jun 6, 45(5), 659 - 66
Induction of beta 2-interferon by tumor necrosis factor: a homeostatic mechanism in the control of cell proliferation; Kohase M et al.; Earlier studies showed that tumor necrosis factor (TNF) exerts a mitogenic effect in human diploid fibroblasts . Here we demonstrate that purified E . coli-derived recombinant human TNF inhibits encephalomyocarditis virus replication in "aged" human fibroblasts . Addition of neutralizing antibodies to human beta interferon (IFN-beta) blocked the antiviral action of TNF, indicating that this action is mediated by the generation of IFN-beta . We also show that antiserum to IFN-beta enhanced the mitogenic effect of TNF in confluent, serum-starved human fibroblasts, suggesting that induction of IFN-beta by TNF represents a physiological negative feedback mechanism regulating cell proliferation . Blot hybridization analysis of cytoplasmic polyadenylated RNA showed that TNF induced IFN-beta 2 mRNA, whereas no induction of IFN-beta 1 mRNA could be demonstrated . The results suggest that IFN-beta 2 has biological functions distinct from the other interferons.

Science, 1986 Jun 6, 232(4755), 1242 - 4
Plant glutamine synthetase complements a glnA mutation in Escherichia coli; DasSarma S et al.; A glutamine synthetase gene from alfalfa (Medicago sativa) has been expressed in Escherichia coli after fusion of bacterial transcription and translation signals to a complete alfalfa glutamine synthetase coding sequence . Synthesis of the alfalfa glutamine synthetase enzyme in Escherichia coli was demonstrated by functional genetic complementation of a glutamine synthetase-deficient mutant and by immunoblotting analysis . These results should facilitate protein engineering and structure-function analysis of the plant enzyme.

Science, 1986 Jun 6, 232(4755), 1250 - 3
Transforming growth factor-alpha: a more potent angiogenic mediator than epidermal growth factor; Schreiber AB et al.; Transforming growth factor-alpha (TGF-alpha) and epidermal growth factor (EGF) are structurally related peptides . Purified human TGF-alpha produced in Escherichia coli and pure natural mouse EGF were compared for their ability to bind to target cells in vitro and to promote angiogenesis in the hamster cheek pouch bioassay . Both polypeptides were found to bind in vitro to several target cells, including endothelial cells, and to stimulate their DNA synthesis in an equipotent fashion . In vivo, however, TGF-alpha was more potent than EGF in promoting angiogenesis and, because TGF-alpha is known to be secreted by a variety of human tumors, it is suggested that this growth factor may contribute to tumor-induced angiogenesis.

J Biol Chem, 1986 Jun 5, 261(16), 7491 - 500
Characterization of ribosomal frameshift events by protein sequence analysis; Dayhuff TJ et al.; In cell-free protein synthesis studies with RNA from phage MS2 as template, normal Escherichia coli tRNASer3 promotes two base translocation at GCA alanine codons with a resultant shift of ribosomes to the minus one reading frame . Similarly, normal tRNAThr3 promotes two base translocation at CCG proline codons . These conclusions were reached by amino acid sequencing of tryptic peptides or cyanogen bromide fragments that contained the reading frame shift site . It is proposed that these frameshift events occur by a two-base pair interaction between the anticodons of these exceptional tRNAs and the noncognate codons.

J Mol Biol, 1986 Jun 5, 189(3), 449 - 55
Periplasmic accumulation of truncated forms of outer-membrane PhoE protein of Escherichia coli K-12; Bosch D et al.; In order to localize the information within PhoE protein of Escherichia coli K-12 required for export of the protein to the outer membrane, we have generated deletions throughout the phoE gene . Immunocytochemical labelling on ultrathin cryosections revealed that the polypeptides encoded by the mutant alleles are transported to, and accumulate in, the periplasm . These results show that, except for the signal sequence, there is no specific sequence within the PhoE protein that is essential for transport through the cytoplasmic membrane . The overall structure of the protein, rather than a particular sequence of amino acids, seems to be important for assembly into the outer membrane.

J Mol Biol, 1986 Jun 5, 189(3), 435 - 48
Mnemonic aspects of Escherichia coli DNA polymerase I . Interaction with one template influences the next interaction with another template; Papanicolaou C et al.; When Escherichia coli DNA polymerase I (Pol I) replicates a homopolymer, the excision/polymerization (exo/pol) ratio varies with enzyme and initiator concentration . The study of this effect in the case of poly(dA).oligo(dT) replication led us to propose a mnemonic model for Pol I, in which the 3' to 5' excision activity warms up when the enzyme is actively polymerizing, and cools down when it dissociates from the template . The model predicts that the exo/pol ratio must increase with processivity length and initiator concentration and decrease with enzyme concentration . It predicts also that contact of the enzyme with one template alters its excision efficiency towards another template . The exo/pol ratio and processivities of Pol I and its Klenow fragment were studied on four templates: poly(dA).(dT)10, poly(dT).(dA)10, poly(dC).(dG)10 and poly(dI).(dC)10 . We show that the Klenow fragment is usually much less processive than Pol I and when this is the case it has a much lower exo/pol ratio . At equal processivity, the exo/pol ratios are nearly equal . Furthermore, many factors that influence processivity length (e.g . manganese versus magnesium, inorganic pyrophosphate, ionic strength) influence the exo/pol ratio in the same direction . The study of deaminated poly(dC) replication, where we followed incorporation and excision of both G and A residues, allowed us to assign the origin of the dNMP variations to changes in the 3' to 5' proof-reading activity of Pol I . Similarly, the lower dNMP turnover of the Klenow fragment observed with deaminated poly(dC) was specifically assigned to a decreased 3' to 5' exonuclease activity . The exo/pol ratio generally increased with initiator and decreased with enzyme concentration, in agreement with the model, except for poly(dI).oligo(dC), where it decreased with initiator concentration . However, by terminating chain elongation with dideoxy CTP, we showed directly that, even in this system, excision is relatively inefficient at the beginning of synthesis . Interaction of Pol I with poly(dA).(dT) or with poly(dC).(dG) modifies its exo/pol characteristics in the replication of poly(dI).(dC) and poly(dA).(dT), respectively . The Klenow enzyme is not sensitive to such influences and this correlates with its reduced processivity on the influencing templates . Our results reveal the existence of differences between Pol I and its Klenow fragment that are more profound than has been thought previously.(ABSTRACT TRUNCATED AT 400 WORDS)

J Biol Chem, 1986 Jun 5, 261(16), 7550 - 7
The beta subunit of the Escherichia coli DNA polymerase III holoenzyme interacts functionally with the catalytic core in the absence of other subunits; LaDuca RJ et al.; We have previously demonstrated that the addition of a stoichiometric excess of the beta subunit of Escherichia coli DNA polymerase III holoenzyme to DNA polymerase III or holoenzyme itself can lead to an ATP-independent increase in the processivity of these enzyme forms (Crute, J . J., LaDuca, R . J., Johanson, K . O., McHenry, C . S., and Bambara, R . A . (1983) J . Biol . Chem . 258, 11344-11349) . Here, we show that the beta subunit can interact directly with the catalytic core of the holoenzyme, DNA polymerase III, generating a new form of the enzyme with enhanced catalytic and processive capabilities . The addition of saturating levels of the beta subunit to the core DNA polymerase III enzyme results in as much as a 7-fold stimulation of synthetic activity . Two populations of DNA products were generated by the DNA polymerase III X beta enzyme complex . Short products resulting from the addition of 5-10 nucleotides/primer fragment were generated by DNA polymerase III in the presence and absence of added beta subunit . A second population of much longer products was generated only in beta-supplemented DNA polymerase III reactions . The DNA polymerase III-beta reaction was inhibited by single-stranded DNA binding protein and was unaffected by ATP, distinguishing it from the holoenzyme-catalyzed reaction . Complex formation of the DNA polymerase III core enzyme with beta increased the residence time of the enzyme on synthetic DNA templates . Our results demonstrate that the beta stimulation of DNA polymerase III can be attributed to a more efficient and highly processive elongation capability of the DNA polymerase III X beta complex . They also prove that at least part of beta's normal contribution to the DNA polymerase III holoenzyme reaction takes place through interaction with DNA polymerase III core enzyme components to produce the essential complex necessary for efficient elongation in vivo.

J Biol Chem, 1986 Jun 5, 261(16), 7136 - 43
Processivity and kinetics of the reaction of exonuclease I from Escherichia coli with polydeoxyribonucleotides; Brody RS et al.; The enzyme exonuclease I from Escherichia coli hydrolyzes successive nucleotides from the 3'-termini of single-stranded deoxyribonucleotide homopolymers . When the reaction is stopped after partial hydrolysis, only intact starting material and small oligomers can be isolated . The distribution of oligomeric products varies with the base composition of the polymer but the largest oligomer that can be isolated from the reaction of exonuclease I with homopolymers of deoxyadenylate, deoxythymidylate, or deoxycytidylate is a decamer . These results suggest a model in which exonuclease I possesses at least two nucleotide binding sites . When both sites are filled, with 11-mers and longer polymers, the enzyme does not dissociate from the polymer during hydrolysis . When, with smaller oligomers, only a single site is filled, the reaction partitions at each oligomer between hydrolysis and dissociation . The kinetics of the reactions of exonuclease I with purified polydeoxyriboadenylates of defined size distributions have been investigated . The maximum rates of hydrolysis are nearly independent of polymer size while the apparent Michaelis constants are inversely proportional to the polymer size . A simple steady state model yields a kinetic equation that is consistent with our results . Competition experiments indicate that the rate at which exonuclease I associates with the 3'-terminus of a polydeoxyribonucleotide is independent of the polymer's chain length.

J Mol Biol, 1986 Jun 5, 189(3), 477 - 86
Intermediates in the assembly of the TonA polypeptide into the outer membrane of Escherichia coli K12; Jackson ME et al.; The tonA gene of Escherichia coli K12 was cloned into a multicopy plasmid, leading to substantial overproduction of the corresponding 78,000 Mr polypeptide in the outer membrane . The approximate size of the tonA gene and its direction of transcription were established by Tn1000 mutagenesis . A family of tonA deletions was constructed in vitro by Bal31 exonuclease digestion, followed by splicing of an "oligo stop" sequence to each 3' terminus in order to ensure prompt termination of translation of the truncated polypeptides in vivo . All these polypeptides proved to be extremely unstable in exponentially growing cultures but were relatively stable in maxicells . Under these conditions the truncated polypeptides, unlike wild-type TonA, fractionated with the Sarkosyl-soluble fraction of the cell envelope, indicating that these proteins are blocked in assembly as inner membrane (translocation) intermediates or as outer membrane (maturation) intermediates unable to form Sarkosyl-resistant complexes . We have also examined the kinetics of assembly of wild-type TonA into the outer membrane and the results indicate that, following cleavage of the N-terminal signal peptide, the protein passes through an apparently membrane-free intermediate form and only appears in the outer membrane after a delay of at least 50 seconds, following the completion of synthesis . From these results, we propose that the assembly of TonA involves translocation (with concomitant cleavage of the signal sequence) directly into the periplasm, followed by partitioning into the outer membrane . We further propose that the C terminus of TonA is essential for final maturation in the outer membrane in Sarkosyl-resistant form but that the C-terminal half of the molecule probably does not contain any topogenic sequences required for partitioning to the outer membrane.

J Mol Biol, 1986 Jun 5, 189(3), 389 - 99
Concatemer formation of ColE1-type plasmids in mutants of Escherichia coli lacking RNase H activity; Subia NL et al.; rnh mutants harboring pBR322 were found to contain several slowly migrating DNA species when examined by agarose gel electrophoresis . The plasmid DNA from rnh mutants included large molecules, i.e . plasmids two, three or four times the size of a single plasmid unit . That this DNA contained concatemeric plasmid joined in a head-to-tail fashion was determined by digestion with restriction endonucleases that cleaved the monomeric plasmid DNA at a unique site . This treatment resulted in migration of the plasmid DNA at a mobility identical to that of linearized monomeric plasmid by agarose gel electrophoresis . This was confirmed by electron microscopy . Plasmid concatemer formation was detected with several high-copy-number (relaxed type) plasmids but not with low-copy-number (stringent) plasmids . Concatemer formation was dependent on RecA+ and RecF+ functions since several recA and recF mutations abolished concatemer formation . ColE1-type plasmids were previously shown to replicate in rnh mutants in the absence of DNA polymerase I (PolI) activity . This DNA PolI-independent plasmid replication was also examined for its dependence on the recF and recA gene products . rnh- polA(Ts) recF- strains were efficiently transformed with these plasmids at 30 degrees C and 42 degrees C, indicating the presence of DNA PolI-independent replication under recF- conditions . The presence or absence of plasmid replication in rnh- polA- recA(Ts) strains was also examined by measuring the increase in total amounts of plasmid . The results indicated that DNA PolI-independent replication occurred in these triple mutants at 42 degrees C as well as at 30 degrees C . It was concluded that the recombination event giving rise to concatemer formation was not essential for DNA PolI-independent replication in rnh mutants.

J Biol Chem, 1986 Jun 5, 261(16), 7253 - 6
Different conformations of the 3' termini of initiator and elongator transfer ribonucleic acids . An EPR study; Pscheidt RH et al.; The 3' ends of transfer ribonucleic acids were covalently labeled with a nitroxide spin label . The 3' end of initiator tRNA (tRNAMetf) from Escherichia coli shows different motional behavior than the 3' terminus of elongator tRNAs as monitored by EPR . The line shapes of the EPR spectra are quite sensitive to the buffer conditions, as shown by measurements in 4 different buffers . The data are consistent with a constrained or folded back 3' terminus in the initiator tRNA as opposed to the freely rotating elongator 3' terminus . The EPR spectra are also sensitive to aggregation of the tRNA.

