Cell, 1984 Jan, 36(1), 203 - 9 Maintenance of multicopy plasmid Clo DF13 in E . coli cells: evidence for site-specific recombination at parB; Hakkaart MJ et al.; Certain derivatives of copy-control mutants of plasmid Clo DF13 are not stably inherited in E . coli . These plasmids, predominantly present as multimeric DNA molecules, lack a specific region, designated parB . Here we present the nucleotide sequence of this parB region spanning 328 bp between 46% and 49% on the plasmid genome . parB is a noncoding region with extensive internal symmetry . A recA-independent, site-specific resolution process occurs between two intramolecular parB sites present in direct orientation relative to each other . A gene located in the direct vicinity of parB, gene L, is not essential for parB functioning . However, our genetic data indicate that transcription from the gene L-containing operon into parB is required . We conclude that the efficient maintenance of Clo DF13 cop derivatives containing parB is provided by resolution of mutimeric molecules . Because Clo DF13 wt and cop derivatives have a different response to the deletion of parB we postulate that two different recombination systems, a parB-dependent and a parB-independent system, operate in the efficient maintenance of Clo DF13 plasmids. Environ Mutagen, 1984, 6(1), 59 - 69 The genetic toxicology of metal compounds: I . Induction of lambda prophage in E coli WP2s(lambda); Rossman TG et al.; A number of metal compounds have been shown to be human carcinogens . Others, while not proven human carcinogens, are able to cause tumors in laboratory animals . Short-term bacterial assays for genotoxic effects have not been successful in predicting the carcinogenicity of metal compounds . We report here the ability of some metal compounds to cause the induction of lambda prophage in E coli WP2s(lambda) . By far the strongest inducing ability was observed with K2CrO4, followed by Pb(NO3)2 greater than MnCl2 greater than Ni(OOCCH3)2 greater than CrCl2 greater than NaWO4 greater than Na2MoO4 greater than KMnO4 . With the exception of chromate, long-term exposures in a narrow, subtoxic dose range were required in order to demonstrate phage induction . A new microtiter assay for lambda prophage induction, which incorporates these features, is described . This system also was able to detect very small amounts of organic carcinogens. Mol Gen Genet, 1984, 195(3), 391 - 401 Promotion, termination, and anti-termination in the rpsU-dnaG-rpoD macromolecular synthesis operon of E . coli K-12; Lupski JR et al.; The regulatory regions for the rpsU-dnaG-rpoD macromolecular synthesis operon have been fused to a structural gene whose product is readily assayed (the Cmr structural gene coding for chloramphenicol acetyl transferase, CAT) . The promoters (P1, P2, P3, Pa, Pb, Phs) for the macromolecular synthesis operon have different strengths as shown by their relative abilities to drive expression of the CAT gene . Promoter occlusion by P1 can be demonstrated within this operon . Regions 5kb upstream have a profound effect on operon gene expression . There is a thermoinducible promoter located within the dnaG structural gene . One of the macromolecular synthesis operon promoters is under lexA control . Although the operon structure allows coordinate expression of rpsU, dnaG and rpoD these additional features suggest that expression of individual genes can be independently regulated in response to altered growth conditions. Basic Life Sci, 1984, 27, 25 - 45 Structural elements of the 50 S subunit of E . coli ribosomes; May RP et al.; The large (50 S) subunit from E . coli ribosomes consists of 32 different proteins and two RNA molecules of different length . In an attempt to determine the three-dimensional arrangement of the proteins in the subunit, we are also interested in obtaining direct information on the shape of the proteins within the subunit . This is possible only with ribosomal subunits which, unlike natural protonated subunits, are homogeneous for neutrons . These homogeneous particles are produced by reconstituting 50 S particles from RNA and proteins isolated from bacteria grown at different levels of D2O in the culture medium, 76% D2O for RNA and 84% D2O for proteins . Model calculations and test experiments reveal that the pursued strategy allows direct determination of radii of gyration of 50 S components within the particle with reasonable precision . Data evaluation and interpretation are significantly facilitated by contrast variation of the reconstituted particles . The determination of protein shape parameters is only one aspect of the new strategy . The pair distance measurements are completely independent of its success . Data on radii of gyration of five ribosomal proteins in situ are reported: L1 (26 +/- 2 A), L2 (22 +/- 2 A), L3 (22 +/- 2 A), L4 (20 +/- 2 A), and L23 (13 +/- 2 A). Acta Chir Scand, 1984, 150(7), 535 - 9 Release of gastrointestinal peptides during E . coli endotoxinaemia; Revhaug A et al.; We have earlier reported an elevation of vasoactive intestinal polypeptide (VIP) plasma levels during endotoxinaemia in pigs (Revhaug et al., 1983) . In the present study the plasma levels of pancreatic polypeptide (PP), somatostatin, gastric inhibitory peptide (GIP) and insulin have been measured by sensitive Ria methods during E . coli endotoxin (Difco) shock in six anaesthetized pigs . The plasma samples were drawn from the vena porta, cava superior, internal jugular vein and the aorta . A significant increase in plasma levels of PP, somatostatin, and insulin was observed in this model . The increased plasma levels were higher in the portal vein than in the other sampling site . GIP did not present any change in this model. Biomed Biochim Acta, 1984, 43(11), K25 - 9 A well transformable E . coli tdk-strain--suitable for direct rescue of tk gene plasmids from mammalian cells; Kaehler R et al.; An E . coli C600 tdk strain which is suitable for the direct rescue of the eukaryotic HSV-1 tk gene by functional selection in E . coli is described . This E . coli tdk mutant failed to grow on fluorouracil selection plates and showed values for thymidine kinase activity smaller than in the tdk negative KY 895 strain . It is genetically well transformable, i.e . the transformation efficiency with plasmid DNA from pSK1 amounts to 5 X 10(6) or 3 X 10(8) depending on the transformation procedure applied. Mol Gen Genet, 1984, 196(3), 546 - 9 Mechanism of illegitimate recombination: common sites for recombination and cleavage mediated by E . coli DNA gyrase; Ikeda H et al.; Illegitimate recombination dependent on DNA gyrase in a cell-free system has previously been described . We have now mapped DNA gyrase cleavage sites in the vicinity of known recombination sites in pBR322 . Among five recombination sites examined, three were found to coincide with a DNA gyrase cleavage site . This result suggests that the cleavage of DNA by DNA gyrase has a central role in the recombination process. Mol Gen Genet, 1984, 196(3), 508 - 12 Native supercoiling of DNA: the effects of DNA gyrase and omega protein in E . coli; Mirkin SM et al.; This study deals with the effects of a temperature-sensitive (ts) mutation at the gene encoding the DNA gyrase B subunit (gyrBts) and a deletion of the top gene encoding the omega protein upon the superhelical density of the pAO3 plasmid in E . coli cells . The alteration of the DNA gyrase B subunit is shown to lead to a partial relaxation of DNA . On the other hand, the lack of omega protein due to the top gene deletion leads to an abnormally high degree of DNA supercoiling . In a double gyrBts delta top mutant the DNA supercoiling is greater than native at the permissive temperature, while under nonpermissive conditions a partial relaxation is observed . However, the pattern of DNA relaxation in the latter case is quite different from that in a single gyrBts mutant . The conclusion is that the native supercoiling of DNA in the cell is maintained through the counter-activities of DNA gyrase and the omega protein. Mol Gen Genet, 1984, 196(1), 129 - 34 Functional characterization of a cloned haemolysin determinant from E . coli of human origin, encoding information for the secretion of a 107K polypeptide; Mackman N et al.; We have recently reported the secretion of a 107K polypeptide by an E . coli strain containing the haemolytic plasmid pHly167 (Mackman and Holland 1984) . In this paper we show that a large number of haemolytic E . coli strains, apparently including both plasmid and chromosomally located haemolysin genes, secrete similar large molecular weight proteins . Partial purification of one haemolysin suggests that activity co-purifies with a 107K polypeptide . These results were confirmed by cloning the corresponding haemolysin determinant in the form of a recombinant plasmid pLG570, containing chromosomal DNA prepared from a human isolate of E . coli, LE2001 . Tn5 was used as a mutagen to localize the haemolysin genes to a 7-kilobase region of pLG570 . Structural and export functions were identified by assaying cell sonicates of non-haemolytic mutants . At least one structural gene was identified which coded for a 107K polypeptide . Insertions into this gene completely eliminated haemolysin activity and resulted in truncation of the 107K protein whereas insertions into the adjacent 4-kb region resulted in intracellular haemolytic activity . This internal haemolysin appeared to accumulate in the periplasm which suggests that factors encoded by the 4-kb region are involved in exporting the 107K polypeptide across the outer membrane. J Mol Appl Genet, 1984, 2(5), 485 - 96 Construction of E . coli expression plasmid libraries: localization of a pseudorabies virus glycoprotein gene; Robbins AK et al.; We describe the use of a combined cloning/expression protocol to identify a gene encoding a Pseudorabies virus (PRV) glycoprotein . Prior to this study, the genome locations of PRV glycoproteins had not been described . We first identified PRV glycoproteins using antibodies directed against PRV virions . Using affinity chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the PRV glycoproteins were separated and antibodies were made against them in rabbits . Using DNase digestion, PRV genomic DNA was fragmented into approximately 500-base-pair regions . These random fragments were inserted in an expression vector and the PRV DNA sequences were expressed as proteins fused to beta-galactosidase . The antibodies made in rabbits were then used as probes in a Western blot analysis to screen for the presence of PRV-specific glycoprotein sequences in these PRV-beta-galactosidase fusion proteins . Two expression plasmids were isolated that specified fusion proteins that reacted in the Western blot analysis with rabbit antibodies directed against a 74,000 molecular weight PRV glycoprotein . The PRV DNA sequences represented in the expression plasmids were mapped within a single BamHI fragment of PRV genomic DNA, but were not overlapping . PRV-beta-galactosidase fusion proteins produced by these two expression plasmids were used to inoculate rabbits . Antibodies produced against both fusion proteins recognized PRV-specific glycoproteins of 74,000 and 92,000 molecular weight . This protocol should have general application in localizing genes within large DNA virus genomes. Nucleic Acids Res, 1983 Dec 20, 11(24), 8809 - 16 Nucleotide sequence of the xanthine guanine phosphoribosyl transferase gene of E . coli; Richardson KK et al.; The nucleotide sequence of the gpt coding for the enzyme xanthine guanine phosphoribosyl transferase has been determined . The gene codes for a protein of molecular weight 16,950 . The construction of deletions in the gpt gene which can be used for the genetic analysis of mutations in the gpt gene, is described. Nucleic Acids Res, 1983 Dec 20, 11(24), 8691 - 701 Mechanistic study of E . coli DNA topoisomerase I: cleavage of oligonucleotides; Tse-Dinh YC et al.; E . coli DNA topoisomerase I catalyzes DNA topoisomerization by transiently breaking and rejoining single DNA strands (1) . When an enzyme-DNA incubation mixture is treated with alkaline or detergent, DNA strand cleavage occurs, and the enzyme becomes covalently linked to the 5'-phosphoryl end of the cleaved DNA (2) . Using oligonucleotides of defined length and sequence composition, this cleavage reaction is utilized to study the mechanism of E . coli DNA topoisomerase I . dA7 is the shortest oligonucleotide tested that can be cleaved by the enzyme . dT8 is the shortest oligo(dT) that can be cleaved . The site of cleavage in both cases is four nucleotides from the 3' end of the oligonucleotide . No cleavage can be observed for oligo(dC) and oligo(dG) of length up to eleven bases long . dC15 and dC16 are cleaved at one tenth or less the efficiency of oligo(dA) and oligo(dT) of comparable length. Nucleic Acids Res, 1983 Dec 10, 11(23), 8237 - 51 Nucleotide sequence and stability of the RNA component of RNase P from a temperature-sensitive mutant of E . coli; Sakamoto H et al.; The gene coding for the RNA component of RNase P was cloned from a temperature-sensitive mutant of Escherichia coli defective in RNase P activity (ts709) and its parental wild-type strain (4273), and the complete nucleotide sequences of the gene and its flanking regions were determined . The 5'- and 3'-terminal sequences of the RNA component were determined and mapped on the DNA sequence . The mutant gene has GC-to-AT substitutions at positions corresponding to 89 and 365 nucleotides downstream from the 5' terminus of the RNA sequence . Comparing to the wild-type RNA, the mutant RNA is less stable and rapidly degraded in vivo and in vitro. J Biochem (Tokyo), 1983 Dec, 94(6), 2079 - 81 Identification and cloning of the gene coding for lysophospholipase L2 of E . coli K-12; Homma H et al.; E . coli bearing hybrid plasmid pKOl (Oeda et al . (1981) Mol . Gen . Genet . 