J Mol Biol, 1986 Jun 5, 189(3), 413 - 9
rho factor-dependent transcription termination . Interference by a mutant rho; Richardson JP et al.; The rho protein isolated from a strain of Escherichia coli with the rho1 (suA1) mutant allele is defective in interactions with RNA that are coupled to ATP hydrolysis . Here we show that the rho1 allele is partially dominant over wild-type and demonstrate that the mechanism of that dominance is due to an interference of wild-type rho factor function by the defective rho factor . The rho1 mutant protein can inhibit transcription termination and RNA-dependent ATPase activities of normal rho protein . Inhibition of the ATPase activity with excess RNA occurs by exchange of subunits to form hybrid hexamers in which the defective subunits apparently disrupt co-operative interactions essential for wild-type subunit function.

J Biol Chem, 1986 Jun 5, 261(16), 7472 - 8
The homologous recombination system of phage lambda . Pairing activities of beta protein; Muniyappa K et al.; The red genes of phage lambda specify two proteins, exonuclease and beta protein, which are essential for its general genetic recombination in recA- cells . These proteins seem to occur in vivo as an equimolar complex . In addition, beta protein forms a complex with another polypeptide, probably of phage origin, of Mr 70,000 . The 70-kDa protein appears to be neither a precursor nor an aggregated form of either exonuclease or beta protein, since antibodies directed against the latter two proteins failed to react with 70-kDa protein on Ouchterlony double diffusion analysis . beta protein promotes Mg2+-dependent renaturation of complementary strands (Kmiec, E., and Holloman, W . K . (1981) J . Biol . Chem . 256, 12636-12639) . To look for other pairing activities of beta protein, we developed methods of purification to free it of associated exonuclease . Exonuclease-free beta protein appeared unable to cause the pairing of a single strand with duplex DNA; however, like Escherichia coli single strand binding protein (SSB), beta protein stimulated formation of joint molecules by recA protein from linear duplex DNA and homologous circular single strands . Like recA protein, but unlike SSB, beta protein promoted the joining of the complementary single-stranded ends of phage lambda DNA . beta protein specifically protected single-stranded DNA from digestion by pancreatic DNase . The half-time for renaturation catalyzed by beta protein was independent of DNA concentration, unlike renaturation promoted by SSB and spontaneous renaturation, which are second order reactions . Thus, beta protein resembles recA protein in its ability to bring single-stranded DNA molecules together and resembles SSB in its ability to reduce secondary structure in single-stranded DNA.

J Biol Chem, 1986 Jun 5, 261(16), 7109 - 11
The beta-subunit of the F1F0-ATPase is conserved in mycoplasmas; Zilberstein D et al.; Monospecific polyclonal antibodies that were generated against the beta-subunit of Escherichia coli ATPase (F1Fo) cross-reacted with a protein present in the cells of several Mycoplasma and Acholeplasma species . In Mycoplasma gallisepticum, the reactive protein was found almost exclusively in the cell membrane . This protein had an apparent molecular mass of approximately 52 kDa and could not be released from the membranes by repeated washings with either low or high salt solutions in the presence or absence of EDTA . The reactive protein was found to be catalytically active, exhibiting up to 44% of the total membrane-bound ATPase activity . We suggest that mycoplasmas possess a F1Fo-ATPase which undergoes structural modification(s) allowing its integration into the membrane.

J Biol Chem, 1986 Jun 5, 261(16), 7115 - 8
Production of native, correctly folded bovine pancreatic trypsin inhibitor by Escherichia coli; Marks CB et al.; A gene for bovine pancreatic trypsin inhibitor (BPTI) was fused to the coding sequence for the Escherichia coli alkaline phosphatase signal peptide and expressed in E . coli under the control of the alkaline phosphatase promoter . When induced in phosphate-depleted medium such cells produced a trypsin inhibitor that was indistinguishable from native, properly folded BPTI . In particular, the BPTI produced by E . coli had three disulfide bonds that appeared to be identical to those found in native BPTI, as assayed by sensitivity to iodoacetate, dithiothreitol, and urea . This expression/secretion system will make possible the production of variant BPTI molecules, thus allowing the perturbing effects of amino acid substitutions on BPTI folding, structure, and function to be assessed.

Biochemistry, 1986 Jun 3, 25(11), 3245 - 55
Affinities of tRNA binding sites of ribosomes from Escherichia coli; Lill R et al.; The binding affinities of tRNAPhe, Phe-tRNAPhe, and N-AcPhe-tRNAPhe from either Escherichia coli or yeast to the P, A, and E sites of E . coli 70S ribosomes were determined at various ionic conditions . For the titrations, both equilibrium (fluorescence) and nonequilibrium (filtration) techniques were used . Site-specific rather than stoichiometric binding constants were determined by taking advantage of the varying affinities, stabilities, and specificities of the three binding sites . The P site of poly(U)-programmed ribosomes binds tRNAPhe and N-AcPhe-tRNAPhe with binding constants in the range of 10(8) M-1 and 5 X 10(9) M-1, respectively . Binding to the A site is 10-200 times weaker, depending on the Mg2+ concentration . Phe-tRNAPhe binds to the A site with a similar affinity . Coupling A site binding of Phe-tRNAPhe to GTP hydrolysis, by the addition of elongation factor Tu and GTP, leads to an apparent increase of the equilibrium constant by at least a factor of 10(4) . Upon omission of poly(U), the affinity of the P site is lowered by 2-4 orders of magnitude, depending on the ionic conditions, while A site binding is not detectable anymore . The affinity of the E site, which specifically binds deacylated tRNAPhe, is comparable to that of the A site . In contrast to P and A sites, binding to the E site is labile and insensitive to changes of the ionic strength . Omission of the mRNA lowers the affinity at most by a factor of 4, suggesting that there is no efficient codon-anticodon interaction in the E site . On the basis of the equilibrium constants, the displacement step of translocation, to be exergonic, requires that the tRNA leaving the P site is bound to the E site . Under in vivo conditions, the functional role of transient binding of the leaving tRNA to the E site, or a related site, most likely is to enhance the rate of translocation.

Biochemistry, 1986 Jun 3, 25(11), 3397 - 404
Topography of oligomycin sensitivity conferring protein in the mitochondrial adenosinetriphosphatase-ATP synthase; Archinard P et al.; The topographical organization of oligomycin sensitivity conferring protein (OSCP) in the mitochondrial adenosinetriphosphatase (ATPase)-ATP synthase complex has been studied . The accessibility of OSCP to monoclonal antibodies has been qualitatively visualized by using the protein A-gold electron microscopy immunocytochemistry or quantitatively estimated by immunotitration of OSCP in depolymerized or intact membranes . Besides, OSCP cannot be labeled by 3-(trifluoromethyl)-3-(m-{125I}iodophenyl)diazirine ({125I}TID) which selectively labels the hydrophobic core of membrane proteins . These observations demonstrate an external location of OSCP on the inner face of the inner mitochondrial membrane . The position of OSCP relative to other peptides of the complex has been analyzed by cross-linking experiments using either zero length N-(ethoxycarbonyl)-2-ethoxydihydroquinoline or 11-A span dimethyl suberimidate cross-linkers in the ATPase-ATP synthase complex . The OSCP cross-linked products were identified either by immunocharacterization with anti-alpha, anti-beta, or anti-OSCP monoclonal antibodies or by their molecular weight . OSCP was cross-linked with either the alpha- or beta-subunits of F1 or to a subunit of Mr 24 000 . Other types of cross-linking were obtained by the labeling of OSCP with {cysteamine-35S}-N-succinimidyl 3-{{2-((2-nitro-4-azidophenyl)amino)ethyl}dithio}propionate ({35S}SNAP) and reconstitution of SNAP-OSCP with F1 in urea-treated submitochondrial particles . Under these conditions, OSCP is found to be adjacent to two other peptides of molecular weight close to 30 000 . A comparison is made between the topology and the organization of the b-subunit of Escherichia coli and OSCP, suggesting an analogy between OSCP and the hydrophilic part of the b-subunit.

Eur J Biochem, 1986 Jun 2, 157(2), 427 - 32
Cross-linking between 16S ribosomal RNA and protein S4 in Escherichia coli ribosomal 30S subunits effected by treatment with bisulfite/hydrazine and bromopyruvate; Nitta N et al.; Cytosine in nucleic acids can be modified by treatment with a mixture of bisulfite and hydrazine . The reaction is specific for single-stranded regions of nucleic acids and the product is N4-aminocytosine . Bromopyruvate has been used for alkylation of protein SH groups and through its 2-oxo group it can form a hydrazone with N4-aminocytosine . Escherichia coli ribosomal 30S subunits were treated with 1 M sodium bisulfite + 2 M hydrazine in the presence of 10 mM MgCl2 at pH 7.0 and 37 degrees C for 30 min . By this treatment, 2.4 cytosine residues/molecule 16S rRNA were derivatized into N4-aminocytosines . 35S-labeled 30S subunits were modified in this way and then treated with 10 mM bromopyruvate at pH 8.0 and 37 degrees C for 5 min . Analysis in sodium dodecyl sulfate/sucrose density gradient centrifugation showed co-sedimentation of a part of the 35S radioactivity with the RNA . The co-sedimentation was dependent on both the bisulfite/hydrazine and the bromopyruvate treatments . The RNA-protein complex was prepared from unlabeled 30S subunits . The protein portion was labeled with 125I, the RNA portion was digested with nucleases, and then the hydrazone linkage between the protein and oligonucleotides was cleaved by treatment with 0.2 M HCl . The oligonucleotides formed were removed by dialysis and the protein was identified as S4 by two-dimensional electrophoresis and by sodium dodecyl sulfate/polyacrylamide gel electrophoresis . The results indicate that the cysteinyl residue of protein S4 at position 31 from the N-terminus is located close to a cytosine residue which is non-base-paired and easily accessible by the externally present bisulfite/hydrazine reagent.

Eur J Biochem, 1986 Jun 2, 157(2), 405 - 13
The interaction of the trp repressor from Escherichia coli with L-tryptophan and indole propanoic acid; Lane AN; The binding of the corepressor, L-tryptophan, and an inducer, indole propanoic acid, to the trp repressor from Escherichia coli was studied by absorbance, fluorescence, circular dichroic and proton NMR spectroscopy . The two ligands bind to the same site on the repressor in the same orientation; they are molecular competitors . The binding site is of relatively low polarity and contains at least one methyl group that lies 0.3 nm over the indole moiety near the C5 proton of the bound ligand, and an aromatic residue, probably tyrosine . The dissociation constant was determined as a function of temperature and pH . At 25 degrees C in 0.1 M phosphate buffer, pH 7.6, the dissociation constant is 18 +/- 2 microM for both ligands . In the same buffer system, the van't Hoff enthalpy for dissociation is 35.5 +/- 1 kJ/mol for tryptophan, and 30.5 +/- 2 kJ/mol for indole propanoic acid . The affinity of the repressor for indole propanoic acid is independent of pH in the range 7 less than 10, but decreases four fold for tryptophan in the same range . The amino group of tryptophan makes a significant contribution to its binding affinity . Difference NMR spectra showed that there are few changes of protein resonances on binding ligands . The NMR signals of the bound resonances were assigned by difference and nuclear Overhauser effect spectroscopy . The properties of the bound resonances are consistent with the ligands being largely immobilised within the binding site . The difference spectra, and the known functional differences of the two ligands, suggest that tryptophan induces a slightly different conformational state in the repressor from that induced by indole propanoic acid . There is no evidence for a global transition . The rate of dissociation of ligands is relatively large, being in the range 400-600 s-1.

Vet Immunol Immunopathol, 1986 Jun, 12(1-4), 29 - 38
Primary in vitro stimulation of antibody production by rainbow trout lymphocytes; Kaattari SL et al.; Trinitrophenylated (TNP) forms of E . coli lipopolysaccharide (LPS) and keyhole limpet hemocyanin (KLH) were used to produce antigen specific plaque-forming cell (PFC) responses with rainbow trout (Salmo gairdneri) splenocytes from unprimed fish in vitro . The culture system that was developed is described and characterized with respect to the kinetics and dose responses for both the haptenated and unhaptenated forms of the carriers . The induction of the PFC response to TNP-LPS was inhibited with TNP-lysine . Exposure to graded levels of gamma-radiation demonstrated a low dose augmentation of the PFC response with both antigens . Antigen addition experiments reveal that both antigens appear to stimulate the same population of antibody-producing B lymphocytes.

Jpn J Antibiot, 1986 Jun, 39(6), 1494 - 503
{Effects of astromicin on a host defence mechanism}; Yoneyama H et al.; We investigated the effect of astromicin (ASTM) on a defence mechanism . The existence of ASTM (100 micrograms/ml) did not influence the ability of phagocytizing and killing by the mouse peritoneal exudated polymorphonuclear leukocyte (PMN) . We observed no change of either phagocytizing and killing or phagocytizing ability of PMN by a pretreatment with ASTM (100 micrograms/ml) . When the luminol-dependent chemiluminescence of PMN was examined, a slight decrease of relative light intensity in the presence of ASTM was observed at a concentration of 100 micrograms/ml or 50 micrograms/ml . There was, however, no change of relative light intensity in the presence of 20 micrograms/ml of ASTM . The chemotaxis of PMN was not influenced in the presence of even 100 micrograms/ml of ASTM . We also examined whether a combination effect existed between ASTM and either fresh human serum or fresh mouse serum in vitro . Considerable combination effects were observed against E . coli, P . aeruginosa, S . marcescens, K . pneumoniae and S . aureus . From these results, we concluded that ASTM did not exert a detrimental effect on examined functions of PMN which is important in the nonspecific host defence mechanism in the early phase of bacterial infections . We also concluded that ASTM, which had a synergic effect with a human serum factor, was a safe and effective chemotherapeutic agent.

Thromb Res, 1986 Jun 1, 42(5), 661 - 71
Platelet activating factor (PAF) involvement in endotoxin-induced thrombocytopenia in rabbits: studies with FR-900452, a specific inhibitor of PAF; Okamoto M et al.; PAF (1 ug/kg) injected intravenously (i.v.) into anesthetized rabbits resulted in marked loss of circulating platelets and leukocytes . Administration of FR-900452 1-methyl-3-(1-(5-methylthiomethyl-6-oxo-3-(2-oxo-3-cyclopenten-1-y lidene)- 2-piperazinyl) ethyl)-2-indolinone, a specific PAF inhibitor, at a dose of 10 mg/kg i.v . with 10 min prior to the PAF injection significantly prevented both changes . On the other hand, PAF has been considered as a mediator of endotoxin shock . Therefore, in order to determine whether endogenous PAF contributes to the occurrence of thrombocytopenia or leukopenia in endotoxin shock, we assessed the effect of FR-900452 on the thrombocytopenia and the leukopenia following bolus i.v . injection of E.coli endotoxin (0.03 mg/kg) in rabbits . As a result, pretreatment with the compound (10 mg/kg, i.v.) significantly reduced the thrombocytopenia at 60 and 180 min after the endotoxin injection . In contrast, FR-900452 did not reduced the leukopenia at any time of after endotoxin . These results indicate that PAF might be involved in the occurrence of thrombocytopenia in rabbit endotoxemia and the contribution of PAF to the leukopenia is much less extent than that to the thrombocytopenia.

Arch Biochem Biophys, 1986 Jun, 247(2), 346 - 54
Modification of bovine heart succinate dehydrogenase with ethoxyformic anhydride and rose bengal: evidence for essential histidyl residues protectable by substrates; Hederstedt L et al.; Purified and membrane-bound succinate dehydrogenase (SDH) from bovine heart mitochondria was inhibited by the histidine-modifying reagents ethoxyformic anhydride (EFA) and Rose Bengal in the presence of light . Succinate and competitive inhibitors protected against inhibition, and decreased the number of histidyl residues modified by EFA . The essential residue modified by EFA was not the essential thiol of SDH, but modification of the essential thiol abolished the protective effect of malonate against inhibition of SDH by EFA . The EFA inhibition was reversed by hydroxylamine nearly completely when the inhibition was less than or equal to 35%, and only partially when the inhibition was more extensive . The uv spectrum of EFA-modified SDH before and after hydroxylamine treatment suggested that extensive inhibition of SDH with EFA may result in ethoxyformylation at both imidazole nitrogens of histidyl residues . Such a modification is not reversed by hydroxylamine . Succinate dehydrogenases and fumarate reductases from several different sources have similar compositions, and the two enzymes from Escherichia coli have considerable homology in the amino acid composition of their respective flavoprotein and iron-sulfur protein subunits . In the former, there is a short stretch containing conserved histidine, cysteine, and arginine residues . These residues, if also conserved in the bovine enzyme, may be the essential active site residues suggested by this work (histidine) and previously (cysteine, arginine).

J Clin Invest, 1986 Jun, 77(6), 1812 - 6
Effects of anti-C5a antibodies on the adult respiratory distress syndrome in septic primates; Stevens JH et al.; In vitro and in vivo studies have suggested that human complement component C5a plays a key role in neutrophil injury in the adult respiratory distress syndrome (ARDS) . First, using leukocyte aggregometry, we demonstrated that the addition of a recently developed rabbit anti-human polyclonal antibody to C5a des arg to endotoxin-activated plasma prevented leukocyte aggregation in vitro . We then administered the anti-C5a des arg antibody to septic primates (Macaca fascicularis) . Three groups of primates, control, septic, and anti-C5a antibody treated septic, were studied (n = 4 in each group) . A 30-min infusion of Escherichia coli (1 X 10(10)/kg) resulted in severe sepsis and ARDS . Primates were killed 4 h after completion of the E . coli infusion . Septic animals not treated with anti-C5a antibody had 75% mortality (3/4), decreased oxygenation, severe pulmonary edema, and profound hypotension . Septic primates treated with anti-C5a antibodies did not die and did not develop decreased oxygenation (P less than 0.05) or increased extravascular lung water (P less than 0.05) . They also had a marked recovery in their mean arterial blood pressure (P less than 0.05) . This study demonstrates that treatment with rabbit anti-human C5a des arg antibodies attenuates ARDS and some of the systemic manifestations of sepsis in nonhuman primates.

J Immunol, 1986 Jun 1, 136(11), 4122 - 7
A monosaccharide precursor of Escherichia coli lipid A has the ability to induce tumor-cytotoxic factor production by a murine macrophage-like cell line, J774.1; Amano F et al.; A monosaccharide precursor of Escherichia coli lipid A, designated lipid X, which is a diacylglucosamine 1-phosphate with beta-hydroxymyristoyl groups at positions 2 and 3, was shown to have the ability to induce the production of tumor necrosis factor (TNF)-like tumor-cytotoxic factor by a murine macrophage-like cell line, J774.1 . This cytotoxic factor was released from J774.1 cells grown in the presence of lipid X and related compounds, and it was assayed as to its lytic activity against {3H}thymidine-labeled L929 cells . Dose-response studies revealed that lipid X induced the production of smaller amounts of the tumor-cytotoxic factor than LPS at low concentrations, but it induced that of considerable amounts at and over 1 microgram/ml . Elimination of 1-phosphate or 3-O-beta-hydroxymyristoyl group from lipid X completely prevented the induction of producing this factor by the macrophages . Therefore, it is suggested that both 1-phosphate and 3-O-beta-hydroxymyristoyl groups are essential for the biologic activity of lipid X, as to the induction of the tumor-cytotoxic factor production in the macrophages.

J Gen Microbiol, 1986 Jun, 132 ( Pt 6), 1753 - 62
Cloning and expression of the succinyl-CoA synthetase genes of Escherichia coli K12; Buck D et al.; The genes encoding both subunits of the succinyl-CoA synthetase of Escherichia coli have been identified as distal genes of the suc operon, which also encodes the dehydrogenase (Elo; sucA) and succinyltransferase (E2o; sucB) components of the 2-oxoglutarate dehydrogenase complex . The newly defined genes express polypeptides of 41 kDa (sucC) and 31 kDa (sucD), corresponding to the beta and alpha subunits of succinyl-CoA synthetase, respectively . The genes are thus located at 16.8 min in the E . coli linkage map, together with the citrate synthase (gltA) and succinate dehydrogenase (sdh) genes, in a cluster of nine citric acid cycle genes: gltA-sdhCDAB-sucABCD . Four deletion strains lacking all of these citric acid cycle enzymes were characterized . The succinyl-CoA synthetase activities of strains harbouring plasmids containing the sucC and sucD genes were amplified some fourfold . Further enzymological studies indicated that expression of succinyl-CoA synthetase is coordinately regulated with 2-oxoglutarate dehydrogenase.

J Gen Microbiol, 1986 Jun, 132 ( Pt 6), 1525 - 39
Haemoprotein b-590 (Escherichia coli), a reducible catalase and peroxidase: evidence for its close relationship to hydroperoxidase I and a 'cytochrome a1b' preparation; Poole RK et al.; A reducible hydroperoxidase, haemoprotein b-590, has been purified 16-fold from a soluble fraction of Escherichia coli K12, grown anaerobically with glycerol and fumarate . The Mr of the native protein, determined by gel filtration, was 331,000 although a minor, smaller species with a Mr of 188,000 was also detected; both had catalase activities . Based on the subunit Mr, determined from SDS gel electrophoresis to be 75,000, the above species are tentatively identified as tetramers and dimers, respectively . The isoelectric point of both species was 4.4 . The absorption spectrum of the isolated haemoprotein is typical of ferric, high-spin haem . The A405/A280 ratio never exceeded 0.27, a value half of that obtained for E . coli hydroperoxidase I . On reduction with dithionite, the gamma, beta, and alpha bands were at 441, 559 and 590 nm respectively, the alpha-band being unusually distinct . Treatment of the reduced form with CO gave a sharp prominent gamma-band at 426 nm and caused significant shifts of the alpha and beta bands to shorter (574 and 545 nm) wavelengths . The pyridine haemochrome spectra showed the haem to be protohaem IX; the spectra were featureless between 580 and 630 nm, thus excluding the presence of haem a . However, some features of the difference spectra of the haemoprotein were reminiscent of cytochrome a1, notably the maxima in reduced minus oxidized spectra at 444 and 593 nm and the peaks and troughs in CO difference spectra at 426 and 446 nm respectively . The haemoprotein had high catalase activity: Vmax was 2.3 X 10(6) mol H2O2 (mol haem)-1 min-1 and the Km was 11 mM . At 10 mM-H2O2 the first order rate constant was 0.3 X 10(7) M-1 s-1 . The haemoprotein was also a peroxidase with o-dianisidine or 2,3',6-trichloroindophenol as substrates; for the latter substrate, the Km was 0.18 mM . It is concluded that haemoprotein b-590 strongly resembles the hydroperoxidase I purified by Claiborne & Fridovich (Journal of Biological Chemistry 254, 4245-4252, 1979) and that a similar haemoprotein was mistaken for a cytochrome a1 b complex by Barrett & Sinclair (Abstracts of the 7th International Congress of Biochemistry, Tokyo, H-107, p . 907, 1967).

J Gen Microbiol, 1986 Jun, 132 ( Pt 6), 1429 - 39
Isolation and characterization of the tubular organelles induced by fumarate reductase overproduction in Escherichia coli; Elmes ML et al.; Strains of Escherichia coli amplifying the intrinsic membrane enzyme fumarate reductase accommodate the overproduced enzyme by increasing the amount of membrane material, in the form of intracellular tubular structures . These tubules have been observed in strains harbouring multicopy frd plasmids and in ampicillin hyper-resistant strains . A procedure has been developed for isolation of tubules nearly free of cytoplasmic membrane . Using protein A-gold labelling and optical diffraction of electron micrographs, a model for tubule structure is proposed . The tubules have a lower lipid/protein ratio than the cytoplasmic membrane, with the enzyme accounting for greater than 90% of the protein in the tubules . Both cytoplasmic membranes and tubules from amplified strains are enriched in cardiolipin and have a more fluid fatty acid composition than wild-type strains . Mutants defective in cardiolipin synthesis produce tubules in response to excess fumarate reductase, but these tubules have an altered appearance, indicating that lipid-protein interactions may be important for tubule assembly.

Mol Cell Biol, 1986 Jun, 6(6), 1983 - 90
Substrate specificity of a mammalian DNA repair endonuclease that recognizes oxidative base damage; Helland DE et al.; The substrate specificity of a calf thymus endonuclease on DNA damaged by UV ligh, ionizing radiation, and oxidizing agents was investigated . End-labeled DNA fragments of defined sequence were used as substrates, and the enzyme-generated scission products were analyzed by using DNA sequencing methodologies . The enzyme was shown to incise damaged DNA at pyrimidine sites . The enzyme incised DNA damaged with UV light, ionizing radiation, osmium tetroxide, potassium permanganate, and hydrogen peroxide at cytosine and thymine sites . The substrate specificity of the calf thymus endonuclease was compared to that of Escherichia coli endonuclease III . Similar pyrimidine base damage specificities were found for both enzymes . These results define a highly conserved class of enzymes present in both procaryotes and eucaryotes that may mediate an important role in the repair of oxidative DNA damage.

J Nutr Sci Vitaminol (Tokyo), 1986 Jun, 32(3), 279 - 86
Reinvestigation of compound X, a suspected biotin intermediate: identification of N-formyl derivatives of biotin and dethiobiotin; Osakai M et al.; Compound X, reported as an intermediate in the biosynthesis of biotin from dethiobiotin (DTB) (Biochem . Biophys . Res . Commun., 88, 312 (1979)), was found to contain N-formyl DTB and biotin . The methyl ester of N-formyl biotin was considered to be a product from the biotin, which was biosynthesized from DTB by resting cells of E . coli C 124, through treatment with diazomethane in the presence of a trace amount of formic acid after Dowex 1X2 column chromatography . NMR analysis revealed that biotin was formylated at 1'-N . N-Formylated DTB and biotin are new biotin derivatives.

Biochem J, 1986 Jun 1, 236(2), 453 - 60
Pea chloroplast DNA encodes homologues of Escherichia coli ribosomal subunit S2 and the beta'-subunit of RNA polymerase; Cozens AL et al.; The nucleotide sequence has been determined of a segment of 4680 bases of the pea chloroplast genome . It adjoins a sequence described elsewhere that encodes subunits of the F0 membrane domain of the ATP-synthase complex . The sequence contains a potential gene encoding a protein which is strongly related to the S2 polypeptide of Escherichia coli ribosomes . It also encodes an incomplete protein which contains segments that are homologous to the beta'-subunit of E . coli RNA polymerase and to yeast RNA polymerases II and III.

Mol Gen Genet, 1986 Jun, 203(3), 520 - 3
In vivo cloning ad characterization of mutations of the regulatory locus ompR of Escherichia coli K12; Pirhonen M et al.; The product of the ompR gene of E . coli K12 is a positive regulatory protein, which is needed for the expression of the major outer membrane proteins OmpC and OmpF in E . coli K12 . A simple in vivo technique was used to transfer three ompR mutations (ompR101, ompR472, ompR4) onto a multicopy plasmid carrying the wild-type ompR gene . The resulting clones were transformed into wild type and corresponding mutant backgrounds to analyze their effects on ompC and ompF expression . All of the cloned ompR mutant alleles exhibited a dominant OmpC- phenotype in an ompR+ background . In addition negative complementation of ompF expression was observed between chromosomal ompR4 and multicopy ompR101 alleles . The results suggest an interaction between different OmpR molecules and thereby support the idea that OmpR can exist as a multimeric protein.