184, 191-199) expressed a large amount of lysophospholipase L2 activity . When a mutant which was defective in lysophospholipase L2 activity was transformed with plasmid pKOl, it overproduced lysophospholipase L2 activity . The gene responsible for the lysophospholipase L2 activity was designated as pld B . On the same hybrid plasmid another gene (pld A) coding for detergent-resistant phospholipase A (DR-phospholipase A) was also identified . These facts together with the results of a Pl transduction experiment revealed that the pld B gene must be between the pld A and met E genes on the E . coli chromosome. Mutat Res, 1983 Dec, 122(3-4), 293 - 8 UV-induced imbalance of the deoxyribonucleoside triphosphate pool in E . coli; Suzuki K et al.; The effect of UV irradiation on the intracellular DNA precursor pool in E . coli was investigated . UV irradiation of E . coli, followed by post-incubation for 1-1.5 h, altered the relative sizes of the deoxyribonucleoside triphosphate (dNTP) pool . The total amount of dNTPs increased: both dATP and dTTP increased several-fold, dCTP about twofold, while dGTP remained almost unchanged . In recA- and umuC- strains, which are defective in UV-induced mutagenesis, the pattern of nucleotide pool alterations was similar to that of wild-type strains. Int J Radiat Biol Relat Stud Phys Chem Med, 1983 Dec, 44(6), 607 - 13 Effect of gamma radiation on E . coli ribosomes, II . Efficiencies of inactivation by free radicals; Singh H et al.; Efficiencies for the inactivation of ribosomes by the hydrogen atom, the hydrated electron, the hydroxyl radical and radicals derived from isopropanol and tertiary-butanol, have been calculated . The relative inactivation efficiencies of the hydrogen atom, the hydrated electron and the hydroxyl radical are 4.2, 0.8 and 1, respectively . The oxygen enhancement ratio for damage by the hydroxyl radical is 2.5. Int J Radiat Biol Relat Stud Phys Chem Med, 1983 Dec, 44(6), 601 - 6 Effect of gamma radiation on E . coli ribosomes . I . Inactivation by hydrogen atoms, hydroxyl radicals, hydrated electrons and secondary radicals; Singh H et al.; E . coli ribosomes in aerated solutions were inactivated with a D37 dose of 144 Gy . In comparison, deaerated solutions bubbled with N2 or N2O showed a slight enhancement of the inactivation with D37 doses of 115 and 125 Gy, respectively . Isopropanol and tertiary-butanol provided partial protection . Results show that hydroxyl radicals, hydrated electrons and hydrogen atoms are involved in the inactivation process . Secondary radicals derived from the alcohols also inactivate ribosomes, with reduced efficiency. Cell, 1983 Dec, 35(2 Pt 1), 503 - 10 Transposable element IS50 improves growth rate of E . coli cells without transposition; Hartl DL et al.; Insertion sequence IS50R, which encodes the transposase and an inhibitor of transposition of the kanamycin-resistance transposon Tn5, increases the growth rate of E . coli K12 cells relative to that of their otherwise isogenic counterparts during competition in continuous culture . Most clones isolated from chemostats in which selection had occurred retain their original number of copies of IS50R at their original genomic locations, implying that the increased growth rate is not mediated by transposition . The selective advantage due to a single IS50R element averages about 5% per hour . When the number of copies of IS50R is small, the growth-rate advantage is approximately proportional to the number of copies of IS50R . These results imply that IS50R has effects on cells that are independent of both position and transposition and may be important in the initial selection leading to the appearance of such elements in bacterial populations. Pediatr Res, 1983 Dec, 17(12), 1017 - 20 The effect of E . coli endotoxin on the developing rat liver . II . Immunohistochemical localization of alpha-fetoprotein in rat liver multinucleated giant cells; Andres JM et al.; Immunoperoxidase histochemical staining techniques have been used previously to localize alpha-fetoprotein (AFP) in hepatocytes from rat and human liver tissues . In this study, we extended these observations by examining liver tissues from rat fetuses exposed to E . coli endotoxin in order to document the presence of AFP in hepatic multinucleated giant cells . Liver sections were examined under light microscopy after incubation with purified antibody-peroxidase conjugates and histochemical stains . These sections showed a positive reaction for AFP in giant cells and hepatocytes that appeared as granular, brown intracytoplasmic deposits in cells throughout the hepatic lobule . Furthermore, a direct correlation was found between the number of positively stained giant cells and the serum concentration of AFP . The findings demonstrated that AFP distribution in endotoxin-induced liver injury is confined to isolated hepatocytes and multinucleated giant cells . This observation provides evidence that the origin of the giant cell in toxin-exposed fetal rat liver may be the hepatocyte. Boll Ist Sieroter Milan, 1983 Nov 30, 62(5), 420 - 5 A study on the erythrocyte structures involved in the interaction with mannose-resistant E . coli adhesins; Chiarini F et al.; Research has been carried out on the role of different chemical groups present on human group A erythrocyte membranes and involved in the interaction with mannose-resistant (MR) adhesins of a uropathogenic E . coli strain . For this purpose several enzymes specifically acting on protein, lipid and carbohydrate components have been used to modify the erythrocyte surface structures and the effect produced by these treatments on the agglutinability of red blood cells by E . coli has been studied . Moreover different biological compounds have been tested as possible competitive inhibitors for erythrocyte receptors involved in MR agglutination by E . coli. Radiat Res, 1983 Nov, 96(2), 309 - 21 Measurements of DNA double-strand break yields in E . coli after rapid irradiation and cell inactivation: the effects of inactivation technique and anoxic radiosensitizers; Tilby MJ et al.; A study has been made of methods for rapidly inactivating cells of E . coli at neutral pH to prevent enzymatic, chemical, or physical modification to DNA damaged by irradiation . The radiation was delivered in a fraction of a second using an electron accelerator . Cell inactivation was with ethanol or a solution (CSE) containing detergent, EDTA, and chloroform . It was found that DNA could be released from irradiated and inactivated cells simply by incubating them with the protease Pronase, and this DNA appeared to be in a form suitable for centrifugational analysis in neutral sucrose gradients . When cells were irradiated in the presence of oxygen, inactivation with ethanol gave a radiation-induced double-strand break (dsb) yield 1.8-fold higher than when inactivation was with CSE . Possible explanations of this are discussed . Using CSE inactivation, an oxygen enhancement ratio for dsb formation of 4.3 was observed and yields of dsb per Gray were in good agreement with results from other laboratories . At concentrations which enhanced cell killing 2.6-fold the "electron-affinic" type anoxic radiosensitizer misonidazole enhanced dsb formation 2.0-fold, whereas the nitroxyl free radical anoxic radiosensitizer norpseudopelletierine-N-oxyl had no significant effect on dsb yield although there was a possible slight enhancement at the higher doses used. Radiat Res, 1983 Nov, 96(2), 275 - 83 Radiation sensitization of E . coli B/r by nitrous oxide; Ewing D; E . coli B/r have been used to study radiation sensitization by nitrous oxide (N2O) . Cells suspended in Sorensen's phosphate buffer show a large amount of sensitization by N2O (relative to the response in 100% N2) . Cells in McIlvaine's phosphate-citric acid buffer, however, show no sensitization by N2O . Sensitization in Sorensen's buffer can be prevented by hydroxyl radical (.OH) removal or by catalase . Chemical assays for the amounts of H2O2 formed under various conditions provide the basis for the conclusion that the high concentration of the citrate ion in McIlvaine's buffer does not allow the build-up of H2O2 . Sensitization by N2O requires that both H2O2 and OH radicals be present. Int J Radiat Biol Relat Stud Phys Chem Med, 1983 Nov, 44(5), 519 - 22 The oxygen effect in E . coli WP2: the role of the photoreactivatable component in modification of the oxygen effect in E . coli WP2 uvrA6; Selyova NG et al.; The role of photo-products induced by secondary U.V . which accompanies high-energy gamma-radiations has been studied in bacteria in the presence and absence of oxygen . The photodamage makes an essential contribution to the lethal effect of ionizing radiation in U.V.-sensitive strains in anoxia and thus can influence the value of the oxygen effect . When the secondary U.V.-contribution was minimized (irradiation by X-rays or gamma-rays in the presence of 1 per cent nigrosine) the oxygen enhancement ratio was shown to increase and approximate to that for E . coli WP2 cells. Immunology, 1983 Nov, 50(3), 497 - 502 Opsonization of various capsular (K) E . coli by the alternative complement pathway; Stevens P et al.; A virulence factor of E . coli K-1 is its capacity to avoid opsonization via the alternative complement pathway (ACP) . Since it is not known if E . coli with other capsular (K) antigens have similar properties we examined various capsular E . coli for opsonization by the ACP . To assess opsonization we used whole blood luminol-dependent chemiluminescence (CL) and the magnesium salt of ethyleneglycol tetraacetic acid to block the classical pathway . E . coli K-types 6, 7, 27, 30, 42, 53, 57 and 75 were effectively opsonized via the ACP (greater than 65% of CL obtained with unchelated normal serum) . K types 2 and 13 were opsonized by the ACP in both the high range greater than 65% and intermediate range 36-65% . Only K-types 1, 3, 5, 12 and 92 were poorly opsonized (less than 35%) by the ACP . The data demonstrate that most E . coli K-types were opsonized via the ACP . The poor opsonization of E . coli K3, 5, 12 and 92 by the ACP may be virulence factor for these bacteria. Carcinogenesis, 1983 Nov, 4(11), 1379 - 84 Differences in transformation of repair-deficient mutants of E . coli with BPDE- or chlorozotocin-modified plasmid DNA; Vadi HV; Plasmid pBR322 was alkylated with either chlorozotocin or with r-7,t-8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydrobenzo-{a}pyrene (BPDE) before it was transformed into various strains of Escherichia coli . Plasmid survival was determined as ability to convert the bacteria to tetracycline and ampicillin resistance . Increased levels of alkylation caused a decrease in transforming activity in all strains studied . This decrease did not seem to be a result of alkylation induced strand scission, but rather some other biochemical or conformational change induced by the alkylating event . In E . coli AB1157 transformation was decreased by 50% with 6 alkylations/plasmid molecule for BPDE and 8-9 alkylations for chlorozotocin . At these levels of alkylation the loss in supercoiled DNA due to strand scission was less than 5% . Alkylated pBR322 was also transformed into repair-deficient strains of E . coli . In strain JC2924 (recA6) the survival of both BPDE- and chlorozotocin-modified DNA was similar to survival in the repair proficient strain AB1157, which would indicate that postreplicational repair of BPDE- or chlorozotocin-modified plasmid DNA was not significant under these conditions . Chlorozotocin-modified pBR322 did not seem to be repaired by the bacterial uvr-endonucleases as determined by plasmid survival in strains AB1884 (uvrC34), AB1885 (uvrB5) and AB1886 (uvrA6) . With BPDE-alkylated plasmid DNA the results were strikingly different . Strains AB1884 and AB1886 were more sensitive to BPDE modified DNA than the wild type strain AB1157 . Strain AB1885 was similar to AB1157 in sensitivity to BPDE-alkylated plasmid . These findings suggest that bacterial uvr-endonucleases may be able to recognize and repair BPDE-alkylated pBR322 . The role of the uvrB protein in repair of alkylated DNA needs to be further investigated. Cell, 1983 Nov, 35(1), 265 - 73 RNA polymerase interactions with the upstream region of the E . coli tyrT promoter; Travers AA et al.; The rate of in vivo transcription from the E . coli tRNA and rRNA promoters depends on both cellular growth rate and aminoacid availability . To investigate the molecular mechanisms involved we determined the extent of interaction of RNA polymerase with the promoter of the tyrT stable RNA gene . We show that the enzyme can protect from DNAase I digestion a region of at least 85 bp of the wild-type tyrT promoter and only approximately 62 bp of the lacUV5 mRNA promoter, the protected region extending on the antisense strand to approximately 65 and 42 bp respectively upstream of the transcription startpoint . A mutant tyrT promoter, tyrTp27, is protected more extensively, RNA polymerase interactions extending to at least approximately -130 . We propose that these upstream interactions of RNA polymerase perform two functions; activating initiation by polymerase bound at the primary binding site and increasing the concentration of polymerase in the vicinity of the tyrT promoter, thus allowing a high rate of maximal expression and enabling the promoter to be regulated over a wide range of activity. Nucleic Acids Res, 1983 Oct 11, 11(19), 6723 - 32 Nucleotide sequence of metF, the E . coli structural gene for 5-10 methylene tetrahydrofolate reductase and of its control region; Saint-Girons I et al.; The nucleotide sequence of the E.coli metF gene (888 nucleotides), coding for 5-10 methylene tetrahydrofolate reductase, has been determined . The metF gene product was identified in maxicells and found to be a protein of subunit molecular weight 33,000, in agreement with the size of the coding region . The starting point for metF transcription was determined by S1 nuclease mapping . No structural evidence was found for an attenuation mechanism regulating the independent metF transcriptional unit . Comparison of the regulatory region preceding the metF structural gene with the 5' flanking region of the metBL operon shows some homology spanning 24 nucleotides . These homologous sequences could be operator structures belonging to the two transcriptional units, metF and metBL, and recognized by the same regulatory protein. Nucleic Acids Res, 1983 Oct 11, 11(19), 6667 - 78 Identification and amplification of the E . coli phr gene product; Sancar GB et al.; We have constructed a series of multicopy plasmids that complement mutations in the phr gene of Escherichia coli . By subcloning into a tac plasmid vector we obtained a phr plasmid that upon induction overproduces two proteins of Mr's 49,000 and 20,000 . Tn1000 insertions into the phr gene caused the disappearance of the 49,000 dalton protein, thus demonstrating this protein to be the phr gene product, DNA photolyase . The photolyase encoded by the phr gene makes up about 15% of total cellular proteins after induction of cells carrying a tac-phr plasmid . This protein binds specifically to UV (254 nm) irradiated DNA and upon exposure to near UV (300-500 nm) illumination repairs the UV damage and dissociates from DNA. Nucleic Acids Res, 1983 Oct 11, 11(19), 6597 - 609 Cloning and expression of a porcine prorelaxin gene in E . coli; Stewart AG et al.; A porcine prorelaxin gene has been constructed partly by synthetic means and partly from its natural messenger RNA . A gene coding for the 32 N-terminal amino acids including a chain initiator methionine codon (B gene) was synthesised and inserted in a plasmid at a site downstream from a tryptophan promoter in such a way that its expression is under the control of the trp promoter . DNA corresponding to the rest of the prorelaxin was prepared using reverse transcriptase extension of a primer complementary to relaxin mRNA and joined at a suitable restriction site to the B gene . Transformation of E . coli with this plasmid followed by suitable induction resulted in the synthesis of a new protein identified as prorelaxin by its size and its antigenic similarity to relaxin. J Biomol Struct Dyn, 1983 Oct, 1(2), 383 - 94 Physical studies on a nucleoprotein from the ribosome of E . coli; Moore PB et al.; Bacterial 5S RNA and its cognate proteins constitute an attractive system to study nucleoprotein interactions . The molecular weights of the components involved are modest and they can be prepared in the quantities necessary to permit the application of material-intensive techniques like NMR and X-ray crystallography . 