Mol Gen Genet, 1986 Jun, 203(3), 492 - 5
Genetic studies on the beta subunit of Escherichia coli RNA polymerase . IX . The role of the carboxy-terminus in enzyme assembly; Glass RE et al.; The assembly of RNA polymerase was studied in Escherichia coli mutants encoding large N-terminal amber fragments of the beta subunit . Whereas the removal of up to 20% of the carboxy-terminus does not prevent the formation of premature core enzyme, the amber fragments seem to interfere with holoenzyme production . These studies permit, therefore, the localization of a region on the beta polypeptide involved in sigma binding.

Mol Gen Genet, 1986 Jun, 203(3), 487 - 91
Genetic studies on the beta subunit of Escherichia coli RNA polymerase . VIII . Localisation of a region involved in promoter selectivity; Glass RE et al.; We have previously isolated an E . coli derivative carrying a small internal deletion (delta(rpoB)1570-1) of the beta structural gene . This RNA polymerase deletion mutant has no noticeable phenotype other than a slightly increased generation time in minimal medium . The deletion, which removes about 165 bp, has been localised to between codons 965 and 1,083, indicating it excises part of a tandem repeat structure present in the C-terminal region of beta . Analysis in vitro of purified RNA polymerase from the deletion mutant indicates that this enzyme has an altered promoter selectivity . These observations allow localisation of a site on the beta polypeptide of E . coli RNA polymerase involved in transcriptional initiation.

Mol Gen Genet, 1986 Jun, 203(3), 479 - 86
The pentafunctional FAS1 gene of yeast: its nucleotide sequence and order of the catalytic domains; Schweizer M et al.; FAS1, the structural gene of the pentafunctional fatty acid synthetase subunit beta in Saccharomyces cerevisiae has been sequenced . Its reading frame represents an intron-free nucleotide sequence of 5,535 base pairs, corresponding to a protein of 1,845 amino acids with a molecular weight of 205,130 daltons . In addition to the coding sequence, 1,468 base pairs of its 5'-flanking region were determined . S1 nuclease mapping revealed two transcriptional initiation sites; 5 and 36 base pairs upstream of the translational start codon . Within the flanking sequences two TATATAAA boxes, several A-rich and T-rich blocks and a TAG...TATGTT...TATGTT...TTT sequence were found and are discussed as transcriptional initiation and termination signals, respectively . The order of catalytic domains in the cluster gene was established by complementation of defined fas1 mutants with overlapping FAS1 subclones . Acetyl transferase (amino acids 1-468) is located proximal to the N-terminus of subunit beta, followed by the enoyl reductase (amino acids 480-858), the dehydratase (amino acids 1,134-1,615) and the malonyl/palmityl transferase (amino acids 1,616-1,845) domains . One major inter-domain region of about 276 amino acids with so far unknown function was found between the enoyl reductase and dehydratase domains . The substrate-binding serine residues of acetyl, malonyl and palmityl transferases were identified within the corresponding domains . Significant sequence homologies exist between the acyl transferase active sites of yeast and animal fatty acid synthetases . Similarly, a putative sequence of the enoyl reductase active site was identified.

J Biochem (Tokyo), 1986 Jun, 99(6), 1773 - 9
Further characterization of trimethylamine N-oxide reductase from Escherichia coli, a molybdoprotein; Yamamoto I et al.; Escherichia coli trimethylamine N-oxide (TMAO) reductase I, the major enzyme among inducible TMAO reductases, was purified to homogeneity by an improved method including heat treatment, ammonium sulfate precipitation, and chromatographies on Bio-Gel A-1.5m, DEAE-cellulose, and Reactive blue-agarose . The molecular weight was estimated by gel filtration to be approximately 200,000 . A single subunit peptide with a molecular weight of 95,000 was found by sodium dodecyl sulfate-polyacrylamide gel electrophoresis . This enzyme contained 1.96 atoms of molybdenum, 0.96 atoms of iron, 1.52 atoms of zinc, and less than 0.4 atoms of acid-labile sulfur per molecular weight of 200,000 . The absorption spectrum of the enzyme showed a peak at 278 nm and a shoulder at 288 nm, but no characteristic absorption was found from 350 to 700 nm . A fluorescent derivative of molybdenum cofactor was found when the enzyme was boiled with iodine in acidic solution; its fluorescence spectra were almost the same as those of the form A derivative of molybdopterin found in sulfite oxidase . The molybdenum cofactor released from heated TMAO reductase I reconstituted nitrate reductase in the extracts of Neurospora crassa mutant strain nit-1 lacking molybdenum cofactor . Thus, TMAO reductase I contains molybdopterin, which is a common constituent of some molybdenum-containing enzymes . Some kinetic properties were also determined.

J Biochem (Tokyo), 1986 Jun, 99(6), 1707 - 12
Isolation and expression in Escherichia coli of a cDNA clone encoding porcine pancreatic elastase; Shirasu Y et al.; We have cloned a DNA that is complementary to the messenger RNA that encodes porcine pancreatic elastase 1 from pancreas using rat pancreatic elastase 1 cDNA as a probe . This complementary DNA contains the entire protein coding region of 798 nucleotides which encodes an elastase of 266 amino acids, and 22 and 136 nucleotides of the 5' and 3'-untranslated sequences . When this deduced amino acid sequence was compared with known amino acid sequences, a carboxy-terminal 240 amino acids long peptide was found to be identical with a mature form of porcine pancreatic elastase 1, except for two amino acids . The porcine enzyme contains the same number of amino acid residues as the rat enzyme, and their amino acid sequences are 85% homologous . Taking the above findings together, we conclude that the cloned cDNA encodes a mature enzyme of 240 amino acids including a leader and activation peptide of 26 amino acids . We expressed the cloned porcine pancreatic elastase 1 cDNA in E . coli as a lac-fused protein . The resulting fused protein showed enzymatic activity and immunoreactivity toward anti-elastase serum.

Biochem Cell Biol, 1986 Jun, 64(6), 509 - 14
The amino acid sequence encompassing the active-site histidine residue of lipoamide dehydrogenase from Escherichia coli labelled with a bifunctional arsenoxide; Holmes CF et al.; Pyruvate dehydrogenase multienzyme complex (PD complex) in the presence of pyruvate, thiamine pyrophosphate, coenzyme A, and Mg2+ (or NADH) was irreversibly inhibited with the radiolabelled bifunctional aresenoxide p-{(bromoacetyl)amino}phenyl arsenoxide (BrCH2 14CONHPhAsO) . The initial reaction of the reagent was with a reduced lipoyl group of the lipoamide acetyltransferase component to form a dithioarsinite complex . Following the normal catalytic reactions, the anchored reagent was delivered into the active site of the lipoamide dehydrogenase (E3) component where an irreversible alkylation ensued via the bromoacetamidyl moiety . Treatment with 2,3-dithiopropanol (to break dithioarsinite bonds) caused the radiolabelled reagent to reside with E3 . E3 was isolated from the inhibited PD complex and CNBr cleavage of the inhibited enzyme yielded a single radiolabelled peptide that was purified on a cyanopropyl silica column using high performance liquid chromatography . The radiolabelled amino acid was identified (after acid hydrolysis) as N3-{14C}carboxymethyl histidine in agreement with earlier studies . The radiolabel was located in residue 14 of the peptide for which the sequence was determined as GCDAEDIALTIHAHPTL-EIVGLAAEVFEG . This sequence agrees with the amino acid sequence determined from the gene sequence of E3 . The histidine alkylated in the E3 component of the PD complex by BrCH2 14CONHPhAsO is residue-444 and further establishes its active site role.

J Antimicrob Chemother, 1986 Jun, 17(6), 821 - 5
The assay of chloramphenicol acetyltransferase activity by high performance liquid chromatography; Lovering AM et al.; A high performance liquid chromatographic method is described for the assay of chloramphenicol acetyltransferase activity which detects the formation of both 3-acetoxy chloramphenicol and 1,3-diacetoxy chloramphenicol . In experiments performed at pH 6.8 only 3-acetoxy chloramphenicol was detected while in experiments performed at pH 7.8 both 3-acetoxy and 1,3-diacetoxy chloramphenicol were detected.

EMBO J, 1986 Jun, 5(6), 1373 - 6
A temperature-sensitive mutant in the gene rplX for ribosomal protein L24 and its suppression by spontaneous mutations in a 23S rRNA gene of Escherichia coli; Nishi K et al.; A temperature-sensitive mutant with an altered ribosomal protein L24 was analysed . Revertant analysis showed that the temperature-sensitive growth was correlated with the altered protein . A DNA segment containing the mutant rplX gene was cloned and sequenced . The GGC codon for glycine at the amino acid position 84 of the protein was found to be altered to a GAC codon for aspartic acid . By transforming the rplX mutant with a plasmid carrying the rrnB operon and by selecting for temperature-resistant transformants we obtained two spontaneous suppressor mutants in the gene for 23S rRNA . DNA sequence analysis of the region corresponding to the 5' end of the 23S rRNA showed a C to T alteration at position 33 in both mutants and an additional A to G alteration at position 466 in one of them . The results suggest intimate interaction of protein L24 and the 5' end of 23S rRNA in vivo and support a secondary structure model of the 23S rRNA which brings these mutational points into a close contact.

Appl Environ Microbiol, 1986 Jun, 51(6), 1364 - 6
Toxicity of organic acids for repair-deficient strains of Escherichia coli; Sinha RP; The wild-type strain and four DNA repair-deficient strains (uvrA6, uvrB5, recA56, and polA1) of Escherichia coli K-12 were treated with acetic acid, lactic acid, and p-aminobenzoic acid at pH 3.5 during their stationary phase of growth . All three acids were highly toxic to the polymerase-deficient strain . The greater sensitivity of the strain carrying the polA1 gene than its isogenic pol+ derivatives suggested that damage caused by acidity requires polA+ gene products for repair.

Antimicrob Agents Chemother, 1986 Jun, 29(6), 1040 - 6
Role of a novel antidiarrheal agent, BW942C, alone or in combination with trimethoprim-sulfamethoxazole in the treatment of traveler's diarrhea; Ericsson CD et al.; The efficacy of BW942C, a novel enkephalinlike pentapeptide antidiarrheal agent, was compared with the efficacy of trimethoprim-sulfamethoxazole (TMP-SMX) and the combination of the two agents in a placebo-controlled trial of the 72-h treatment of acute diarrhea . Subjects with diarrhea but without bloody stools or fever greater than 102 degrees F (38.9 degrees C) were enrolled . Administered to 134 U.S . adults with diarrhea that developed shortly after their arrival in Guadalajara, Mexico, BW942C was more efficacious than TMP-SMX in relieving diarrhea and cramps in the first 12 h of therapy, especially among subjects with diarrhea caused by enterotoxigenic E . coli . In the BW942C treatment group, 25% of subjects eventually took additional therapy because their diarrhea did not respond to BW942C alone . Neurological side effects such as dizziness and light-headedness occurred more frequently among BW942C-treated subjects . Therapy for 3 days with TMP-SMX provided lasting relief comparable with previously reported 5-day therapy . Use of the combination of both agents provided the benefits of prompt relief afforded by BW942C and lasting relief afforded by TMP-SMX . BW942C might prove to be an agent suitable for the treatment of acute diarrhea, with TMP-SMX reserved for treatment of those who do not respond adequately . The empiric use of the combination of BW942C and TMP-SMX appears appropriate for the treatment of severe nondysenteric disease.

Antimicrob Agents Chemother, 1986 Jun, 29(6), 1005 - 9
Autoradiography of tobramycin uptake by the proximal and distal tubules of normal and endotoxin-treated rats; Bergeron MG et al.; Multiple factors may modify the pharmacokinetics of aminoglycosides and increase their nephrotoxic potential . In the present study, the influence of Escherichia coli endotoxin on the renal handling of {3H}tobramycin was investigated . The accumulation of {3H}tobramycin in proximal tubules, distal tubules, and collecting ducts was compared in both normal and endotoxin-injected (0.25 mg/kg) rats . Histological observations were also made . Blood pressure and cardiac frequency were recorded, and renal function was evaluated with labeled inulin and p-aminohippuric acid . Following administration of endotoxin, disturbed intrarenal localization of the drug was noted . Grain counts were affected in both proximal and distal tubules . Increased labeling was observed at all time intervals in the proximal tubules . In the distal tubules of endotoxemic animals we could detect higher amounts of drug at 10 and 60 min in the medulla and at 10 min in the cortex . Not all of the tubules were labeled to the same extent . No histological lesion was noted on light microscopy in animals receiving either normal saline or endotoxin . The dose of endotoxin used resulted in very fine physiological disturbance . Both blood pressure and cardiac frequency were minimally affected by endotoxin . Decreases in glomerular filtration rate and renal plasma flow were observed . However, none of these changes was statistically significant . The present study has shown that low doses of endotoxin alter the renal handling of aminoglycosides in the absence of any major physiological disturbance or histological changes . By increasing the total amount of drug within the kidney, endotoxin might increase the nephrotoxic potential of aminoglycosides.

Am J Vet Res, 1986 Jun, 47(6), 1366 - 72
Efficacy of flunixin meglumine for the treatment of endotoxin-induced bovine mastitis; Anderson KL et al.; The clinical effect of flunixin meglumine administration was determined in cows with acute mastitis induced by intramammary administration of endotoxin . In 12 lactating cows, 10 micrograms of Escherichia coli 026:B6 endotoxin were administered via a teat cannula into the teat cistern of single randomly selected rear quarters . Cows were challenge exposed as pairs . One cow in each pair was administered parenteral flunixin meglumine (6 cows) and 1 cow per pair was administered saline solution (6 cows) . Multiple doses (7) of 1.1 mg of flunixin meglumine/kg of body weight or saline solution were administered at 8-hour intervals beginning 2 hours after endotoxin . Cow and quarter clinical signs as well as milk somatic cell concentrations, bovine serum albumin, electrical conductivity, and milk production were determined before and for 14 days after endotoxin inoculation . Intramammary endotoxin produced signs characteristic of acute coliform mastitis . Quarter and systemic abnormalities occurred and milk production was reduced by approximately 50% at 12 hours after endotoxin . Flunixin meglumine therapy significantly (P less than or equal to 0.05) reduced rectal temperatures and quarter signs of inflammation and improved clinically graded depression when compared with these signs in saline solution-treated controls . Milk production and laboratory indicators of inflammation in milk were not significantly (P greater than 0.05) different for flunixin meglumine vs saline solution controls . The clinical response observed was consistent with the antipyretic, analgesic, and anti-inflammatory properties of flunixin meglumine.

Am J Physiol, 1986 Jun, 250(6 Pt 2), F1098 - 106
Endotoxemic acute renal failure in awake rats; Kikeri D et al.; The sequence of changes in renal function in endotoxemic acute renal failure (ARF) and the role of hypotension and systemic hemodynamics were evaluated in awake female Sprague-Dawley rats given an intravenous bolus of Escherichia coli endotoxin (20-40 mg/kg) . After endotoxin ARF was abrupt in onset as glomerular filtration rate (GFR) fell promptly and progressively by 53% within 3.5 h, whereas renal blood flow decreased by 42% and renal vascular resistance nearly doubled . Systemic hemodynamics remained stable, including mean arterial blood pressure, cardiac output, and total peripheral resistance . Early endotoxemic ARF was associated with oliguria and sodium retention, a finding consistent with intact tubular function . Three and one-half hours after endotoxin, however, fractional water and sodium excretion were significantly increased . Ultrastructural studies then demonstrated sequestration of phagocytic leukocytes and intracellular edema in the peritubular capillaries with normal glomeruli . The decrease in GFR was spontaneously reversible within 7-9 days . Extracellular fluid volume expansion with saline either before or 24 h after administration of endotoxin failed to prevent the decrease in GFR or to normalize renal function . The data suggest that endotoxin has direct renal effects . The endothelial cells may be the primary target of endotoxin in the kidney.

Proc Natl Acad Sci U S A, 1986 Jun, 83(12), 4423 - 7
Host participation in plasmid maintenance: dependence upon dnaA of replicons derived from P1 and F; Hansen EB et al.; Nonparticipation of the bacterial dnaA gene in plasmid replication has been assumed to be the general rule . In conditional dnaA mutants of Escherichia coli, only plasmid pSC101 has been shown to have a dnaA requirement . Experiments with dnaA null mutants of E . coli, presented here, show that dnaA plays a critical and direct role in the replication of miniplasmids derived from P1 and F as it does in the initiation of bacterial replication . Evidence is also presented for the existence of a dnaA-independent secondary replicon of P1 that is able to drive bacterial chromosome replication but is inadequate to support the maintenance of P1 as a plasmid in E . coli.

Proc Natl Acad Sci U S A, 1986 Jun, 83(12), 4134 - 7
lac Repressor blocks transcribing RNA polymerase and terminates transcription; Deuschle U et al.; Operator sequences are essential elements in many negatively controlled operons . By binding repressors, they prevent the formation of active complexes between RNA polymerase and promoters . Here we show that the Escherichia coli lac operator-repressor complex also efficiently interrupts ongoing transcription . This observation suggests a mechanism of action for operators located distal to promoter sequences.

Proc Natl Acad Sci U S A, 1986 Jun, 83(12), 4129 - 33
Translational regulation is responsible for growth-rate-dependent and stringent control of the synthesis of ribosomal proteins L11 and L1 in Escherichia coli; Cole JR et al.; The physiological importance of translational regulation in controlling the synthesis of ribosomal proteins from the L11 ribosomal protein operon was determined for the classical regulatory phenomena of growth rate dependence and stringent control . Translational regulation of the L11 operon by ribosomal protein L1, the L11 operon-specific translational repressor protein, was abolished by introducing a chromosomal mutation that causes an alteration of the site where L1 interacts with L11 operon mRNA . It was found that abolishing translational regulation of the L11 operon also abolished growth-rate-dependent regulation and stringent control of the L11 operon ribosomal proteins without affecting the normal regulation of ribosomal proteins from other operons that are not regulated by L1 . These results show that both growth-rate-dependent control and stringent control of ribosomal protein synthesis in the L11 operon are a direct result of translational regulation.

Proc Natl Acad Sci U S A, 1986 Jun, 83(12), 4109 - 13
Purification and properties of the mini-F plasmid-encoded E protein needed for autonomous replication control of the plasmid; Tokino T et al.; Mini-F plasmid encodes a protein, E protein, that is indispensable for its autonomous replication . We have constructed a plasmid that overproduces the E protein and have purified the protein to apparent homogeneity . Using nitrocellulose filter binding and nuclease digestion assays, we demonstrated that the E protein binds to three unique regions of the mini-F DNA sequence: the replication origin (ori2) and an incompatibility locus (incB), another incompatibility locus (incC), and the promoter for the E gene . These binding sites have a common 8-base-pair sequence . These findings suggest the direct role of the E protein in initiation of mini-F replication and copy number control . They are also in line with the in vivo evidence that the incompatibility phenotype caused by incB and incC DNA is due to titration of a factor(s) indispensable for replication and that the production of the E initiator protein of the mini-F plasmid is under autoregulatory control.

Proc Natl Acad Sci U S A, 1986 Jun, 83(11), 3654 - 8
The DNA loop model for ara repression: AraC protein occupies the proposed loop sites in vivo and repression-negative mutations lie in these same sites; Martin K et al.; Two sets of experiments have been performed to test the DNA loop model of repression of the araBAD operon of Escherichia coli . First, dimethyl sulfate methylation protection measurements on normally growing cells show that the AraC regulatory protein occupies the araI site in the presence and absence of the inducer arabinose . Similarly, the araO2 site is shown to be occupied by AraC protein in the presence and absence of arabinose; however, its occupancy by AraC is greatly reduced when araI and adjacent sequences are deleted . Thus, AraC protein binds to araO2 cooperatively with some other component of the ara system located at least 60 base pairs away . Second, the mutational analysis presented here shows that the DNA components required for repression of araBAD are araI, araO2, and perhaps the araBAD operon RNA polymerase binding site.

J Urol, 1986 Jun, 135(6), 1259 - 60
Percutaneous drainage of prostatic abscesses; Kadmon D et al.; Percutaneous catheter drainage of intra-abdominal abscesses currently is a well established technique . We report on 2 anuric patients on maintenance hemodialysis who presented with a prostatic abscess . We elected to use a transperineal, percutaneous drainage technique . Adequate drainage was documented by pelvic computerized tomography scans and followup confirmed satisfactory long-term results.

J Clin Invest, 1986 Jun, 77(6), 1734 - 9
Multiple biological activities of human recombinant interleukin 1; Dinarello CA et al.; Complementary DNA coding for human monocyte interleukin 1 (IL-1), pI 7 form, was expressed in Escherichia coli . During purification, IL-1 activity on murine T cells was associated with the recombinant protein . Homogeneous human recombinant IL-1 (hrIL-1) was tested in several assays to demonstrate the immunological and inflammatory properties attributed to this molecule . hrIL-1 induced proliferative responses in a cloned murine T cell in the presence of suboptimal concentrations of mitogen, whereas no effect was observed with hrIL-1 alone . At concentrations of 0.05 ng/ml, hrIL-1 doubled the response to mitogen (5 X 10(6) half maximal units/mg) . Human peripheral blood T cells depleted of adherent cells underwent a blastogenic response and released interleukin 2 in the presence of hrIL-1 and mitogen . hrIL-1 was a potent inflammatory agent by its ability to induce human dermal fibroblast prostaglandin E2 production in vitro and to produce monophasic (endogenous pyrogen) fever when injected into rabbits or endotoxin-resistant mice . These studies establish that the dominant pI 7 form of recombinant human IL-1 possesses immunological and inflammatory properties and acts on the central nervous system to produce fever.

J Bacteriol, 1986 Jun, 166(3), 878 - 83
Suppression of growth and protein secretion defects in Escherichia coli secA mutants by decreasing protein synthesis; Lee CA et al.; We devised a new selection for conditionally lethal suppressors of secA mutants . This selection allows the isolation of both temperature-sensitive and cold-sensitive suppressor mutations, whereas previous studies were limited to nonlethal or cold-sensitive suppressor mutations . Two temperature-sensitive suppressor mutations lie in genes required for protein synthesis: asnS, the gene for the asparaginyl-tRNA synthetase, and divE, which encodes the tRNASer1 . A previously characterized mutation in alaS, the gene for the alanyl-tRNA synthetase, suppresses the growth and secretion defects of a secA mutant . Although the primary effects of these suppressor mutations are different, it is likely that they cause suppression of secA mutations by altering the rate of protein synthesis, since the protein synthesis inhibitors, chloramphenicol and tetracycline, also suppress secA mutations . Chloramphenicol also suppresses the growth defect of certain other sec mutants . We postulate that the impaired secretory capacity of sec mutants can be offset by decreasing the rate of elongation of secreted proteins or by decreasing the total amount of secreted proteins per cell . The results indicate that our initial goal to identify cellular secretory components as suppressors of secA mutations might be difficult to achieve because of a high frequency of nonspecific suppressors that alter protein synthesis . Unexpectedly, the suppressor approach provides a direct genetic selection for mutants in protein synthesis.

J Bacteriol, 1986 Jun, 166(3), 872 - 7
Characterization of Escherichia coli mutants completely defective in synthesis of cyclopropane fatty acids; Grogan DW et al.; The synthesis of cyclopropane fatty acids (CFA) in bacteria represents a biochemically and physiologically unique membrane modification whose importance for the cell remains unknown, despite extensive study of a Cfa- mutant of Escherichia coli and of the cloned cfa gene . Recently we reported the isolation of new Cfa- mutants (D . W . Grogan and J . E . Cronan, Jr., Mol . Gen . Genet . 196:367-372, 1984) . Molecular-genetic and biochemical analysis indicated that these were null mutants of the E . coli cfa locus which were formed by inversions of a chromosomal segment . Isogenic Cfa+ and Cfa- strains were constructed from one such mutant and subjected to various stress conditions . In nearly all cases, both strains responded equally, but certain treatments, such as repeated freezing and thawing, favored the survival of Cfa+ strains over Cfa- strains . Though not essential, CFA thus appeared to play some beneficial role (or roles) in the bacterial cell.

J Bacteriol, 1986 Jun, 166(3), 866 - 71
Consequences of reduced intracellular coenzyme A content in Escherichia coli; Jackowski S et al.; Escherichia coli beta-alanine auxotrophs (panD2) were used to manipulate the specific cellular content of coenzyme A (CoA) and assess the associated physiological effects . Growth-limiting concentrations of CoA resulted in an increase in phospholipid/protein ratio in relA1 mutants, but not in their rel+ counterparts, indicating that protein biosynthesis was more severely affected by CoA deprivation than phospholipid biosynthesis . Acetyl-CoA was the dominant component (79.8%) of the CoA pool in cells exponentially growing in glucose-minimal medium, with significant concentrations of CoA (13.8%) and succinyl-CoA (5.9%) also detected . Malonyl-CoA was a minor species (0.5%), and the mixed disulfide of CoA and glutathione was not present . Acetyl-CoA was also the major constituent in cells depleted of CoA . On the other hand, succinyl-CoA was absent, suggesting that the protein synthesis defect may be due to the inability to generate sufficient quantities of precursors via the tricarboxylic acid cycle to support amino acid biosynthesis . Production of acyl carrier protein was controlled in part by the availability of CoA, and the lower concentration of acyl carrier protein in CoA-depleted cells was associated with a concomitant decrease in the saturated/unsaturated fatty acid ratio.

J Bacteriol, 1986 Jun, 166(3), 1141 - 3
New role for photoreversible pyrimidine dimers in induction of prototrophic mutations in excision-deficient Escherichia coli by UV light; Ruiz-Rubio M et al.; UV mutagenesis to His+ in certain recA441 lexA51 bacteria was not photoreversible, indicating that pyrimidine dimers are not target lesions . Photoreversibility was observed in recA+ lexA51 bacteria, showing that pyrimidine dimers are needed to activate the recA+ protein (unlike the recA441 protein) to perform a function in UV mutagenesis distinct from cleavage of the lexA repressor.

J Bacteriol, 1986 Jun, 166(3), 1007 - 12
Sequence of the flaA (cheC) locus of Escherichia coli and discovery of a new gene; Kuo SC et al.; The flaA (cheC) locus from Escherichia coli is important in controlling the rotational direction of flagella during chemotaxis . The locus was sequenced, and a site of transcriptional initiation was determined . Two reading frames, flaAI and flaAII, span the locus . flaAII corresponds to certain flaA and cheC mutations, and has some unusual features in the predicted secondary structure . flaAI, however, has not been identified previously, but a flaAI deletion, which produced a truncated FlaAI peptide in minicells, clearly identified the FlaAI protein.