5S RNA is being examined by proton NMR at 500 MHz with special attention paid to the downfield NH proton region . A substantial number of assignments can be suggested in this region based on nuclear Overhauser results . The binding of protein L25 (E . coli) to the RNA gives rise to a highly characteristic set of perturbations in the spectrum of the RNA . The data suggest a localized and assignable alteration in RNA structure upon formation of the complex . In addition we have grown large crystals of RNAs related to 5S RNA and their complexes with a cognate protein . The properties of these crystals and the progress made in analyzing their structure are discussed. Biokhimiia, 1983 Oct, 48(10), 1752 - 4 {The ability of E . coli DNA methylases to modify denatured DNA}; Bur'ianov IaI et al.; Adenine and cytosine DNA methylases from different strains of E . coli are able to methylate denaturated and single-stranded DNAs. Int J Radiat Biol Relat Stud Phys Chem Med, 1983 Oct, 44(4), 399 - 404 Modification of radiation response of E . coli B/r cells by phenothiazines; Maniar HS et al.; Promethazine and trimeprazine sensitized anoxic E . coli B/r cells to 60Co gamma-rays, but both drugs showed a radioprotective effect under euoxic conditions . Their radiosensitizing effect was found to be due to the reaction of radiolytically induced hydroxyl radicals with the sensitizers . The radioprotective effect of these drugs is attributed to changes in the membrane structure conducive with chemical repair of the damaged sites in the gel region of the cellular membrane by intracellular sulphydryl compounds . Pre-irradiation depletion of sulphydryls from E . coli B/r by treatment with N-ethyl maleimide abolished the radio-protective effect of these drugs under euoxic conditions. Mutat Res, 1983 Oct, 112(5), 261 - 73 Expression of eight unrelated Muc+ plasmids in eleven DNA repair-deficient E . coli strains; Upton C et al.; 23 plasmids from different incompatibility groups were tested for their ability to increase post-UV survival and UV-induced reversion to Arg+ in Escherichia coli strain AB1157 argE3 8 plasmids increased mutagenesis, of which 7 increased UV resistance . The exception, plasmid R391, sensitized AB1157 to UV . All 8 plasmids were absolutely dependent upon host recA+ and lexA+ genotypes for expression of these functions, but were independent of uvrA+, uvrB+, umuC+, recF+, polA+, uvrD+ or recL+ . E . coli KMBL91 uvrE was sensitized to UV by R391, but protected by only 3 plasmids . All 8 plasmids restored mutation frequency in the non-mutable TK501 uvrB umuC strain to levels found in the JC3890 uvrB umuC+ parent strain . R391 sensitized TK501 to UV, but all other plasmids increased survival in the strain by over 1000-fold to levels found in the JC3890 uvrB umuC+ R+ strains . Plasmid R391 reduced the UV-protecting effect of R46 when both were present in strain TK501 . Mutation frequencies were higher in TK501 (R46) than in TK501 (R391); in TK501 (R46/R391) they were slightly lower than in TK501 (R46). Genetics, 1983 Oct, 105(2), 265 - 80 The pleiotropic ts15 mutation of E . coli is an IS1 insertion in the rho structural gene; Gulletta E et al.; Rho protein regulates transcription termination in E . coli . Some of the temperature-sensitive mutants defective in Rho protein, e.g., ts15, show remarkable pleiotropic phenotypes . The ts mutations map between the ilv and cya loci on the E . coli chromosome . We have cloned the gene that restores the wild-type phenotypes of these mutants . Genetic and biochemical characterizations have shown that the cloned DNA segment carries the structural gene for the Rho polypeptide . Analysis of the rhots15 mutation has revealed the presence of an IS1 insertion in the carboxy terminal segment of the rho cistron, thereby truncating the 52-kilodalton (kd) Rho polypeptide to a 50-kd size and also making it thermolabile . This provides an example of how an IS1 insertion mutation can cause a TS phenotype . We have also shown that the multiple phenotypes of the mutant cell, including the temperature sensitivity, are caused by a single mutation (rhots15::IS1) in the rho structural gene . How a rho structural gene mutation may cause such pleiotropy is discussed. Cell, 1983 Oct, 34(3), 931 - 9 Synapsis and the formation of paranemic joints by E . coli RecA protein; Bianchi M et al.; E . coli RecA protein promotes the homologous pairing of a single strand with duplex DNA even when certain features of the substrates, such as circularity, prohibit the true intertwining of the newly paired strands . The formation of such nonintertwined or paranemic joints does not require superhelicity, and indeed can occur with relaxed closed circular DNA . E . coli topoisomerase I can intertwine the incoming single strand in the paranemic joint with its complement, thereby topologically linking single-stranded DNA to all of the duplex molecules in the reaction mixture . The efficiency of formation of paranemic joints, the time course, and estimates of their length, all suggest that they represent true synaptic intermediates in the pairing reaction promoted by RecA protein. Nucleic Acids Res, 1983 Sep 24, 11(18), 6157 - 66 Nucleotide sequence of the promoter region of the E . coli lysC gene; Cassan M et al.; The regulatory region of the lysC gene (that encodes the lysine-sensitive aspartokinase of Escherichia coli) has been identified and purified by the use of lysC-lacZ fusions . Its regulatory sequence has been determined . No signals similar to those described in the case of an attenuation mechanism could be found in the long leader sequence existing between the starts of transcription and of translation. Nucleic Acids Res, 1983 Sep 24, 11(18), 6531 - 9 Flexibility in RNA priming of Okazaki pieces at the E . coli replication fork; Thomas KR et al.; We present results which suggest considerable flexibility in the RNA priming of Okazaki pieces at the E . coli replication fork . Using film lysates on cellophane discs, we have identified RNA at the 5' ends of Okazaki pieces . All four ribonucleotides are found to be present at the RNA-DNA junction if all four ribonucleoside triphosphates are used . However, if only ATP, or ATP and GTP are used, then only 2' (3')AMP, or 2' (3')AMP and 2' (3')GMP are found at the RNA-DNA junction . A nearest neighbor analysis of RNA associated with Okazaki pieces using alpha 32P-CTP as a probe shows a similar dependence of nearest neighbor composition on the ribonucleoside triphosphate composition of the incubation mixture . Thus, the nucleotide composition of the RNA primers at the ends of Okazaki pieces varies as a function of the ribonucleoside triphosphates available. Biochem Biophys Res Commun, 1983 Sep 15, 115(2), 567 - 76 Mechanism of o-phenanthroline mediated inhibition of E . coli DNA polymerase I : formation of template-primer-metal-phenanthroline complexes with resultant loss of catalytic activity; Abraham KI et al.; Inhibition of E . coli DNA polymerase I activity by 1,10 phenanthroline in the absence of reducing agents requires a high concentration of inhibitor (1-10 mM) depending upon the template primer used to direct the synthesis . We find that o-phenanthroline, unlike its non-chelating analogue, forms a divalent cation mediated complex with template-primers . Enzyme bound to such complexes is unable to catalyse either polymerization or nuclease functions. Nucleic Acids Res, 1983 Sep 10, 11(17), 5855 - 64 Essential structure of E . coli promoter: effect of spacer length between the two consensus sequences on promoter function; Aoyama T et al.; A promoter with the consensus sequence(TTGACA and TATAAT) at -35 and -10 regions was constructed, and the distance between the two consensus sequences was independently altered at two restriction sites . In an in vitro transcription system, a maximum activity was observed at the spacer length of 17 base-pairs regardless of the sites of space adjustment . Evidence was also provided that the start point of transcription was fixed by the consensus sequence at the -10 region. Bioorg Khim, 1983 Sep, 9(9), 1285 - 9 {Plasmid vectors with a semi-synthetic beta-galactosidase gene of E . coli}; Korobko VG et al.; A partial synthesis of a structural gene for beta-galactosidase and construction of a series of pLZ plasmids for quantitative study of E . coli promoters are reported . The gene was assembled of two short synthetic DNAs and of a 3000 bp long EcoRI fragment (comprising the lacZ sequence 16-3013) isolated from plasmid p198/1 of B . Gronenborn . Among the plasmids constructed, pLZ4 is a promoter-probe vector that contains the semi-synthetic gene fused with a synthetic Shine-Dalgarno sequence and preceded by unique EcoRI and KpnI cleavage sites . On cloning a promoter into these sites, its signal strength in vivo could easily be measured by assaying beta-galactosidase activity . The use of pLZ4 vector was demonstrated by quantifying the effect of T7 early promoters A1 and A2, the latter being found 4,5 times more active under the conditions employed. Biofizika, 1983 Sep-Oct, 28(5), 835 - 7 {Water exchange in E . coli cells in the presence of D20 using IR-spectroscopy}; Bagriantseva EA et al.; Time dependence of isotopic composition of intracellular water was studied by the method of infrared spectroscopy after D2O adding into outer medium of E . coli, M-17 . These changes were shown to be periodic with the time of repeating of extremums near two min . Such oscillations are connected with periodic processes which take place in the cell itself under the influence of D2O and characterized a degree of viability of organisms. Thromb Haemost, 1983 Aug 30, 50(2), 557 - 9 Contribution of adrenaline to the fibrinolytic activity evoked by E . coli endotoxin in the rat; Fracasso JF et al.; Intravenous injection of E . coli endotoxin (ETX), of adrenaline (AD) or of carbamylcholine (CBCH), caused fibrinolytic activity (FA), directly detectable on plasminogen-rich fibrin plates, to appear in the plasma of the rat . Adrenodemedullation abolished responses to ETX or CBCH, but enhanced those to AD . Rats given ETX exhibited marked hypotension, followed by a compensatory phase of normotension abolished by adrenodemedullation and significantly attenuated by phenoxybenzamine, an alpha-adrenergic blocking agent which however failed to block FA caused by either ETX or AD . Aspirin, but not indomethacin, inhibited FA evoked by ETX, AD or CBCH . These results suggest that FA evoked by ETX in the rat is caused by AD released from the adrenal gland and does involve the fatty acid cyclooxygenase system. Nucleic Acids Res, 1983 Aug 25, 11(16), 5661 - 9 Formation of genes coding for hybrid proteins by recombination between related, cloned genes in E . coli; Weber H et al.; We describe a method for the formation of hybrid genes by in vivo recombination between two genes with partial sequence homology . DNA structures consisting of plasmid vector sequences, flanked by the alpha 2 interferon gene on the one side and a portion of the alpha 1 interferon gene (homology about 80%) on the other, were transfected into E . coli SK1592 . Appropriate resistance markers allowed the isolation of colonies containing circular plasmids which arose by in vivo recombination between the partly homologous interferon gene sequences . Eleven different recombinant genes were identified, six of which encoded new hybrid interferons not easily accessible by recombinant DNA techniques. FEBS Lett, 1983 Aug 22, 160(1-2), 239 - 42 {125I}Iodonaphtylazide labeling selectively a cysteine residue in the F0 of the ATP-synthase from E . coli is unsuitable for topographic studies of membrane proteins; Hoppe J et al.; The ATP synthase from E . coli was reacted with the hydrophobic photolabel {125I}iodonaphtylazide . Subunit b in the F0-part was selectively labelled . Label was traced back to the single cysteine21 in subunit b . Thus the reactive intermediate of INA generated by photolysis had a high preference for nucleophiles . Due to this high selectivity the detection of membrane spanning peptide segments by labelling with INA is not reliable. Nucleic Acids Res, 1983 Aug 11, 11(15), 5235 - 42 Different levels of induction