Infect Immun, 1986 Jun, 52(3), 905 - 7
Protection of mice against lethal endotoxemia by a lipid A precursor; Proctor RA et al.; Lipid X, the major biosynthetic precursor of lipid A, has recently been described . Although lipid X is a mitogen and coagulates the Limulus amebocyte lysate, we found that it is not lethal for mice, even when given in large doses (2 X 10(6) micrograms/kg) . Furthermore, lipid X was found to give partial protection against a 100% lethal dose of endotoxin, even if the lipid X was given as late as 6 h after endotoxin challenge.

Exp Hematol, 1986 Jun, 14(5), 372 - 9
Aging and hematopoiesis . VI . Neutrophilia and other leukocyte changes in aged mice; Boggs D et al.; The concentration of blood leukocytes rose progressively as mice aged . All blood leukocytes increased, although a greater degree of increase was seen in neutrophils and monocytes than in lymphocytes and eosinophils . The total number of nucleated cells per marrow cavity of the humerus was also higher in aged than in young adult mice, the increase primarily reflecting peroxidase-positive cells . Both blood and marrow neutrophils of aged mice responded to perturbations induced by bleeding or by endotoxin injection in a manner qualitatively similar to that seen in young adults . When hematopoietic chimeras were produced by marrow transplantation, blood and marrow neutrophils were characteristic of the age of the recipient, not the cells; i.e., young mice kept a "young" neutrophil pattern and old mice kept an "old" neutrophil pattern when given marrow from either old or young mice . Colonies of granulocytes and/or monocyte-macrophages were grown from young marrow cells placed in plasma clots in Millipore chambers in the peritoneal cavity . The number of colonies was the same in young and in old mice, suggesting that long-range humoral stimulation of cell growth was similar in young and old . Thus, neutrophilia due to increased neutrophil production appears to be a normal part of the aging process in the mouse . The increase in neutrophil production may be due to a changing hematopoietic microenvironment.

Mutat Res, 1986 Jun, 161(1), 9 - 17
The relative cytotoxicity and mutagenicity of cyclobutane pyrimidine dimers and (6-4) photoproducts in Escherichia coli cells; Tang MS et al.; In order to calculate the relative cytotoxicity and mutagenicity of (5-6) cyclobutane pyrimidine dimers and (6-4) photoproducts, we have measured survival and mutation induction in UV-irradiated excision-deficient E . coli uvrA cells, with or without complete photoreactivation of the (5-6) dimers . Radioimmunoassays with specificity for (5-6) dimers or (6-4) photoproducts have shown that maximum photoreactivation eliminates all of the (5-6) dimers produced up to 10 Jm-2 254-nm light, while it has no effect on (6-4) photoproducts . These results were confirmed by measuring the frequency of T4 endonuclease V-sensitive sites . Based on the best fit equations for survival and mutation induction, we have found that the calculated cytotoxicity of (6-4) photoproducts is similar to that of (5-6) dimers; however, the former is much more mutagenic than the latter.

Mutat Res, 1986 Jun, 161(1), 1 - 7
Mutagenic and comutagenic action of 5'-deoxy-5'-(methylthio)adenosine; Naslund M et al.; 5'-Deoxy-5'-(methylthio)adenosine (MTA) alone and in combination with methionine was found to increase frequencies of background HGPRT mutation and SCE in V79 Chinese hamster cells . The same agents exert a comutagenic action on these effects as well as upon mutation induction in Escherichia coli stain AB1157 (but not in its non-adaptable derivative ada-6) following treatment with N-methyl-N-nitrosourea (MNU) . MTA plus methionine also enhanced the lethal action of MNU on hamster cells . The effects observed may tentatively be ascribed to hypomethylation due to inhibition of DNA methylase by MTA.

Proc Soc Exp Biol Med, 1986 Jun, 182(2), 263 - 71
The biological activity in vivo of recombinant murine interleukin 1 in the rat; Tocco-Bradley R et al.; The present study summarizes the biological response of rats to infusion with recombinant murine IL-1 (rIL-1) cloned in Escherichia coli . Thirty-seven male rats (135-180 g) were infused over a 6-hr period with either 0.008 M guanidine hydrochloride (the vehicle) or E . coli product (both groups are controls) or 1000, 3750, 7500, 15,000, or 37,500 LAF units/hr of rIL-1 . The controls and the group receiving 1000 LAF units/hr of rIL-1 did not exhibit a change in body temperature during the experiment . A mild fever was noted with 3750 LAF units/hr which became significantly elevated with 7500 and 15,000 LAF units/hr . At a dose of 37,500 LAF units/hr of rIL-1 (in 0.08 M guanidine hydrochloride) the rats became hypothermic and died . An equivalent dose of guanidine hydrochloride alone (0.08 M) was not fatally toxic although the rats did become hypothermic . Plasma zinc levels were significantly depressed and white blood cell count elevated at 6 hr postinfusion onset . Resting energy expenditure (REE) was significantly depressed during an infusion of 7500 and 15,000 LAF units/hr of rIL-1 despite a concurrent elevation in body temperature . Whole-body leucine kinetics were unchanged by infusion with rIL-1 . Plasma fibrinogen and serum haptoglobin and copper levels were not altered by rIL-1 . In conclusion, murine rIL-1 is similar to monocytic-derived IL-1 in that it produces a fever, hypozincemia, and leukocytosis; however, rIL-1 does not induce changes in protein metabolism.

Proc Natl Acad Sci U S A, 1986 Jun, 83(11), 3634 - 8
NMR studies of a complex of deuterated calmodulin with melittin; Seeholzer SH et al.; Completely deuterated calmodulin ({2H}CaM) has been prepared by expressing the chicken gene for CaM in Escherichia coli grown in 2H2O on a deuterated medium . The structural and dynamic properties of a 1:1 CaM/melittin (Mel) complex have been investigated by proton NMR . The spectrum of bound Mel is obtained directly from the spectrum of the {2H}CaM X Mel complex and is found to resemble strongly the spectrum of the helical species in methanol rather than that of the random coil species in water . The spectrum of bound CaM is obtained indirectly from the difference spectrum between {1H}CaM X Mel and {2H}CaM X Mel . Many changes are observed between free and bound CaM and they are distributed in both halves of the molecule, indicating that the binding of Mel affects the structure in both parts of the molecule . The rates of exchange of the amide protons of {2H}CaM with 2H2O were compared to those of {2H}CaM X Mel . The results showed that most, but not all, of the protons exchanged more slowly in the complex; after 40 hr, the residual peaks number 7 in CaM and greater than 20 in the complex . Again, changes in rates in CaM due to binding of Mel occurred in both halves of the molecule . The relative rates of amide proton exchange in CaM and its complex with Mel prove to be a sensitive criterion of differences in conformational stability and/or structure.

J Clin Oncol, 1986 Jun, 4(6), 883 - 7
Phase I-II study of recombinant alpha-2 interferon against advanced leukemia and lymphoma in children; Ochs J et al.; Alpha-2 interferon, produced in Escherichia coli using recombinant DNA techniques, was administered to 17 children with refractory acute lymphoblastic leukemia (ALL) in relapse, two children with TdT-positive, Philadelphia chromosome-positive chronic myelocytic leukemia (CML) in blast crisis, and one child with B cell (SIg+) non-Hodgkin's lymphoma (NHL) in a second extramedullary relapse . An initial 2-week intravenous (IV) phase of interferon was followed by a 3-month subcutaneous (SC) maintenance phase if patients had an objective response or disease stabilization without significant bleeding or infectious complications . When interferon dosages were escalated from 3 to 100 X 10(6) U/m2 in the first phase of therapy, there was rapid progression of disease in the first four patients treated, prompting a modification of the treatment plan . The last 16 patients enrolled received fixed dosages of interferon (ie, 10, 20, 30, and 50 X 10(6) U/m2 administered to four subjects each) . One child with T cell ALL had an 11-month complete remission; the patient with lymphoma had a dramatic but brief response; three others (one CML and two ALL) showed disease stabilization for 3 to 6 months with a definite oncolytic effect in two of the three patients . The remaining 15 patients had progressive disease within 2 months and were removed from the study . Acute toxicity included a flu-like syndrome in all patients, increased serum transaminase levels in five, seizures in three (two cases temporally related to fever and one to a thrombocytopenic subarachnoid hemorrhage), and prolonged activated partial thromboplastin times in seven . This phase I-II trial of recombinant alpha-2 interferon demonstrated definite activity without dose-limiting toxicity.

Biochimie, 1986 Jun, 68(6), 813 - 34
Photosensitized reactions of nucleic acids; Cadet J et al.; The main effects of near-ultraviolet and visible light on cellular DNA are reviewed with emphasis on base lesions, oligonucleotide single-strand breaks and DNA-protein cross-links . Model system photosensitization reactions of DNA are also discussed . This includes photodynamic effects, menadione-mediated photooxidation, photoionization of antibiotics, the photochemistry of 5-halogenopyrimidines and urocanic acid.

J Interferon Res, 1986 Jun, 6(3), 313 - 20
A sensitive radioimmunoprecipitation assay for the detection of antibody to recombinant human gamma-interferon: comparison to a bioassay neutralization test; Chen AB et al.; This report describes a specific radioimmunoprecipitation (RIP) assay for the detection of antibodies to recombinant DNA (rDNA) derived human gamma-interferon (rHuIFN-gamma) . The assay was shown not to detect antibodies to rHuIFN-alpha, rHuIFN-beta, human lymphotoxin, or E . coli proteins and was reproducible with intraassay and interassay coefficients of variation of 1.6 and 3%, respectively, for the log titer of a high positive control . Comparison of this assay with a standard bioassay for detection of neutralizing antibody (abrogation of the inhibitory effect of rHuIFN-gamma on EMC virus replication in A549 cells) demonstrated that the RIP assay was more sensitive for detection of HuIFN-gamma neutralizing monoclonal antibody . Nonneutralizing monoclonal antibody was detectable in the RIP assay but not in the bioassay neutralization test . Examination of polyclonal antisera (rabbit and monkey) that contained neutralizing antibodies also demonstrated the RIP system to be a more sensitive indicator of the presence of antibodies than the bioassay neutralization test . In preliminary studies of human samples (86 patients) from clinical trials using an assay precipitation system capable of detecting antibody of the IgG, IgM, IgA, and IgE classes, no antibody to rHuIFN-gamma was observed . These patients were also found negative for neutralizing antibody to rHuIFN-gamma.

Biochem Cell Biol, 1986 Jun, 64(6), 523 - 7
A detailed examination of the iodination of beta-galactosidase: stoichiometric inactivation by nonspecific iodination; Edwards LA et al.; The incorporation of 125I, using lactoperoxidase, and the subsequent inactivation of beta-galactosidase in the period when incorporation and inactivation were stoichiometric were investigated in detail . The high pressure liquid chromatographic (HPLC) radioactive profiles of the tryptic peptides of samples taken in the stoichiometric period showed that, although two labelled peptides predominated, there were other labelled peptides . The predominating peptides were shown to be the mono- and di-iodinated forms of the peptide containing Tyr-253 . This confirmed the result of an earlier study, but quantitation showed that this iodination accounted for only 15-18% of the total . To show that the other labelled peptides in the HPLC profiles were not merely oxidized or partially digested forms of the peptide containing Tyr-253, two experiments were carried out . In one of the experiments, two of the other labelled peptides were isolated and identified as iodinated forms of the peptide containing Tyr-285 (5-7% of the incorporation) . In the other experiment, four additional labelled fractions from the HPLC eluate were treated further with trypsin . No further digestion was observed and thus these peptides did not result from incomplete digestion of the sequence containing Tyr-253 . Overall, these results show that, although the incorporation of 125I was stoichiometric with inactivation, no single Tyr was responsible for the inactivation as was tentatively suggested previously . The competitive inhibitor isopropyl-beta-D-thiogalactopyranoside (IPTG) was effective in reducing the rates of inactivation of the enzyme and incorporation of 125I, but the same peptides were labelled in the presence of IPTG as in its absence.

Biochem Int, 1986 Jun, 12(6), 897 - 903
Lipoic acid provokes inhibition and aggregation of the maltose binding protein of Escherichia coli; Richarme G; Lipoic acid provokes aggregation of the monomeric maltose binding protein of Escherichia Coli into dimers and tetramers, and inhibits maltose binding . The sigmoidal shape of the curves showing the dependence of maltose binding versus lipoic acid concentration, and versus maltose concentration (in the presence of lipoic acid) suggests that the inhibition of the maltose binding protein by lipoic acid is a consequence of its aggregation . These results are discussed in relation to recent studies describing dimers of the maltose binding protein purified under certain conditions, and in relation to results suggesting an implication of lipoic acid in the binding protein-dependent transports.

Am J Vet Res, 1986 Jun, 47(6), 1373 - 7
Endotoxin-induced bovine mastitis: arachidonic acid metabolites in milk and plasma and effect of flunixin meglumine; Anderson KL et al.; Arachidonic acid metabolites (AAM) were measured in milk and plasma during the course of acute endotoxin-induced mastitis in 12 lactating cows . Mastitis was induced by intramammary challenge exposure with 10 micrograms of Escherichia coli (026:B6) endotoxin . Endotoxin was injected into the teat cistern via the teat canal of a single randomly selected rear quarter of each cow . Concentrations of prostaglandin (PG) F2 alpha and thromboxane (Tx) B2 in fat-free unextracted milk and of 15-keto-13,14-dihydro-PGF2 alpha in plasma were measured by radioimmunoassay . Total production of AAM in milk was determined by measuring quarter milk production . The AAM were compared in 6 cows administered flunixin meglumine (1.1 mg/kg of body weight) and in 6 cows administered saline solution . Concentrations of TxB2 in milk were significantly (P less than 0.001) increased during the early course of acute mastitis in endotoxin-treated quarters of cows not administered flunixin meglumine . Peak concentrations of TxB2 in milk occurred at 8 hours after endotoxin inoculation . Flunixin meglumine treatment produced significant (P less than 0.05) reductions in milk TxB2 and plasma 15-keto-13,14-dihydro-PGF2 alpha concentrations . Concentrations of PGF2 alpha in milk and total PGF2 alpha and TxB2 production per quarter per milking were not significantly influenced by endotoxin challenge or by flunixin meglumine treatment.

J Appl Physiol, 1986 Jun, 60(6), 2094 - 100
Endotoxemia causes increased lung tissue lipid peroxidation in unanesthetized sheep; Demling RH et al.; Our purpose was to determine whether lipid peroxidation of lung tissue, a reflection of O2 radical injury, occurs with endotoxin, and whether the degree of tissue change corresponds with the degree of increased protein permeability . Unanesthetized adult sheep with lung lymph fistulas were given Escherichia coli endotoxin at a dose of 2 micrograms/kg (n = 34) . Tissue lipid peroxidation was measured using the thiobarbituric acid assay for malondialdehyde (MDA) . The MDA content of lung tissue in nanomoles per gram increased from a control value of 48 +/- 8 to 98 +/- 18 at 5 h postendotoxin (2 micrograms/kg), whereas lung lymph protein transport (Cp), was increased 3- to 4-fold . The MDA content returned to base line with Cp by 24 h postendotoxin . Six sheep given endotoxin were pretreated with 12.5 mg/kg of ibuprofen, and six were infused with dimethylthiourea (DMTU) 0.75 g/kg . With ibuprofen, Cp was only increased 2.5- to 3-fold and MDA was increased to 69 +/- 15 nmol/g . With DMTU, the increase in Cp was comparable to that with endotoxin alone, as was the MDA of lung tissue with a value of 92 +/- 12 nmol/g . The correlation of tissue MDA with Cp in all animals was 0.83 . We conclude that lipid peroxidation occurs in lung tissue after a moderately severe endotoxin injury with the degree of change corresponding to the degree of increased Cp.

Biophys J, 1986 Jun, 49(6), 1205 - 14
Quasi-elastic light scattering from migrating chemotactic bands of Escherichia coli . III . Studies of band formation propagation and motility in oxygen and serine substrates; Wang PC et al.; A series of light scattering experiments have been performed to study both macroscopic aspects of band formation and propagation and microscopic motility parameters of Escherichia coli in the combined substrate gradients of oxygen and serine . From the band formation experiment the conclusion is drawn that a minimum threshold gradient of the substrate is required for bacteria to form a band . From the band propagation experiment in the serine substrate the motility coefficient mu and chemotactic coefficient delta are determined . A separate quasi-elastic scattering experiment has been made with a propagating band to obtain three microscopic motility parameters: mean twiddle time tau 1, mean run time tau 2, and mean run speed V2 . Finally, a scaling argument is made to connect the macroscopic parameters mu and delta with the microscopic parameters tau 1, tau 2, and V2, thus achieving a unified understanding of macroscopic and microscopic aspect of chemotaxis.

Proc Natl Acad Sci U S A, 1986 Jun, 83(12), 4384 - 8
Genetic definition of the translational operator of the threonine-tRNA ligase gene in Escherichia coli; Springer M et al.; The Escherichia coli gene thrS that codes for threonine-tRNA ligase (tRNAThr ligase, formerly threonine-tRNA synthetase, EC 6.1.1.3) has previously been shown to be negatively autoregulated at the level of translation . Here we describe the use of several thrS-lac gene fusions to isolate cis-acting regulatory mutations that increase the translation but not the transcription of the thrS gene . These mutations lead to a total loss of control of repression and derepression of thrS . DNA sequence analysis locates the mutations between 10 and 40 base pairs upstream of the translation initiation codon of thrS and more than 100 base pairs downstream of the transcription initiation site . The mRNA region where these mutations are located shares primary and secondary structure homologies with specific parts of several isoacceptor tRNAThr species . These findings suggest that the ligase regulates its translation by binding to its mRNA at a place that shares some homology with its natural substrate.

Sci Sin {B}, 1986 Jun, 29(6), 609 - 17
Coliphage M13 cloning system and ddXTP chain-termination method for DNA sequencing; Qi YP; Two DNA fragments of cytochrome b gene in yeast mitochondrial DNA were sequenced by Messing's M13 cloning system and Sanger's ddXTP chain-termination method . M13mp8 and M13mp9 serve as vectors for insert fragment . The recipient strain is E . coli JM103 for transfection . When the ratio of insert/vector was 3:1, high frequencies of recombination and positive recombination were obtained . The two fragments, which have 575 bp and 709 bp, were sequenced . To read more bases, the ratio of ddXTP/dXTP must be reduced . On one X-ray film of 80 x 17 cm 394 bp were read.

J Gen Microbiol, 1986 Jun, 132 ( Pt 6), 1739 - 52
The expression of the Escherichia coli lacZ gene in Streptomyces; King AA et al.; The Escherichia coli lacZ gene was stably introduced into phi C31-based phage cloning vectors in Streptomyces lividans . However, lacZ could not be stably introduced into S . lividans on the plasmid vectors pIJ702 or pIJ41 . Studies of the expression of lacZ in S . lividans and S . coelicolor were facilitated by the use of mutants and/or growth conditions in which endogenous beta-galactosidase activity was low or absent . Plaques and lysogens of phi C31::lacZ constructs involving transcriptional fusions to the pBR322 tet gene contained beta-galactosidase activity . Activities were markedly higher in S . lividans than in S . coelicolor . Insertion of the major transcriptional terminator of coliphage fd between the tet promoter and lacZ reduced lacZ expression more in lysogens than in lytically infected cultures, suggesting the existence of a phage-specified anti-termination function . Several phi C31 derivatives suitable for the construction of different kinds of transcriptional and translational fusions in Streptomyces were derived . The expression of lacZ in one transcriptional fusion vector (KC659) was increased both in plaques and in lysogens by the appropriately positioned insertion of a previously characterized promoter from the aph gene of S . fradiae . When phi C31 DNA fragments were inserted into KC659, at least one recombinant phage gave high beta-galactosidase activity in lytic infections, but not in lysogens . The potential usefulness of lacZ fusions in Streptomyces is discussed.

J Gen Microbiol, 1986 Jun, 132 ( Pt 6), 1729 - 37
Expression of the Aspergillus nidulans argB gene in Escherichia coli; Dmochowska A et al.; The Aspergillus nidulans argB gene coding for ornithine carbamoyltransferase (OTCase) is not expressed in Escherichia coli . However, E . coli OTCase-deficient strains transformed with plasmids carrying the argB gene from A . nidulans reverted to prototrophy at a high frequency . In these derivatives the argB gene became functional due to DNA rearrangements upstream of the coding sequence . Two types of rearrangement were characterized . One was identified as an insertion of IS2 . The second was a deletion that resulted in transcription of the argB gene from the TcR gene promoter and translation from a newly created ribosome-binding site formed at the junction between the A . nidulans and vector DNA sequences.

Bioorg Khim, 1986 Jun, 12(6), 836 - 8
{A new specific endodeoxyribonuclease from Escherichia coli RFL 72}; Kazlauskene R et al.; A restriction endonuclease Eco72I with a novel substrate specificity has been isolated from Escherichia coli strain RFL 72 . The enzyme recognizes (Formula: see text) hexanucleotide palindromic sequence and cleaves it, as indicated by the arrows, to produce blunt-ended fragments.

Biochimie, 1986 Jun, 68(6), 857 - 64
Photosensitized UVA light induction of the SOS response in Escherichia coli; Favre A et al.; Several of the factors controlling the extent of the ultraviolet light (and particularly of UVA 320 nm less than lambda less than 380 nm) induced SOS response in E . coli have been studied using a sfiA::lacZ fusion . The decreased 254 nm induced sfiA expression level triggered by a UVA-induced growth delay (Caldeira de Araujo A . & Favre A . (1986) Embo J., 5, 175-179), is closely mimicked by a transient chloramphenicol protein synthesis inhibition . In a nuvA mutant strain (lacking the growth delay effect), UVA light triggers a 30-40% lower SOS response at temperatures higher than 20 degrees C when illumination is performed under anaerobic conditions: endogenous oxygen-mediated photosensitized reactions appear to contribute to the SOS response . In contrast to the temperature independence of the sfiA induction levels obtained after 254 nm irradiation, the UVA induced response is 30-60% lower when the temperature (T) increases from a value lower than 10 degrees C to a value higher than 20 degrees C . This indicates that detoxifying enzymes play a role at T greater than 20 degrees C . Also the in vitro photooxydation of NADH to give NAD+ is described and its possible role in endogenous photosensibilizations discussed . To explain the contrasted mutagenic efficiencies of UVA light treatment when applied to cells in buffer at high fluences, and to growing cells at low fluence rates, we propose that intrinsically the UVA-induced DNA damages are able to trigger the SOS response (cyclobutyl pyrimidine dimers and some O2-dependent lesions) but also constitute premutagenic sites (some lesions leading to alkali-labile DNA breaks).(ABSTRACT TRUNCATED AT 250 WORDS)

Mol Gen Genet, 1986 Jun, 203(3), 496 - 504
Polar mobilization of the Escherichia coli chromosome by the ColE1 transfer origin; Boyd AC et al.; Mobilization of the plasmid ColE1 from cells containing a conjugative plasmid (such as F) requires the synthesis of ColE1 mob proteins, and the presence, in cis, of bom (basis of mobility), a region of ColE1 containing the origin of transfer (oriT) . The process of ColE1 transfer is thought to resemble that of the conjugative plasmid F, although the plasmids share little sequence homology . In F, conjugation is preceded by a strand-specific nicking event at oriT . The nicked strand is then conducted to the recipient with the 5' end leading . This is believed also to occur with ColE1, but direct biochemical confirmation has been precluded by its small size (6.65 kb) . To test this hypothesis genetically, a novel method, using a lambda dv-based vector, has been devised to site-specifically integrate bom (or any other cloned sequence) into the chromosome of Escherichia coli . When provided with suitable mobilizing plasmids, such strains were found to transfer the chromosome in a polar way . From these data, the orientation of transfer of ColE1 was deduced and shown to be analogous to F.

Mol Gen Genet, 1986 Jun, 203(3), 468 - 78
Construction and characterisation of a series of multi-copy promoter-probe plasmid vectors for Streptomyces using the aminoglycoside phosphotransferase gene from Tn5 as indicator; Ward JM et al.; Several versatile, multi-copy, promoter-probe plasmid vectors have been constructed that replicate in a wide range of Streptomyces species . Transcriptional activity is detected by the expression of a promoter-less aminoglycoside phosphotransferase gene (neo) derived from the transposon Tn5; expression of this gene confers kanamycin and neomycin resistance on Streptomyces lividans . An efficient transcriptional terminator from E . coli phage fd has been inserted upstream of the neo coding region to prevent significant transcriptional read-through from vector promoters . A translational stop codon situated downstream from the site(s) used for cloning and preceding and in frame with the ATG start codon of the neo gene ensures the detection of transcriptional, rather than translational, fusions . Relative promoter strengths can be determined by gradient plate assays of kanamycin resistance, by measuring the amount of aminoglycoside phosphotransferase produced or by estimating neo mRNA synthesised . The high copy number of the vectors facilitates the rapid isolation and characterisation of promoter-active fragments and convenient restriction sites are available for DNA sequencing and S1 mapping of cloned inserts . Some derivatives contain a polylinker that facilitates the insertion, excision and analysis of cloned fragments and which enhances the use of these plasmids as general cloning vectors.

J Bioenerg Biomembr, 1986 Jun, 18(3), 195 - 224
The pathway of electron transfer in the dimeric QH2: cytochrome c oxidoreductase; de Vries S; The experimental data currently available suggest that QH2:cytochrome c oxidoreductase functions according to a Q-cycle type of mechanism . The molecular weight of the enzyme in a natural or artificial phospholipid bilayer or in solution corresponds to that of a dimer . The pre-steady state kinetics of reduction of the prosthetic groups indicate that the enzyme is functionally dimeric . A double Q cycle is proposed, describing the pathway of electron transfer in the dimeric QH2:cytochrome c oxidoreductase . According to this scheme, the two monomeric halves of the enzyme act in a cooperative fashion to complete the catalytic cycle . It is proposed that high-potential cytochrome b-562 and low-potential cytochrome b-562 act cooperatively, viz . as a functional pair, in the antimycin-sensitive reduction of ubiquinone to ubiquinol.

Genetika, 1986 Jun, 22(6), 914 - 9
{Mutants of ColE1 plasmid with altered expression of the colicin E1 immunity gene}; Krashennikova LV et al.; Mutagenesis with Tn1 transposon was used to isolate mutants of ColE1 plasmid with inactivated gene responsible for immunity to colicin E1 . Cells containing such mutants synthesized active colicin E1 and were sensitive to its action . Spontaneous and UV-induced colicin synthesis was strongly changed in the mutants, as compared to the control . Mutations occurring outside the immunity gene, including those within the structural gene for colicin E1, could also affect the immunity gene expression.