of RecA protein in E . coli (PQ 10) after treatment with two related carcinogens; Salles B et al.; By means of an immunoradiometric assay the induction of protein RecA in E . coli PQ 10 was measured after treatment by two related carcinogens . On an adduct basis N-Acetoxy-N-2-acetylaminofluorene was shown to induce the protein RecA at a similar level as U.V . On the other hand, N-hydroxy-N-2-aminofluorene shows only a poor induction capacity of the RecA protein . The difference in the SOS inducing potential of the aminofluorene and acetylaminofluorene adducts is discussed in relation to the major difference in the local conformational change the two adducts induce in DNA. Nucleic Acids Res, 1983 Aug 11, 11(15), 5165 - 80 Interactions of RNA polymerase and the cyclic AMP receptor protein on DNA of the E . coli galactose operon; Taniguchi T et al.; We have examined the interaction site on gal DNA for the cyclic AMP receptor protein and RNA polymerase when both are present together to form a stable initiation complex at the P1 gal promoter . Substitution of the bases to the left of -60 by unrelated DNA sequences does not change the cyclic AMP concentration dependency for in vitro transcription at P1 and inhibition of P2 . Although the presence of some DNA to the left of -60 appears to be needed for efficient in vitro transcription at P1, the gal sequence to the left of -60 does not provide any specific interactions for transcription initiation at P1 . Similarly, efficient in vitro transcription from P2 also requires non-specific DNA sequences to the left of -60 . We have also examined which bases were protected by RNA polymerase and CRP together from the action of DNAase and dimethylsulfate . Some of the interactions that take place when cAMP-CRP alone interacts with gal DNA appear to be preserved in the cAMP-CRP-RNA polymerase-gal DNA complex, suggesting that CRP occupies the same site in the DNA when it is alone or together with RNA polymerase . Our results suggest that the formation of an open complex at different promoters can result from different interaction patterns between RNA polymerase and promoter DNA. Mol Biol Rep, 1983 Aug, 9(3), 191 - 5 A method for the purification of E . coli plasmid DNA by homogeneous lysis and polyethylene glycol precipitation; Pulleyblank D et al.; A procedure is described for the isolation and purification of E . coli plasmid DNA by polyethylene glycol precipitation . The method is rapid, simple, inexpensive and amenable to both small and large scale manipulation . This procedure involves lysis of bacterial cells by treatment with pronase in sodium dodecyl sulfate, removal of chromosomal DNA by centrifugation, precipitation of residual nucleic acids with polyethylene glycol and removal of RNA by precipitation with LiCl . Plasmid DNA purified as described is pure enough for restriction endonuclease analysis, for use as a vector for the cloning of cDNA or synthetic DNA, or for use as a template in an E . coli transcription-translation cell-free system. Mutat Res, 1983 Aug, 113(5), 403 - 15 A differential DNA-repair test using mixtures of E . coli K12 strains in liquid suspension and animal-mediated assays; Mohn GR et al.; The feasibility of performing tests for repairable DNA damage in animal assay procedures was investigated by using repair-proficient and repair-deficient derivatives of E . coli K12 strain 343/113, including mutations in the uvrB, recA, polA and dam genes . To avoid variations in the relative recovery of viable cells from different samples, the strains were further marked with auxotrophic growth requirements, so that mixtures could be treated and the survival of each strain determined individually on media containing the corresponding growth factors . Spot tests were performed with the various strains to re-assess the necessity of using a combination of repair deficiencies, when genotoxic agents of differing mode of action are to be detected . Liquid suspension tests on mixtures of the different strains, furthermore, confirmed that the survival of the individual strains can be determined separately on selective media after treatment with methyl methanesulfonate (MMS) and methyl nitrosourea (MNU) . These tests were also used to demonstrate that dimethyl nitrosamine (DMNA) is activated by Aroclor-1254-induced rat-liver S9 fractions to genotoxic products, as measured by the low survival of a recA derivative compared with the repair-proficient wild-type strain . Intrasanguineous host-mediated assays using the present derivatives of E . coli K12/343/113 showed that the various strains, injected simultaneously into mice, could be recovered in amounts sufficient for the individual determination of the relative survival in liver, spleen, lungs, kidneys, pancreas and the blood stream of the host animals . Using a mixture of the repair-proficient parent and the recA derivative inoculated into mice that were subsequently treated with MMS, NMU or DMNA, we found that these chemicals induce a larger decrease in survival in the recA strain as compared with the wild-type in cells recovered from the liver and the spleen . The order of genotoxic potency so determined was DMNA greater than MNU greater than MMS; this is similar to the ranking of the carcinogenicity of these compounds in rodents and probably also reflects the various degrees of DNA alkylation in cells of the livers of the treated animals . The general usefulness of the host-mediated differential DNA repair assay for detecting genotoxic factors in various organs of animals remains to be assessed by using chemical mutagens of different modes of action. Cell, 1983 Aug, 34(1), 105 - 13 Regulation of the genes for E . coli DNA gyrase: homeostatic control of DNA supercoiling; Menzel R et al.; DNA gyrase is the bacterial enzyme responsible for converting circular DNA to a negatively supercoiled form . We show that the synthesis of DNA gyrase is itself controlled by DNA supercoiling; synthesis is highest when the DNA template is relaxed . The rates of synthesis in vivo of both the A and B subunits of DNA gyrase are increased up to 10-fold by treatments that block DNA gyrase activity and decrease the supercoiling of intracellular DNA . Similarly, efficient synthesis of both gyrase subunits in a cell-free S-30 extract depends on keeping the closed circular DNA template in a relaxed conformation . The results suggest that DNA supercoiling in E . coli is controlled by a homeostatic mechanism . Synthesis of the RecA protein and several other proteins is also increased by treatments that relax intracellular DNA. Nucleic Acids Res, 1983 Jul 11, 11(13), 4355 - 63 In vivo transcription of the E . coli uvrB gene: both promoters are inducible by UV; van den Berg EA et al.; The transcriptional activity of the tandem promoters of the Escherichia coli uvrB gene was measured in vivo . Both promoters are shown to be inducible by UV irradiation . P1, the most proximal promoter, is responsible for the main part of transcription both in uninduced and induced cells . Plasmids have been constructed carrying small deletions in the lexA binding site that overlaps with P2, the distal promoter . These deletions result in constitutive transcription from P1 . This indicates that the DNA region which contains P2 functions mainly as a target site for regulation of P1 transcription in vivo. Nucleic Acids Res, 1983 Jul 11, 11(13), 4307 - 23 Molecular cloning of human interleukin 2 cDNA and its expression in E . coli; Devos R et al.; A recombinant plasmid containing human interleukin 2 (IL2) cDNA was identified in a cDNA library constructed from mRNA derived from PHA-TPA induced splenocytes . Using this cDNA as a hybridization probe, a DNA fragment containing the IL2 gene was isolated from a collection of hybrid phages derived from human genomic DNA . A unique reading frame was identified from the nucleotide sequence derived from these plasmids coding for a polypeptide of 153 amino acids and containing a putative signal sequence of 20 amino acids . A mature polypeptide starting with either Met-Ala-Pro or Met-Pro was expressed in E . coli under control of the E . coli trp promoter or using a combination of the phage lambda PL promoter and a ribosome binding site derived from phage Mu . The bacterial IL2 polypeptide had a molecular weight of 15,000 daltons and accounted for more than 10% of the total E . coli proteins in fully induced cells; it was biologically active in the T-cell specific DNA synthesis assay, even after recovery from a SDS-containing polyacrylamide gel. Biokhimiia, 1983 Jul, 48(7), 1095 - 102 {Metabolic regulation of threonine operon transcription in E . coli cells}; Kliachko EV et al.; The threonine biosynthetic operon transcription in E . coli cells during balanced growth was studied . The rate of threonine-mRNA synthesis was measured by hybridization of impulse-labelled RNA with pYN 1107 DNA carrying the structural threonine genes A, B, C . It was shown that threonine-mRNA synthesis depends on bacterial growth rate being maximal at mu = 0.8 doublings per hour . The influence of the ppGpp on the bacterial growth rate and threonine-mRNA synthesis rate was demonstrated, using spoT-mutants and strains with several relA gene copies . The rate of threonine-mRNA synthesis is maximal at the ppGpp level of about 50-60 pmole/A450 . The deviation from this ppGpp optimum level results in inhibition of the threonine-mRNA synthesis . Thus, ppGpp appears to be involved in metabolic regulation of operon transcription . A mechanism of negative regulation of threonine-mRNA synthesis by high concentrations of ppGpp is discussed. Mikrobiyol Bul, 1983 Jul, 17(3), 177 - 85 {Spontaneous reversion of polA1 mutation on a mutant strain of E . coli K12 by transfer of ColE1 plasmid}; Kalaycioglu A; Specific polA1 mutation prevented the expression of high mutability in the mutator strain, H2IIO polA1 Spontaneous revertant of H2IIpolA1 was isolated by the transfer of ColE1 plasmid onto the mutator mutant . This spontaneous revertant, H2IIOpol, regained the mutator phenotype. Br J Pharmacol, 1983 Jul, 79(3), 711 - 8 The role of 5-hydroxytryptamine in the feline response to intravenous infusion of live E . coli; Arvidsson S et al.; A standardized septic shock was induced in cats by intravenous infusion of a live E . coli bacteria strain . The bacterial infusion induced a rapid haemodynamic response characterized mainly by a pulmonary arterial hypertension and a late phase characterized by systemic hypotension and hypodynamic circulation . Systemic arterial, pulmonary arterial, portal venous, left atrial pressures, max inspiratory-expiratory pressure difference in the trachea, aortic and intestinal blood flows were monitored . Arterial blood samples were taken for recording the number of circulating platelets and white blood cells and for determining the acid-base balance . The effect of pretreatment with ketanserin, a specific 5-hydroxytryptamine2 (5-HT2)-receptor blocker on these haemodynamic reactions was studied . In short term experiments on non-bacteriaemic control cats, ketanserin prevented the pulmonary hypertension induced by intravenous 5-HT infusions but not the increase in intestinal blood flow . Ketanserin induced a reduction of total peripheral (including intestinal) vascular resistance to blood flow but had no effect on aortic blood flow . After infusion of bacteria, ketanserin pretreated cats were more hypotensive due to a relative peripheral dilatation of the resistance vessels . Ketanserin pretreatment had no effect on the pulmonary vascular reactions, the tracheal pressure difference or the number of circulating platelets or white blood cells . Thus, except for a more pronounced hypotension early after bacterial infusion, ketanserin pretreatment did not influence the haemodynamic response . It is concluded that 5-HT is not of significant importance in the pathogenesis of the haemodynamic reactions following experimental bacteraemia. Prikl Biokhim Mikrobiol, 1983 Jul-Aug, 19(4), 555 - 9 {Determination of the activity of a polynucleotide phosphorylase preparation from E . coli}; Pupkova VI et al.; Thin-layer chromatography was employed to measure the activity of polynucleotide phosphorylase (PNP-ase) from E . coli during purification and in the resultant preparation . The TLC data were verified with respect to released inorganic orthophosphate and to 14C-AMP incorporation rate . It is concluded that TLC can be used to determine PNP-ase activity in crude extract, at various purification stages and in the final preparation, yielding correct results. J Biochem (Tokyo), 1983 Jul, 94(1), 275 - 82 Preliminary chemical and X-ray studies on the interactions of E . coli DNA with putrescine; Takeda Y et al.; The interactions of putrescine, the major diamine in E . coli, with E . coli DNA as a model of phage DNA were studied by melting temperature analysis, equilibrium dialysis and X-ray diffraction with the aid of molecular model building . The chemical analysis of the DNA-putrescine complex shows that the molar binding ratio of putrescine to DNA (phosphate) is nearly 1 to 2 . The equilibrium (or reversible) binding of putrescine to DNA was suggested by the fact that the melting temperature increased according to the concentration of added putrescine, and its elevation was not saturated even at the molar ratio of 6 to 1 . The equilibrium dialysis experiments indicate that the association constant for the complex is a little smaller than, but in the same order (10(3) liter/mol) as, that of DNA-spermine complex . The binding of putrescine stabilizes the B-form of DNA fiber, which is well preserved even at 66% relative humidity . The distance between the neighboring DNA helices in the wet fiber increased with the increasing degree of hydration, as in the case of native DNA . Unlike spermine, putrescine does not form precipitate upon mixing with DNA in the concentration range for UV measurement, suggesting that the cross-bridge formed by putrescine is intra-double helical . The equilibrium binding of putrescine to DNA, seems to be important for the life cycle of lambda-phage. J Biochem (Tokyo), 1983 Jul, 94(1), 243 - 7 Nucleotide sequence of the regulatory region of malB operons in E . coli; Ohsumi M et al.; The nucleotide sequence of a cloned section of the Escherichia coli chromosome containing the promoter regions of the malB divergent operons was determined . The region of the proximal gene, malE of the malEFG operon, was identified on the basis of the known amino acid sequence of the precursor molecule of maltose-binding protein . The region of malK, the proximal gene of the malKlamB operon, was deduced from the observation that a cloned segment contains an amino-terminal portion of the malK gene . The non-coding region between malE and malK is 299 base pairs long and contains two long GC clusters . Another feature of this region that may be related to the regulation of gene expression is the presence of two palindromic structures between the GC clusters . The DNA regions binding to cyclic AMP binding protein were determined by a method using polyacrylamide gel electrophoresis . The sites are thought to be located close to GC clusters. Radiobiologiia, 1983 Jul-Aug, 23(4), 505 - 9 {Toxic and radiosensitizing effect of reduced nitroimidazoles on E . coli B/r cells}; Riabchenko NI et al.; The spectrophotometric method was used to study the rate of chemical reduction of misonidazole and metronidazole by NH4Cl and Zn in the atmosphere of argon and oxygen . Reduction of drugs increased their toxicity for hypoxic and oxygenated E . coli B/r . The reduced metronidazole is a more effective radiosensitizer of hypoxic E . coli B/r than the original compound. Int J Radiat Biol Relat Stud Phys Chem Med, 1983 Jul, 44(1), 87 - 95 Lowered pH eliminates the enhanced hyperthermic killing of E . coli induced by procaine or exposure to N2 gassing; Simone G et al.; Survival of E . coli K1060 is enhanced when they are heated at 47 degrees C in pH 6 medium as compared to pH 7.4 . At pH 6 nitrogen bubbling and 10 mM procaine did not increase hyperthermic killing . The membrane content of phosphatidylethanolamine is about 80 per cent of the total of phosphatidylethanolamine, phosphatidylglycerol and cardiolipin phospholipids . The polar headgroup of this lipid is highly sensitive to pH changes in this pH range . The changes in electrostatic charge with its secondary effects on membrane-protein relationships may explain resistance to hyperthermic killing . Thus, the difference in response to lowered pH of bacterial, compared to mammalian cells may be revealing membrane-related phenomena critical to hyperthermic killing . Also increased levels of cardiolipid were observed in linolenic acid grown cells which could be an 'attempt' to stabilize their membranes. J Biochem (Tokyo), 1983 Jul, 94(1), 315 - 8 A stabilizing factor of yeast mitochondrial F1F0-ATPase-inhibitor complex: common amino acid sequence with yeast ATPase inhibitor and E . coli epsilon and bovine delta subunits; Matsubara H et al.; The amino acid sequence of a stabilizing factor, 9,000 dalton protein, of proton-translocating ATPase (F1F0-ATPase)-inhibitor complex isolated from yeast mitochondria was established . It was highly homologous with that of yeast intrinsic ATPase inhibitor and several structural characteristics were noted . They were compared with those of E . coli epsilon and bovine delta subunits to point out the similarity among them. Biochem Biophys Res Commun, 1983 Jun 29, 113(3), 1018 - 25 Translational control of the expression of the beta subunit gene of E . coli RNA polymerase; Peacock S et al.; Using the plasmid pNF1337 as template, a mRNA preparation has been obtained that directs the in vitro synthesis of fMet-Val, the N-terminal dipeptide of the beta subunit of RNA polymerase . RNA polymerase holoenzyme specifically inhibits the mRNA-directed synthesis of fMet-Val showing that the autoregulation by RNA polymerase of beta, beta 1 synthesis is at the level of translation . L factor (nusA gene product) stimulates the synthesis of fMet-Val from a DNA template but not from mRNA . Rifampicin has no effect on the mRNA-directed synthesis of fMet-Val or the ability of RNA polymerase to inhibit fMet-Val synthesis. Nucleic Acids Res, 1983 Jun 25, 11(12), 3863 - 72 Modification of the anticodon triplet of E.coli tRNAMetf by replacement with trimers complementary to non-sense codons UAG and UAA; Ohtsuka E et al.; E . coli tRNAMetf was hydrolyzed with RNase A using a limited amount of the enzyme to give two half molecules lacking the anticodon trimer and 3'-terminal dimer . Chemically synthesized trimers CUAp and UUAp were joined to the 5'-half molecules by phosphorylation with polynucleotide kinase plus ATP followed by treatment with RNA ligase . These modified tRNAMetf species had anticodons complementary to the termination codons UAG and UAA . Two half fragments were joined by a similar procedure to yield a molecule lacking the anticodon trimer and the 3'-dimer . Methionine acceptor activity of these tRNA was tested under conditions in which the CAU inserted control tRNAMetf accepted methionine . It was found that all three modified molecules were not recognized by the methionyl-tRNA synthetase from E.coli . The other sixteen amino acids were not incorporated with partially purified aminoacyl-tRNA synthetases. Nucleic Acids Res, 1983 Jun 11, 11(11), 3753 - 65 Uracil in deoxyribonucleotide polymers reduces their template-primer activity for E . coli DNA polymerase I; Vilpo JA et al.; The physical and biochemical properties of two pairs of synthetic DNA template-primers were investigated . The copolymer poly(dA-dU) . poly(dA-dU) and the homopolymer duplex poly(dA) . poly(dU) were characterized by a lower Tm and by a higher buoyant density value than the respective thymine polynucleotides poly(dA-dT) . poly(dA-dT) and poly(dA) . poly(dT) . The polymerizing and the primer terminus adding reactions of a homogenous E . coli DNA polymerase I preparation, as measured by incorporation of {3H}dAMP into the acid-insoluble fraction, were significantly poorer with uracil-containing template-primers than with thymine templates . Moreover, the uracil-containing polynucleotides inhibited the polymerizing activity of DNA polymerase I to a greater extent than the thymine polynucleotides, when the enzymatic activity was investigated with a dATP/dTTP/dUTP-free incorporation system making use of poly(dI-dC) . poly(dI-dC) as the template-primer. Nucleic Acids Res, 1983 Jun 11, 11(11), 3581 - 91 Direct expression of hepatitis B surface antigen gene in E . coli; Fujisawa Y et al.; A 809 bp Sau 3A - Hpa I fragment containing a complete HBsAg gene and fragments 744 bp Hinc II - Hpa I and 712 bp Xba I - Hpa I containing a truncated HBsAg gene lacking the sequence encoding the NH2-terminal hydrophobic domain were prepared from a composite plasmid pHBV933 containing the 3.2 kb Eco RI DNA fragment of the entire HBV/adw genome and inserted into an expression vector pTRP801 to give plasmids pTRP SS-6, pTRP SS-39, and pTRP SS-50, respectively . The growth of a recombinant having pTRP SS-6 was greatly inhibited and the transformant expressed a low level of HBsAg, which is reactive to human anti-HBsAg antibody . Interestingly, the growth of transformants harbouring pTRP SS-39 and pTRP SS-50 was not inhibited and these transformants expressed a considerable level of the HBsAg . Minicells harbouring pTRP SS-6, pTRP SS-39, and pTRP SS-50 formed specific polypeptides of about 24 K, 23 K, and 22 K daltons, respectively. Nucleic Acids Res, 1983 Jun 11, 11(11), 3531 - 45 The nucleotide sequence of the rho gene of E . coli K-12; Pinkham JL et al.; We have determined the nucleotide sequence of the rho gene which encodes the E . coli K-12 transcription termination factor . The structural gene was located on a cloned 3.6 kilobase BglII-HindIII restriction fragment by the introduction of the insertion element gamma delta and analysis of the recombinant plasmids by restriction analysis and in maxicells . The coding region consists of 1260 nucleotides directing the synthesis of a polypeptide 419 amino acids in length with a calculated molecular weight of 46,094 . The deduced amino acid composition, amino-terminal protein sequence and calculated molecular weight are consistent with the data from the analysis of purified rho protein (16) . We have shown that the rho genes from E . coli K-12, B and C strains are located on PvuII-HindIII fragments of the same size by hybridization to the rho (K-12) coding sequences. J Immunol Methods, 1983 Jun 10, 60(3), 369 - 77 A new, simple and rapid method for enumerating human T lymphocytes in full blood: the E . coli (ATCC 11303) rosette test; Orye E et al.; A new bacterial rosette technique for enumerating T lymphocytes is described . E . coli (strain B; ATCC 11303), fixed in formaldehyde after overnight growth in thioglycolate medium, are mixed with washed whole blood cells (100 microliters) and after incubation at 4 degrees C, slides are made, stained and counted . The nature of the lymphocytes forming E . coli rosettes was demonstrated by comparing their cytochemical staining characteristics with those of E rosetted lymphocytes, and by mixed E . coli and E, mouse E rosette and Fc receptor tests, and by mixed E . coli rosette tests and anti-Ig staining . E . coli and E rosette tests in controls and pediatric patients were also compared . The results show that T mu and T gamma cells rosette with E . coli. Int J Radiat Biol Relat Stud Phys Chem Med, 1983 Jun, 43(6), 587 - 97 Effect of gamma radiation on E . coli ribosomes, tRNA and aminoacyl-tRNA synthetases; Singh H et al.; Gamma-irradiated E coli ribosomes and tRNA, in aerated solutions, were inactivated with D37 doses of 144 and 77 Gy, respectively . Aminoacyl-tRNA-synthetases were only slightly inactivated under comparable conditions . Effects of additives to ribosome and tRNA solutions suggest that hydroxyl radicals were the major damaging species, that superoxide anions were not damaging and that radiolytically-formed hydrogen peroxide was also unimportant . Part of the damage by hydroxyl radicals is expressed through secondary radicals produced from additives and buffers . Results obtained with three different buffers suggest that (1) acetate ions provide protection by competing for hydroxyl radicals, (2) chloride ions are without effect and (3) inactivation of ribosomes and aminoacyl-tRNA-synthetases in Tris-HCl/MgCl2 and phosphate/MgCl2 buffered solutions was similar but the tRNA inactivation was lower in Tris-HCl/MgCl2 buffer. Cell, 1983 Jun, 33(2), 615 - 22 Sensory transducers of E . coli are composed of discrete structural and functional domains; Krikos A et al.; The tar and tsr genes of E . coli encode functionally analogous transducer proteins that mediate two distinct classes of chemotactic response . The tap gene lies adjacent to tar, and is thought to encode another transducer protein . We present here the complete nucleotide sequence of the tar-tap region of the E . coli genome, together with a comparative analysis of the sequences of the Tar, Tap, and Tsr proteins . The proteins appear to have a simple transmembrane structure consisting of an extracytoplasmic amino-terminal domain, a membrane-spanning domain, and an intracellular carboxy-terminal domain . The carboxy-terminal domains of three proteins possess highly homologous sequences and contain sites of methylation involved in sensory adaptation, while the amino-terminal sequences are only distantly related to one another, consistent with their serving as chemoreceptor domains that have diverged functionally. Biochimie, 1983 Jun, 65(6), 345 - 54 Concerning the thermal stability of E . coli 23S ribosomal RNA; Guerin MF et al.; Total RNA prepared from E . coli by several extraction procedures behaves as a mixture of covalently continuous heat stable 23S, 16S and 4-5S components . 16S rRNA remains heat stable after isolation from such preparations, whereas isolated 23S rRNA is heat labile but becomes heat stable after EDTA treatment . This and other evidence leads to the conclusion that heat lability of purified 23S rRNA is due, not to nuclease contamination of the type observed in earlier studies of the stability of this RNA, but to polyvalent cation catalyzed temperature-dependent scission of phosphodiester bonds . Heat stability of 23S rRNA in total RNA is due to the presence in these preparations of a contaminant which appears to act as a chelator of polyvalent cations . This material is similar or identical to the pyrogenic E . coli lipopolysaccharide described by Westphal and coll. Nucleic Acids Res, 1983 May 25, 11(10), 3227 - 39 Further improvements on the phosphotriester synthesis of deoxyribooligonucleotides and the oligonucleotide directed site-specific mutagenesis of E . coli lipoprotein gene; Hsiung H et al.; Two improvements that greatly enhance the rate of phosphotriester oligonucleotide synthesis are described: 1) use of hindered primary amines, e.g . t-butyl amine for decyanoethylation of oligonucleotide triester intermediates, and 2) a simplified isolation procedure that eliminates the tedious bicarbonate extraction after each condensing reaction . Using the improved procedures, oligonucleotide fragments can be synthesized as rapidly as using solid phase chemistry . The final products are purer than those obtained by solid phase chemistry since each intermediate block is purified by chromatography . The technique has been used to synthesize five oligonucleotide fragments (size 15 to 20) for the purpose of performing guided site-specific mutagenesis on a cloned E . coli lipoprotein gene. Nucleic Acids Res, 1983 May 25, 11(10), 3077 - 85 Expression of human immunoglobulin E epsilon chain cDNA in E . coli; Kurokawa T et al.; Using the cDNA of human epsilon chain, three expression plasmids that code directly the constant portion of the epsilon chain (C epsilon 1-C epsilon 4, C epsilon 2-C epsilon 4 and C epsilon 3-C epsilon 4 domains) were constructed . These epsilon chain peptides were synthesized in E . coli under the control of the trp promoter-operator . The bacterially produced peptides have the antigenicity of human epsilon chain and gave the molecular weights equal to the values calculated from the amino acid sequence of the constructed plasmids. Nucleic Acids Res, 1983 May 11, 11(9), 2701 - 16 Chemical modifications of the sigma subunit of the E . coli RNA polymerase; Narayanan CS et al.; The function of arginine, cysteine and carboxylic