EMBO J, 1986 Jun, 5(6), 1411 - 8
DNA gyrase complex with DNA: determinants for site-specific DNA breakage; Fisher LM et al.; DNA gyrase catalyses DNA supercoiling by making a transient double-stranded DNA break within its 120-150 bp binding site on DNA . Addition of the inhibitor oxolinic acid to the reaction followed by detergent traps a covalent enzyme-DNA intermediate inducing sequence-specific DNA cleavage and revealing potential sites of gyrase action on DNA . We have used site-directed mutagenesis to examine the interaction of Escherichia coli gyrase with its major cleavage site in plasmid pBR322 . Point mutations have been identified within a short region encompassing the site of DNA scission that reduce or abolish gyrase cleavage in vitro . Mapping of gyrase cleavage sites in vivo reveals that the pBR322 site has the same structure as seen in vitro and is similarly sensitive to specific point changes . The mutagenesis results demonstrate conclusively that a major determinant for gyrase cleavage resides at the break site itself and agree broadly with consensus sequence studies . The gyrase cleavage sequence alone is not a good substrate, however, and requires one or other arm of flanking DNA for efficient DNA breakage . These results are discussed in relation to the mechanism and structure of the gyrase complex.

EMBO J, 1986 Jun, 5(6), 1377 - 81
Synthetic lac operator mediates repression through lac repressor when introduced upstream and downstream from lac promoter; Besse M et al.; Plasmids were constructed which carry a synthetic lac operator at various distances from the lac promoter . They were tested in vivo for function in the presence and absence of lac repressor . We found significant repression when the lac operator is situated at the 3' end of the lac I gene or at the 5' end of the lac Z gene . When lac operators are inserted at both sites, we found a greater than 150-fold repression . The complex between lac repressor and DNA carrying these two lac operators is exceedingly stable in vitro suggesting that one tetrameric lac repressor may bind to both lac operators.

EMBO J, 1986 Jun, 5(6), 1351 - 8
Expression of p21 proteins in Escherichia coli and stereochemistry of the nucleotide-binding site; Tucker J et al.; v-Ha-ras encoded p21 protein (p21V), the cellular c-Ha-ras encoded protein (p21C) and its T24 mutant form p21T were produced in Escherichia coli under the control of the tac promoter . Large amounts of the authentic proteins in a soluble form can be extracted and purified without the use of denaturants or detergents . All three proteins are highly active in GDP binding, GTPase and, for p21V, autokinase activity . Inhibition of {3H}GDP binding to p21C by regio- and stereospecific phosphorothioate analogs of GDP and GTP was investigated to obtain a measure of the relative affinities of the three diphosphate and five triphosphate analogs of guanosine . p21 has a preference for the Sp isomers of GDP alpha S and GTP alpha S . It has low specificity for the Sp isomer of GTP beta S . Together with the data for GDP beta S and GTP gamma S these results are compared with those obtained for elongation factor (EF)Tu and transducin . This has enabled us to probe the structural relatedness of these proteins . We conclude that p21 seems to be more closely related to EF-Tu than to transducin.

J Vet Pharmacol Ther, 1986 Jun, 9(2), 192 - 7
Influence of induced disease states on the disposition kinetics of imidocarb in goats; Salam Abdullah A et al.; The influence of fever, induced by different agents, on the disposition kinetics of imidocarb was determined in goats . Escherichia coli endotoxin (0.2 microgram/kg), Trypanosoma evansi (10(7) in 1 ml sterile glucose citrate), and Infectious Bovine Rhinotracheitis virus (10(6.5)TCID50) were the agents administered to induce the febrile state . In control and febrile animals the two-compartment model was used to describe the disposition kinetics of the drug . Fever caused significant changes to occur in the apparent volume of distribution and the body (systemic) clearance of imidocarb, but the half-life remained unchanged . The statistical significance of the changes in these pharmacokinetic parameters varied with the etiology of the febrile state . E . coli endotoxin and IBR virus caused corresponding decreases in apparent volume of distribution and clearance of imidocarb, while fever induced with T . evansi caused highly significant increases in both pharmacokinetic parameters . It was concluded that the alterations in the disposition kinetics of imidocarb that occurred in the febrile goats were related not only to the febrile reaction per se but also to the pathophysiology of the disease condition.

DNA, 1986 Jun, 5(3), 235 - 8
Isolation and characterization of cDNA clones from mouse skeletal muscle actin mRNA; Leader DP et al.; The sequence corresponding to approximately 98% of mouse skeletal muscle actin mRNA was determined from cDNA clones isolated from a library of recombinants in pBR322 . One of these clones contains DNA corresponding to the complete amino acid coding region and a large part of the 5' and 3' untranslated regions of the mRNA . Comparison of the mouse coding region (conserved at the amino acid level) and noncoding regions with the corresponding regions of the rat skeletal muscle actin gene indicates that the noncoding regions have also been under selective pressure during evolution.

Proc Natl Acad Sci U S A, 1986 Jun, 83(11), 3885 - 9
A phage P1 function that stimulates homologous recombination of the Escherichia coli chromosome; Windle BE et al.; Recombination between two different defective lacZ genes in the Escherichia coli chromosome (lac- X lac- recombination) was stimulated 2- to 8-fold by prophage P1, depending on the nature of the phage c1 repressor . The P1 BamHI restriction fragment B8 in a lambda-P1:B8 hybrid phage, stimulated lac- X lac- recombination 90-fold in the absence of P1 repressor . A gene necessary for recombination enhancement, designated ref, was localized to one end of B8 . Ref expression from lambda-P1:B8 was repressed in trans by a P1 c+ prophage . Two P1 regulatory mutations, bof and lxc, derepressed prophage expression of ref and depressed a prophage function that complemented an E . coli mutant (ssb) deficient in the single-stranded DNA binding protein . Ref stimulation was dependent on preexisting E . coli recombination functions (RecA-RecBC and RecA-RecF) . However, other (phage and plasmid) recombination processes involving these functions were not stimulated . ref::Tn5 phages plated and formed lysogens normally . Thus ref appears to be an integral, but not essential, phage gene that stimulates recombination of the host chromosome specifically.

Proc Natl Acad Sci U S A, 1986 Jun, 83(11), 3664 - 8
Isolation and characterization of the gene encoding Drosophila DNA topoisomerase II; Nolan JM et al.; We have isolated the gene coding for the Drosophila type II DNA topoisomerase by immunochemically screening a Drosophila cDNA library constructed with a phage lambda expression vector, lambda gt11 . The identity of the cloned gene is confirmed by the analysis of an antigenic fusion protein produced in Escherichia coli and by the in vitro translation of its RNA . The gene is 5.1 kilobases in length, the expected size for a gene encoding topoisomerase II (Mr 170,000), and it is divided into five exons . By in situ hybridization to the polytene chromosomes from salivary glands, we have mapped it to chromosome 2L at 37D.

J Bacteriol, 1986 Jun, 166(3), 930 - 6
Escherichia coli K-12 envelope proteins specifically required for ferrienterobactin uptake; Pierce JR et al.; Escherichia coli genes specifically required for transport of iron by the siderophore enterobactin are designated fep . The studies reported here were initiated to identify and localize the fepB product . The plasmid pCP111, which consisted of an 11-kilobase E . coli DNA fragment containing fepB ligated to pACYC184, was constructed . The fepB gene was subcloned; in the process, complementation tests and Tn5 mutagenesis results provided evidence for the existence of a new fep gene, fepC . The order of the transport genes in the ent gene cluster is as follows: fepA fes entF fepC fepB entE . Minicell, maxicell, and in vitro DNA-directed protein synthesizing systems were used to identify the fepB and fepC products . The fepC polypeptide was 30,500 daltons in standard sodium dodecyl sulfate-polyacrylamide gels . The fepB gene was responsible for the appearance of three or four bands (their apparent molecular weights ranged from 31,500 to 36,500) in sodium dodecyl sulfate-polyacrylamide gels, depending on the gel system employed . The largest of these was tentatively designated proFepB, since it apparently had a leader sequence . Localization experiments showed that FepC was a membrane constituent and that mature FepB was present in the periplasm . An additional polypeptide (X) was also encoded by the bacterial DNA of pCP111, but its relationship to iron transport is unknown . The results indicated that ferrienterobactin uptake is mediated by a periplasmic transport system and that genes coding for outer membrane (fepA), periplasmic (fepB), and cytoplasmic membrane (fepC) components have now been identified.

J Bacteriol, 1986 Jun, 166(3), 901 - 4
Role of small subunit (IlvN polypeptide) of acetohydroxyacid synthase I from Escherichia coli K-12 in sensitivity of the enzyme to valine inhibition; Eoyang L et al.; Most of the coding sequence for the IlvN polypeptide subunit of acetohydroxyacid synthase I was deleted from the ilvB+ ilvN+ plasmid pTCN12 by in vitro methods . Several ilvB+ delta ilvN derivatives of pTCN12 were identified among transformants of a strain otherwise lacking any acetohydroxyacid synthase . Deletion derivatives produced an enzymatically active IlvB polypeptide, as shown by the Ilv+ phenotype of transformed cells and by immunologic and enzymatic assays . However, whereas the growth of pTCN12 transformants was sensitive to valine inhibition, growth of the ilvB+ delta ilvN transformants was relatively resistant . Moreover, in vitro analyses confirmed that both acetolactate and acetohydroxybutyrate synthesis in extracts of the ilvB+ delta ilvN transformants was resistant to valine inhibition, in comparison with that in extracts of pTCN12 transformants or with that catalyzed by purified acetohydroxyacid synthase I . The IlvN polypeptide had a minimal effect, if any, on IlvB polypeptide accumulation as measured by immunoprecipitation, but its absence resulted in a greater than 10-fold reduction in enzyme specific activity.

J Bacteriol, 1986 Jun, 166(3), 892 - 900
Mutations in the araC regulatory gene of Escherichia coli B/r that affect repressor and activator functions of AraC protein; Cass LG et al.; Mutations in the araC gene of Escherichia coli B/r were isolated which alter both activation of the araBAD operon expression and autoregulation . The mutations were isolated on an araC-containing plasmid by hydroxylamine mutagenesis of plasmid DNA . The mutant phenotype selected was the inability to autoregulate . The DNA sequence of 16 mutants was determined and found to consist of seven different missense mutations located within the distal third of the araC gene . Enzyme activities revealed that each araC mutation had altered both autoregulatory and activator functions of AraC protein . The mutational analysis presented in this paper suggests that both autoregulatory and activator functions are localized to the same determinants of the AraC protein and that the amino acid sequence within the carboxy-terminal region of AraC protein is important for site-specific DNA binding.

J Bacteriol, 1986 Jun, 166(3), 849 - 56
Suppressors of the secY24 mutation: identification and characterization of additional ssy genes in Escherichia coli; Shiba K et al.; We previously reported (Shiba et al., J . Bacteriol . 160:696-701, 1984) the isolation and characterization of the mutation (ssy) that suppresses the protein export defect due to the secY24(Ts) mutation and causes cold-sensitive growth of Escherichia coli . This report describes more systematic isolation of ssy mutations . Among temperature-resistant revertants of the secY24 mutant, 65 mutants were found to be cold sensitive . These cold-sensitive mutations have been classified by genetic mapping . Twenty-two mutations fell into the ssyA class previously described . The remaining mutations were located at five new loci: ssyB at 9.5 min between tsx and lon; ssyD around 3 min; ssyE at 72.5 min near secY; ssyF at 20.5 min within rpsA; and ssyG at 69.0 min near argG . Two predominant classes, ssyA and ssyB, are probably affected in protein synthesis at the elongation step, whereas the ssyF mutant contained an altered form of ribosomal protein S1 (the gene product of rpsA) . These cold-sensitive ssy mutations which suppress secY24 may define genes whose function is somehow involved in the secY-dependent protein secretion mechanism . However, the existence of multiple suppressor loci makes it unlikely that all of these genes specify additional components of the export machinery . A delicate balance may exist between the systems for synthesizing and exporting proteins.

J Bacteriol, 1986 Jun, 166(3), 756 - 62
Overproduction of FtsZ suppresses sensitivity of lon mutants to division inhibition; Lutkenhaus J et al.; Escherichia coli lon mutants are sensitive to UV light and other DNA-damaging agents . This sensitivity is due to the loss of the lon-encoded ATP-dependent proteolytic activity which results in increased stability of the cell division inhibitor SulA . Introduction of the multicopy plasmid pZAQ containing the ftsZ gene, which is known to increase the level of FtsZ, suppressed the sensitivity of lon mutants to the DNA-damaging agents UV and nitrofurantoin . Alterations of pZAQ which reduced the expression of ftsZ reduced the ability of this plasmid to suppress the UV sensitivity . Examination of the kinetics of cell division revealed that pZAQ did not suppress the transient filamentation seen after exposure to UV, but did suppress the long-term inhibition that is normally observed . lon strains carrying pZAQ could stably maintain a multicopy plasmid carrying sulA (pBS2), which cannot otherwise be introduced into lon mutants . In addition, the increased temperature sensitivity of lexA(Ts) strains containing pBS2 was suppressed by pZAQ . These results suggest that SulA inhibits cell division by inhibiting FtsZ and that this interaction is stoichiometric.

J Bacteriol, 1986 Jun, 166(3), 751 - 5
Cloning and characterization of the dcm locus of Escherichia coli K-12; Bhagwat AS et al.; The dcm locus of Escherichia coli K-12 has been shown to code for a methylase that methylates the second cytosine within the sequence 5'-CC(A/T)GG-3' . This sequence is also recognized by the EcoRII restriction-modification system coded by the E . coli plasmid N3 . The methylase within the EcoRII system methylates the same cytosine as the dcm protein . We have isolated, from a library of E . coli K-12 DNA, two overlapping clones that carry the dcm locus . We show that the two clones carry overlapping sequences that are present in a dcm+ strain, but are absent in a delta dcm strain . We also show that the cloned gene codes for a methylase, that it complements mutations in the EcoRII methylase, and that it protects EcoRII recognition sites from cleavage by the EcoRII endonuclease . We found no phage restriction activity associated with the dcm clones.

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