amino acid (glutamic and aspartic) residues of sigma was studied using chemical modification by group specific reagents . Following modification of 3 arginine residues with phenylglyoxal or 3 cysteine residues with N-ethylmaleimide (NEM) sigma activity was lost . Analysis of the kinetic data for inactivation indicated that one arginine or cysteine residue is essential for sigma activity . At low NEM concentration alkylation was limited to a non-critical cysteine which was identified as cysteine-132 . Modification of arginine or cysteine residues had no observable effect on the binding of the inactivated sigma to the core polymerase . Modification of aspartic and/or glutamic acid residues with the water-soluble carbodiimides 1-ethyl-3-(3-dimethylamino-propyl) carbodiimide hydrochloride (EDC) or 1-cyclohexyl-3-(2-morpholinoethyl) carbodiimide metho-p-toluene sulfonate (CMC) resulted in loss of sigma activity . The inactivation data indicated that one carboxylic amino acid residue is essential for sigma activity . Sigma modified with EDC, CMC or EDC in the presence of glycine was inactive in supporting promoter binding and initiation by core polymerase . Reaction with EDC plus (3H)glycine resulted in the incorporation of glycine into sigma . The (3H)glycine-sigma was unable to form a stable holoenzyme complex. Nucleic Acids Res, 1983 May 11, 11(9), 2617 - 26 The 245 base-pair oriC sequence of the E . coli chromosome directs bidirectional replication at an adjacent region; Tabata S et al.; The replication origin of the E . coli K-12 chromosome has been isolated as autonomously replicating molecules(oriC plasmid), and the DNA region essential for replicating function(oriC) has been localized to a sequence of 232-245 base-pairs(bp) by deletion analysis . In this report, the functional role of oriC was analysed by using an in vitro replication system and various OriC+ and OriC- plasmids previously constructed . The results obtained were summarized as follows: (1) The oriC sequence contained information enough to direct bidirectional replication . (2) The actual DNA replication began at a region near, but outside, oriC and progressed bidirectionally . (3) Initiation of DNA synthesis at the specific region required the dnaA-complementing fraction from cells harboring a dnaA-carrying plasmid. Ital J Biochem, 1983 May-Jun, 32(3), 145 - 51 Induction of pyrimidine nucleoside metabolizing enzymes in E . coli B; Vita A et al.; Induction studies on pyrimidine metabolizing enzymes in E . coli B have shown that the enzymes fall into three distinct groups according to their induction pattern . a) Cytidine deaminase and uridine phosphorylase, are induced by cytidine, CMP and adenosine; no induction was observed with uridine and AMP; b) thymidine phosphorylase is induced by cytidine, adenosine, all deoxyribonucleosides, CMP, deoxyribonucleotides, deoxyribose and deoxyribose-1-phosphate; c) uridine-cytidine kinase, uracil phosphoribosyltransferase, 5'-nucleotidase, thymidine kinase, are uninducible enzymes . Simultaneous addition of cytidine and glucose partially overcomes the cytidine deaminase and uridine phosphorylase induction . Cytidine deaminase reaches its maximum activity levels, in E . coli growing cells in presence of cytidine, two hours before the uridine phosphorylase activity . Maximum glucose repression of cytidine deaminase and uridine phosphorylase was obtained in correspondence of maximum cytidine induction. Prikl Biokhim Mikrobiol, 1983 May-Jun, 19(3), 388 - 95 {Dependence of the efficiency of the fractionation of summary E . coli cell membranes on the method of their disintegration}; Mikhaleva NI et al.; The efficiency of fractionation in the sucrose density gradient of E . coli cell membranes obtained after cell disintegration by ultrasonic, ballistic and extrusion methods was measured . The purity of individual membrane fractions was estimated by electron microscopy and polyacrylamide gel electrophoresis . The application of ballistic disintegration and solid state extrusion did not separate inner and outer membranes . Cell disintegration by means of ultrasonic treatment and liquid state extrusion allowed reproducible separation of membranes of the two types with a sufficiently high degree of purity. Radiobiologiia, 1983 May-Jun, 23(3), 307 - 11 {Theoretical analysis of the effect of the photoreactivation of E . coli cells irradiated with gamma quanta}; Pitkevich VA et al.; The assumption that the formation of pyrimidine dimers in E . coli cells, placed in a transient aqueous medium exposed to gamma-quanta, is conditioned by Cerenkov radiation laid the basis for the development of a biophysical model to explain the photoreactivation effect observed . The dependence of the effect upon gamma-radiation energy and the volume of the exposed suspension is predicted and compared with experimental data. Clin Exp Immunol, 1983 May, 52(2), 293 - 6 Loss of CFA/I expression by enteropathogenic E . coli following exposure to antibody; Dean GS et al.; H-10407 (078, H11) an enteropathogenic strain of Escherichia coli carries a fimbriate plasmid-mediated adherence antigen on the cell surface (CFA/I) which facilitates colonization of human small intestine . This shows strong similarities to the K88 antigen of porcine enteropathogenic E . coli . K88 expression may be suppressed by antibody both in vivo and in vitro . Expression of CFA/I, detected by agglutination of human erythrocytes was progressively lost during in vitro culture with antisera containing antibodies specific for CFA/I but, unlike K88, CFA/I was re-expressed during further culture in the absence of antibody . Antibody to CFA/I seemed to exert a switching effect on expression of the adherence antigen. Pediatr Res, 1983 May, 17(5), 349 - 53 Systemic and neuropathologic effects of E . coli endotoxin in neonatal dogs; Young RS et al.; The acute systemic and neuropathologic effects of E . coli endotoxin were determined in neonatal dogs . Administration of sublethal (LD0), moderate (LD50), or lethal (LD100) doses of endotoxin produced significant arterial hypotension, metabolic (lactic) acidosis, and hypoglycemia . Neuropathologic changes consisted of widespread inflammation in both grey and white matter; however, necrotic lesions were found only in forebrain white matter. Biull Eksp Biol Med, 1983 May, 95(5), 67 - 70 {Effect of ultrasonic treatment on the adhesiveness of E . coli}; Kochemasova ZN et al.; Electron microscopy and the hemagglutination test were used to study the effect of low-frequency and low-amplitude ultrasound (26.5 kHz, 30 m) on the adhesiveness of E . coli cells, strain 815, possessing Type I pili . Mild sonication providing the assanation effect during intraoperational treatment of the infected peritonium was found to remove the majority of flagella and pili, despite the fact that the cells seemed to be unimpaired . After sonication the ability of bacteria to cause mannose-sensitive hemagglutination of guinea-pig red blood cells decreased more than 8-fold . The data obtained suggest that the diminution of adhesiveness might be a possible mechanism of the sonication-induced assanation effect. Mutat Res, 1983 May, 109(2), 143 - 53 Loss of photoreversibility for UV mutation in E . coli using 405 nm or near-UV challenge; Kristoff S et al.; E . coli mutagenized with germicidal ultraviolet light (UV) were incubated to allow for development of mutation-fixation processes . Fixation was estimated from the effects on mutation frequency of photoreactivation challenge during the first 60 min post-UV . Two different light sources were used for photoreactivation, one providing effective light primarily at 405 nm and another providing a broad range of near-UV around 365 nm . Kinetics for the loss of photoreversibility (LOP) were determined . The times for completion of LOP in wild-type cells indicated one fixation process for back mutation and another for de novo or converted suppressor mutation regardless of the light source . Using 405-nm light for photoreactivation, the LOP kinetics for back mutation and de novo suppressor mutation in uvrA cells were similar . Hence, classical observations were confirmed here . Immediately post-UV all mutation frequencies were more sensitive to near-UV than 405-nm light . Experiments with rel cells supported the idea that growth delay and inhibition of induced lexA-coordinated responses may be responsible for this early, pronounced sensitivity to photoreactivation by near UV . For back mutation and de novo suppressor mutation, the sensitivity to 405-nm light was initially small and actually increased for 10-15 min . Possibly genome conformation changes are induced by UV and this affects the efficiency of photoenzymatic monomerization of 405-nm light during the first 10-15 min after irradiation. J Immunol Methods, 1983 Apr 29, 59(2), 245 - 54 An enzymatically active antigen-antibody probe to measure circulating immune complexes . II . E . coli beta-galactosidase in the probe and C1q as the recognition unit; Migliorini P et al.; An enzymatically active probe (beta-galactosidase-anti-beta-galactosidase complex) is used to measure circulating immune complexes (CIC), in a competition assay where probe and CIC are confronted with a 'recognition unit' . The latter is bovine conglutinin in the original description of this method . Here we describe a version utilizing human or bovine C1q . The two techniques are compared for their sensitivity and specificity, on both in vitro formed tetanus toxoid-anti-toxoid complexes and on sera from patients with selected diseases . The results confirm that the two recognition units are sensitive to families of CIC that only partially overlap . The parallel use of conglutinin and C1q yields both quantitative and qualitative information on the nature of CIC in individual sera. Int J Radiat Biol Relat Stud Phys Chem Med, 1983 Apr, 43(4), 451 - 8 Radiosensitization and radioprotection of E . coli by thiourea in nitrous oxide saturated suspensions; Antoku S; N2O did not modify the radiosensitivity of E . coli BH and H/r-30 strains as to colony-forming ability and DNA single-strand breaks . In N2O-saturated suspensions of E . coli, thiourea and thiosemicarbasite sensitized at low concentrations, while cysteamine and cysteine protected at all concentrations . Protection by thiourea in N2O-saturated suspensions was observed only in the frozen state . These results suggest that the conversion of e-aq to OH radicals may be responsible for sensitization and this sensitization is probably due to the thiourea and thiosemicarbasite radicals produced extracellularly. Cell, 1983 Apr, 32(4), 1325 - 35 Information within the mature LamB protein necessary for localization to the outer membrane of E coli K12; Benson SA et al.; It has been proposed that the efficient localization of the outer membrane protein LamB requires a functional signal sequence and at least two additional regions contained within the mature protein . We define these regions more precisely by deletion analysis, and we describe methods for cloning deleterious lacZ fusions onto high-copy-number plasmids and generating in-frame deletions . Analysis of the effects of a series of internal lamB deletions on the export of a LamB-LacZ hybrid protein and of the LamB protein itself indicates that necessary informational signal(s) required for localization lie at the amino-terminal end of the protein . In addition, our analysis indicates that there is a region of information close to or within the fusion joint of the largest lamB-lacZ fusion that increases the efficiency of the export process . A unique deletion that removes a protein segment from amino acid 70 to 200 appears to prevent proteolytic removal of the signal sequence . Nevertheless, the mutant protein is exported to the outer membrane. Cell, 1983 Apr, 32(4), 1355 - 65 Structure of E . coli 16S RNA elucidated by psoralen crosslinking; Thompson JF et al.; E . coli 16S RNA in solution was photoreacted with hydroxymethyltrimethylpsoralen and long wave ultraviolet light . Positions of crosslinks were determined to high resolution by partially digesting the RNA with T1 RNase, separating the crosslinked fragments by two-dimensional gel electrophoresis, reversing the crosslink, and sequencing the separated fragments . This method yielded the locations of crosslinks to +/- 15 nucleotides . Even finer placement has been made on the basis of our knowledge of psoralen reactivity . Thirteen unique crosslinks were mapped . Seven crosslinks confirmed regions of secondary structure which had been predicted in published phylogenetic models, three crosslinks discriminated between phylogenetic models, and three proved the existence of new structures . The new structures were all long range interactions which appear to be in dynamic equilibrium with local secondary structure . Because this technique yields direct information about the secondary structure of large RNAs, it should prove invaluable in studying the structure of other RNAs of all sizes. Cell, 1983 Apr, 32(4), 1337 - 46 Differential stringent control of the tandem E . coli ribosomal RNA promoters from the rrnA operon expressed in vivo in multicopy plasmids; Sarmientos P et al.; The tandem P1, P2 promoter region of the rrnA ribosomal operon has been fused to the t1, t2 terminator region of the rrnB operon in pBR322 plasmid derivatives . This deletes most internal RNA structural elements ordinarily processed out of ribosomal operon transcripts . In vivo as well as in vitro transcripts arising from both promoters terminate predominantly in the t1 terminator region about 40 base pairs beyond the mature rrnB 5S RNA gene . Stringent control of the P1 and P2 promoted transcripts has been assessed in vivo . In these plasmid fusions, the upstream (P1) promoter activity was subject to stringent control, while the downstream (P2) promoter activity was inhibited by amino acid starvation in both stringent and relaxed hosts . A plasmid with an additional deletion of the P2 region also showed stringent regulation of the P1 promoter. Nucleic Acids Res, 1983 Mar 25, 11(6), 1913 - 8 A putative gene of tobacco chloroplast coding for ribosomal protein similar to E . coli ribosomal protein S19; Sugita M et al.; A partial sequence of a cloned 3.2 Md BamHI fragment from tobacco chloroplast DNA revealed the occurrence of a putative gene for ribosomal protein . The putative gene is located on the left margin of the large single-copy region in the chloroplast DNA . The coding region contains 276 bp (92 codons) . The amino acid sequence deduced from the DNA sequence shows 55% homology with that of E . coli S19 (91 amino acid residues). Nucleic Acids Res, 1983 Mar 11, 11(5), 1439 - 55 Base substitutions in the wobble position of the anticodon inhibit aminoacylation of E . coli tRNAfMet by E . coli Met-tRNA synthetase; Schulman LH et al.; Derivatives of E . coli tRNAfMet containing single base substitutions at the wobble position of the anticodon have been enzymatically synthesized in vitro . The procedure involves excision of the normal anticodon, CAU, by limited digestion of intact tRNAfMet with RNase A . RNA ligase is then used to join each of four trinucleotides, NAU, to the 5' half molecule and to subsequently link the 3' and modified 5' fragments to regenerate the anticodon loop . Synthesis of intact tRNAfMet containing the anticodon CAU by this procedure yields a product which is indistinguishable from native tRNAfMet with respect to its ability to be aminoacylated by E . coli methionyl-tRNA synthetase . Substitution of any other nucleotide at the wobble position of tRNAfMet drastically impairs the ability of the synthetase to recognize the tRNA . Measurement of methionine acceptance in the presence of high concentrations of pure enzyme has established that the rate of aminoacylation of the AAU, GAU and UAU anticodon derivatives of tRNAfMet is four to five orders of magnitude slower than that of the native or synthesized tRNA containing C as the wobble base . In addition, the inactive tRNA derivatives fail to inhibit aminoacylation of normal tRNAfMet, indicating that they bind poorly to the enzyme . These results support a model involving direct interaction between Met-tRNA synthetase and the C in the wobble position during aminoacylation of tRNAfMet. Nucleic Acids Res, 1983 Mar 11, 11(5), 1491 - 505 A G43 to U43 mutation in E . coli tRNAtyrsu3+ which affects processing by RNase P; Furdon PJ et al.; A novel mutation in the anticodon stem of E . coli tRNA1Tyrsu3+ (G43 to U43) has been characterized . The gene coding for the mutant tRNA, carried by phage phi 80DHA61.3 a derivative of phi 80psu3+su0, produces only 20% of mature suppressor tRNA as compared with phi 80psu3+ . Both the mutant tRNA precursor and mature tRNA have an altered conformation . The precursor tRNA coded for by phi 80DHA61.3 is processed by RNase P more slowly than the su3+ precursor and does not form as stable an enzyme-substrate complex as does su3+ precursor . phi 80 DHA61.3 also contains a large deletion which begins in the spacer region between the su3+ gene and the su0- gene, extends through the su0- gene and includes most of the repeated region following the tRNA genes. Nucleic Acids Res, 1983 Mar 11, 11(5), 1283 - 94 Expression of chemically synthesized alpha-neo-endorphin gene fused to E . coli alkaline phosphatase; Ohsuye K et al.; An alpha-neo-endorphin (alpha NE) gene, which we previously synthesized chemically and inserted into E . coli beta-galactosidase gene of pK013 plasmid, has been excised and fused to E . coli alkaline phosphatase (APase) gene . One of the transformants was named E15/pA alpha NE1 . Under the APase gene regulation, APase-alpha NE chimeric protein was expressed at 1.3 X 10(6) molecules per cell, and accounted for about 60% of total cellular proteins . The HPLC pattern of CNBr treated E15/pA alpha NE1 was very simple reflecting the high content of the chimeric protein and low numbers of methionine residues in it . A series of genes encoding APase-alpha NE chimeric proteins in which 30 to 94 C-terminal amino acid residues were replaced by (met)-alpha NE, was cloned in E . coli . Transportation of the chimeric proteins to periplasmic space was studied . All chimeric proteins were apparently processed by signal peptidase but few, if any, was transported to the periplasmic space. Nature, 1983 Mar 3, 302(5903), 74 - 6 Functional interrelationship between two tandem E . coli ribosomal RNA promoters; Glaser G et al.; The Escherichia coli chromosome carries seven cistrons encoding ribosomal RNA sequences . In all cases studied, in vitro and in vivo, it has been established that transcription is initiated from two tandem promoters . The expression of the rRNA cistrons is regulated in response to growth rate as well as to aminoacyl tRNA availability . In the present study, a plasmid (pPS1) carrying the promoter region of the rrnA cistron fused to the terminator region of rrnB has been used for in vitro transcription experiments . The presence of the terminators (T1 and T2) together with the fact that supercoiled DNA is found to be a highly efficient template, provide an ideal in vitro system in which to study the functional interrelationship between the two tandem promoters of E . coli rRNA cistrons . The results suggest that the rate of rRNA synthesis in E . coli cells growing in various conditions, as reflected by the availability of RNA polymerase, is primarily dependent on the properties of the two tandem rRNA promoters. Biochimie, 1983 Mar, 65(3), 221 - 5 Effect of the overproduction of phenylalanyl- and threonyl-tRNA synthetases on tRNAPhe and tRNAThr concentrations in E . coli cells; Fayat G et al.; Transformation of an E . coli strain with a recombinant plasmid DNA (pB1) encoding the genes for phenylalanyl- and threonyl-tRNA synthetases causes overproduction of these enzymes by about 100- and 5-fold, respectively . A possible effect of the overproduction of the two aminoacyl-tRNA synthetases on intracellular cognate tRNA levels has been searched for by comparing tRNAThr and tRNAPhe aminoacylation capacities in the RNA extracts from strains carrying pB1 or pBR322 plasmid DNA . The answer is that the levels of these tRNAs are not changed by selective increase of the cognate synthetases. Br J Pharmacol, 1983 Mar, 78(3), 561 - 70 The release of prostanoids during the acute pulmonary response to E . coli endotoxin in anaesthetized cats; Coker SJ et al.; 1 The administration of E . coli endotoxin (2 mg/kg i.v.) to anaesthetized cats results in a characteristic acute pulmonary response . This consists of increases in pulmonary artery pressure and airways resistance and a reduction in lung compliance . 2 Plasma concentrations of prostaglandin E2 (PGE2), PGF2 alpha, thromboxane B2 and 6-keto PGF1 alpha were measured by radioimmunoassay in aortic and pulmonary arterial blood samples before, during and after the acute pulmonary response to endotoxin . 3 Endotoxin administration resulted in the rapid release of PGF2 alpha and thromboxane B2 from the lungs . The time course of this release was parallel to that of the pulmonary hypertensive and airways responses to endotoxin . PGE2 and 6-keto PGF1 alpha were released more gradually and with greater variations between individual animals . 4 During the delayed shock phase (2 h after endotoxin) the concentrations of PGE2, PGF2 alpha and 6-keto PGF1 alpha were once again elevated in both the aorta and pulmonary artery . Thromboxane B2 concentrations were not increased at this time . 5 These results suggest that PGF2 alpha and thromboxane A2 may be mediators of the acute pulmonary responses to endotoxin. Cell, 1983 Mar, 32(3), 817 - 29 Enzymatic formation of biparental figure-eight molecules from plasmid DNA and their resolution in E . coli; West SC et al.; A key intermediate in general genetic recombination is a structure in which two double-stranded DNA molecules are covalently linked by a single-strand crossover characteristic of a Holliday junction . When the DNA molecules are circular, the recombinant structures take the form of a figure eight . We have used purified E . coli enzymes to construct biparental figure-eight DNA molecules in vitro from the DNA of two partially homologous plasmids . When purified figure-eight structures are transfected into recA- E . coli cells, they are resolved to produce monomeric or dimeric plasmid progeny, apparently by the cutting and joining of the Holliday crossover . The maturation of figure-eight molecules in bacteria is characterized by the formation and recovery of both parental and recombinant types, cross-over at a frequency of up to 50% and the capability for mismatch repair at regions of hybrid DNA . In these three regards, the products of figure-eight maturation resemble recombinant chromosomes formed at meiosis . These observations show that biparental figure eights behave as recombination intermediates that can be resolved into mature recombinants without need for a functional recA+ gene product. Cell, 1983 Mar, 32(3), 789 - 97 A temperature-sensitive mutant of E . coli exhibiting slow processing of exported proteins; Ito K et al.; A temperature-sensitive E . coli mutant with a mutation in the spc ribosomal protein operon was found to have a conditional defect in the processing of precursor proteins destined for the periplasmic space or the outer membrane . At high temperatures, significant amounts of precursor proteins having unprocessed signal sequences are detected in the mutant cell by pulse-labeling . The precursors are processed at very slow rates during a subsequent chase . Genetic analysis indicates that the mutation impairs the function of a gene, termed secY, located at the promoter-distal part of the spc operon . The secY gene is distinct from those genes previously known to specify ribosomal proteins, yet it is within the spc operon . It is suggested that the product of the secY gene is a component of the cellular apparatus that is essential for protein secretion across the cytoplasmic membrane . The gene secY is probably identical with prlA, previously identified as a suppressor of signal sequence mutations. Mutat Res, 1983 Mar, 116(3-4), 179 - 84 Roasting coffee beans produces compounds that induce prophage lambda in E . coli and are mutagenic in E . coli and S . typhimurium; Kosugi A et al.; Freshly brewed blended coffee, instant coffee and instant caffeine-free coffee induced prophage lambda in lysogenic E . coli K12, strain GY5027 . Because coffee prepared from green beans by the same extraction method as used for freshly brewed blended coffee had no prophage-inducing activity, this activity may be attributed to compounds produced in the roasting process . Roasting also produced compounds that were mutagenic in S . typhimurium TA100 and E . coli WP2 uvrA/pKM101. Mutat Res, 1983 Mar, 108(1-3), 13 - 27 Identification of uracil as a major lesion in E . coli DNA following the incorporation of 5-bromouracil, and some of the accompanying effects; Szyszko J et al.; Cultivation of E . coli cells in the presence of 5-bromodeoxyuridine (BUdR) leads to formation of lesions in the cellular DNA which affect its secondary structure, as reflected by changes in temperature profiles . Such DNA contains single-stranded regions susceptible to endonuclease S1 . One of the major sources of the BU-induced lesions appears to be dehalogenation of incorporated 5-bromouracil (BU) residues, with accompanying formation of uracil . The presence of uracil residues in such DNA was demonstrated directly by chromatography of hydrolyzates, and by the susceptibility of such residues to uracil-DNA glycosylase . The number of uracil residues was dependent on the extent of damage in the DNA, and decreased during the DNA repair that accompanied reactivation of bromouracil-inactivated cells . Dehalogenation of incorporated BU presumably results in formation of apyrimidinic sites by uracil-DNA glycosylase, and then single-strand nicks either by AP-endonuclease and/or dehalogenation . The findings are relevant to the mechanism of BU-induced mutagenesis. J Immunol Methods, 1983 Feb 25, 57(1-3), 253 - 64 A new method for assessment of serum-induced damage to E . coli; Zabucchi G et al.; A simple and rapid spectrophotometric assay for the kinetic evaluation of serum-induced damage to E . coli is described, based on changes in the optical density (OD) of a bacterial suspension . Exposure of antibody-coated E . coli to human absorbed serum results in a diphasic response, namely an increase in OD, which reaches a maximum at about 17 min and is followed by a progressive decrease in OD until a minimum value is reached after 45 min . The increase and the decrease in OD are related to bacterial death and bacterial lysis, respectively. Nucleic Acids Res, 1983 Feb 11, 11(3), 605 - 17 Chemical reactivity of E . coli 5S RNA in situ in the 50S ribosomal subunit; Silberklang M et al.; E . coli 50S ribosomal subunits were reacted with monoperphthalic acid under conditions in which non-base paired adenines are modified to their 1-N-oxides . 5S RNA was isolated from such chemically reacted subunits and the two modified adenines were identified as A73 and A99 . The modified 5S RNA, when used in reconstitution of 50S subunits, yielded particles with reduced biological activity (50%) . The results are discussed with respect to a recently proposed three-dimensional structure for 5S RNA, the interaction of the RNA with proteins E-L5, E-L18 and E-L25 and previously proposed interactions of 5S RNA with tRNA, 16S and 23S ribosomal RNAs. Nucleic Acids Res, 1983 Feb 11, 11(3), 575 - 89 Structural investigation of Phe-tRNAPhe from E.coli bound to the ribosomal A-site; Bertram S et al.; Kethoxal modification of guanosines within Phe-tRNAPhe from E . coli was studied for tRNA in the free state and specifically bound to the ribosomal A-site . Complex formation with the ribosome results in a protection from chemical modification of two distant sites in the tRNA molecule . The guanosines affected are G-18 and G-19, located in the D-loop, and G-34 in the anticodon loop . Modification of Phe-tRNAPhe in the absence of ribosomes leads to a destabilisation of the tRNA structure . Our data are consistent with the conclusion that modification of G-34 at the anticodon loop triggers a conformational instability in distant parts of the tRNA molecule. Nucleic Acids Res, 1983 Feb 11, 11(3), 727 - 36 Identification of clones carrying an E . coli tRNAPhe gene by suppression of phenylalanyl-tRNA synthetase thermosensitive mutants; Caillet J et al.; Two libraries of cloned E . coli DNA were screened for plasmids which complemented thermosensitive phenylalanyl-tRNA synthetase mutants . Four plasmids were isolated which complemented pheS and pheT thermosensitive mutations but which do not carry pheS or pheT, the structural genes for phenylalanyl-tRNA synthetase . All these plasmids increased the intracellular tRNAPhe concentration . Three plasmids were shown to carry the structural gene for tRNAPhe which we call pheU . By restriction enzyme analysis, DNA blotting and DNA:tRNA hybridization, pheU was localised to a 280 bp fragment within a 5.6 kb PstI restriction fragment of E.coli DNA. J Inorg Biochem, 1983 Feb, 18(1), 49 - 58 Additional effects of monovalent cations on the lysine-sensitive aspartokinase of E . coli B; Shaw JF et al.; The apparent specificity of activation of lysine-sensitive aspartokinase (E.C.2.7.2.4) from E . coli by monovalent cations differs depending on the assay used and on the Mg2+ concentration . Activity is nearly absolutely dependent on and is highly specific for a monovalent cation in the aspartate semialdehyde dehydrogenase coupled assay or the adenosine triphosphate-adenosine diphosphate exchange assay . Little specificity for monovalent cations is observed using the aspartyl hydroxamate assay . Activation and specificity are also altered by Mg2+ concentrations at a constant 5 mM nucleotide concentration . At a low (1.25 or 1.6 mM)Mg2+ concentration, monovalent cation activation and specificity are nearly absolute . Less dependence on monovalent cations and less specificity are observed at a higher Mg2+ concentration (6 mM) . Li+ inhibits aspartokinase competitively with respect to either K+ or NH4+ . Monovalent cations are also thermoprotective and differential thermal inactivation experiments at 56 degrees C reveal that NH4+ and K+, either of which will produce maximum catalytic activity, interact differently with aspartokinase . K+ interacts with positive cooperativity, whereas NH4+ does not . K+, NH4+, and Na+ are about equally effective in enhancing the dissociation of the aspartokinase-aspartylphosphate complex . Li+ is less effective. Cell, 1983 Feb, 32(2), 335 - 49 The operon that encodes the sigma subunit of RNA polymerase also encodes ribosomal protein S21 and DNA primase in E . coli K12; Burton ZF et al.; The sigma subunit of E . coli RNA polymerase is encoded by the rpoD gene . Within the sequence upstream from rpoD, we have identified the structural genes rpsU and dnaG, which encode the 30S ribosomal protein S21 and DNA primase, respectively . The three genes are in the order rpsU, dnaG rpoD, and are all encoded by the same DNA strand . Analysis of in vivo transcripts from this region shows that these genes are all within the same operon . By correlating the 5' and 3' ends of in vivo transcripts with our DNA sequence, we have identified several regulatory features of the operon . These features include tandem promoters upstream from rpsU, a terminator between rpsU and dnaG, an RNA processing site separating dnaG and rpoD, and the operon terminator just downstream from rpoD . Immediately upstream of the operon promoters is an active promoter for an unidentified gene . We discuss the regulatory significance of the operon features and the biological significance of an operon encoding proteins essential for translation, replication and transcription. Nucleic Acids Res, 1983 Jan 11, 11(1), 225 - 35 Genes for tRNAIle and tRNAAla in the spacer between the 16S and 23S rRNA genes of a blue-green alga: strong homology to chloroplast tRNA genes and tRNA genes of the E . coli rrnD gene cluster; Williamson SE et al.; A 6.3 kbp Eco RI-Bam HI fragment which carries most of one of the two rRNA gene clusters of the blue-green alga Anacystis nidulans was cloned into plasmid pBR322 . Sequence analysis of the spacer region between the 16S and 23S rRNA genes reveals the presence of genes for tRNAIle and tRNAAla . The 16S rRNA gene is separated from the tRNAIle gene by a 162 bp spacer which shows significant homology to the comparable region in Zea mays plastids . The spacer between the two tRNA genes is 33 bp long and can be folded into a 9 bp stem and loop structure . The 5' portion of the tRNAIle gene is 60% homologous to a "pseudogene"-like sequence which maps beyond the 5S rRNA gene. Adv Shock Res, 1983, 9, 157 - 69 Peripheral vascular adrenergic depression during hypotension induced by E coli endotoxin; Bond RF; Previous studies support the concept that most vascular beds exhibit some degree of blood flow autoregulation (ie, vasodilation) in response to a fall in local perfusion pressure . However, when systemic pressure is reduced by acute hemorrhage, this vasodilation is converted to vasoconstriction . This response is due to extrinsic influences (ie, elevated adrenergic activity) which overwhelm the intrinsic autoregulatory response . The objective of the present investigation was to compare the regional vascular behavior in selected tissues (ie, skeletal muscle, skin, mesenteric and renal) during local hypotension and systemic hypotension induced by stepwise hemorrhage and endotoxin . Previously published data on local hypotension accomplished by gradual occlusion of the arterial inflow, and systemic hypotension induced by stepwise hemorrhage were compared to systemic hypotension induced by IV administration of 2 mg/kg E coli endotoxin . The pressure/vascular conductance data suggest the following: 1) Skeletal muscle, mesenteric, and renal vasculature respond to local hypotension by autoregulating while skin exhibits passive vasoconstriction; 2) all four vascular beds respond to hemorrhage by intense vasoconstriction; and 3) all four vascular beds respond to endotoxin by vasodilating . The data are compatible with the concept that endotoxin inhibits the extrinsic compensatory vasoconstrictor response to systemic hypotension in all of these vascular beds. Physiol Chem Phys Med NMR, 1983, 15(5), 417 - 23 Repression of lysine transport in E . coli KL16; Di Girolamo M et al.; Data reported in this paper show that both lysine transport systems in E . coli KL16 can be repressed by lysine and its isologs, thialysine and selenalysine, whereas they are not repressed by ornithine . The repression is specific on lysine transport systems; it is evident with 0.01 mM lysine or isolog concentration and reaches a maximum with 0.1 mM concentration . By comparing the extent of repression by lysine and its isologs, lysine gives the highest and selenalysine the lowest degree of repression . The shift from the repressed to the depressed state is rather immediate once the amino acid is removed from the culture medium. Physiol Chem Phys Med NMR, 1983, 15(2), 121 - 5 Transport systems for lysine, thialysine and selenalysine in E . coli KL16; Busiello V et al.; Two lysine transport systems have been identified in E . coli KL16 . They differ in their affinity for lysine, one showing a KM of 0.36 microM and the other a KM of 4.7 microM . Different compounds with chemical similarities to lysine were tested for their capacity to interfere with lysine transport . Among these only thialysine and selenalysine competitively inhibit lysine transport . The inhibition is on both transport systems . Thialysine shows a KI of 4 microM for the low affinity system and a KI of 8 microM for the high affinity system . Selenalysine shows values of 6 microM and 12 microM respectively. Nucleic Acids Symp Ser, 1983, (12), 87 - 90 Construction of expression plasmids producing high levels of human immuno interferon in E . coli; Nomura M et al.; Improved expression vectors have been constructed which are derived from runaway-replication mutants of plasmid pYM307 and carry the strong hybrid promoter tac with lac iq gene . The activity of this promoter is controlled by lac repressor, product of the lac iq gene . Heat induction leads to amplification of the plasmid copy number . This system was used for high level expression of the chemically synthesized gene for human immune interferon (hIFN-7) . 3 h after induction at 37 degrees C the hIFN-7 amounted to about 20% of total cellular proteins. Ital J Biochem, 1983 Jan-Feb, 32(1), 19 - 27 Thialysine and selenalysine utilization for protein synthesis by E . coli in comparison with lysine; Coccia R et al.; Utilization of thialysine and selenalysine for protein synthesis by a lysine requiring E . coli mutant was studied . Incorporation into proteins of thialysine or selenalysine, added to culture medium together with lysine, becomes evident when the amount of available lysine in the medium is highly reduced, that is the mutant utilizes the isologs only after all the available natural aminoacid has been utilized . Compared to selenalysine, thialysine is better utilized; when both isologs are present in the medium at equal concentrations, up to 46% of protein lysine is substituted by thialysine and only 12% by selenalysine. Mol Cell Biochem, 1983, 52(2), 177 - 89 Soluble pig intestinal cell membrane components with affinities for E . coli K88+ antigen; Staley TE et al.; Pig intestinal brush borders (BB) were radiolabeled by iodination using the lactoperoxidase-hydrogen peroxide procedure . The BB were then detergent solubilized, centrifuged to remove particulate material, and chromatographed on Sepharose CL-4B . The fractions were incubated with K88+ E . coli using an in vitro binding assay . Binding of the iodinated membranes to K88+ E . coli occurred throughout a wide range of molecular weight components, in excess of 690K daltons to near 25K daltons . The system utilizing intact K88+ E . coli and solubilized BB was shown to be saturable . Prior contact of K88+ E . coli with nonradiolabeled membranes or specific antibodies to K88+ pili inhibited binding of the radiolabeled BB . Simple sugars were tested for their ability to block binding of the labeled BB; partial inhibition occurred with galactose (17.9%), galactosamine (32%), glucose (10.6%), and N-acetylglucosamine (32%) . Calcium enhanced binding with as little as 10 microM . A 10 x increase in binding occurred with 500 microM calcium . Affinity chromatography using K88+ pili coupled on agarose beads avidly bound the labeled BB . The receptor membranes were eluted with high molar concentrations of salt, however considerable degradation occurred . Despite low yields from the affinity system, receptor membranes with higher binding activities were recovered . Protein: glycoprotein ratios were 1:4 . Elution with SDS and electrophoresis on 12.5% polyacrylamide gels in the presence of a reducing agent produced two major subunits 35--32K and 23K daltons . These components were recovered from the gels and retained their binding activity . This information suggests that the intestinal receptor responsible for binding of K88+ E . coli is a glycoprotein, that in the native state exists in multimeric forms. G Batteriol Virol Immunol, 1983 Jan-Jun, 76(1-6), 20 - 4 {Bacterial lipopolysaccharides: comparative analysis of the cardiovascular effects of E . coli, S . sonnei and Y . enterocolitica in anesthetized rats}; Altavilla D et al.; Intravenous injection of S . sonnei LPS (640 mcg/kg) and of Y . enterocolitica LPS (1280 mcg/kg) elicited a decrease in mean arterial pressure and heart rate in urethane anaesthetized rats . The bradycardia reached a maximum when hypotension had already reverted, thus suggesting direct and independent effects on the vessels and on the heart. Pediatr Pharmacol (New York), 1983, 3(3-4), 303 - 11 Modified pharmacokinetics of I-asparaginase from E coli by formation of specific antibodies to I-Asparaginase of different immunoglobulin classes in children with acute lymphocytic leukemia; Wahn V et al.; Twenty-four children (2-15 years old) with acute lymphocytic leukemia (ALL) were treated intravenously with 1-Asparaginase (1-Asp) isolated from E coli at a dose of 3,000 U/kg body weight four times every third day as part of a standard chemotherapy protocol . Sera of patients were obtained prior to each infusion, immediately following each infusion, and at defined intervals (2, 4, 12, 24, 36, and 48 hours postinfusion) and assayed for 1-Asp enzymatic activity.1-Asp antigen, and anti-1-Asp antibodies . Results indicate that the in-vivo elimination half-life of 1-Asp activity in patients with no demonstrable specific antibody is approximately 5.5 hours . Half-life of enzymatic activity in patients with a moderately high level of specific antibodies (pre-infusion) was prolonged (approximately 7.0 hours) in comparison to the group with no specific antibodies . In patients with very high levels of specific antibodies several infusions could not be completed because of apparent anaphylactic reactions . In-vitro studies showed that experimental immune complexes made of 1-Asp and the IgG-fraction of a rabbit-anti-1-Asp antibody under conditions of antigen excess still exhibit enzymatic activity . On the basis of this observation we conclude that specific antibodies to 1-Asp in vitro and, most likely, in vivo do not inactivate the drug but may lead to either delayed elimination of enzyme activity or, in the presence of high levels of specific antibodies, anaphylactic reaction. Radiat Environ Biophys, 1983, 22(4), 281 - 91 Radiobiological effect of heparin in Swiss mice, human amnion cells and E . coli B/r irradiated with Co60 gamma-rays; Chaubal KA et al.; The radiobiological action of Heparin was investigated using the test systems E . Coli B/r, Human Amnion (HA) cells and Swiss mice . The Heparin treatment of these systems effected following changes in their response towards irradiation with Co60 gamma-rays: (a) more sensitization of E . coli B/r in hypoxic than in oxic condition, (b) no significant modification for HA cells in oxic condition but their sensitization under hypoxia, (c) larger recovery of anodic electrophoretic mobility of irradiated HA cells, (d) increased life span and smaller reduction in the splenic and thymus weights of irradiated Swiss mice . It seems, therefore, that Heparin, a natural molecule of animal world, possesses the potentiality to modify radiation response of living systems and may find useful application in radiation therapy. Ann Rech Vet, 1983, 14(3), 319 - 31 Infant mouse model of E . coli diarrhoea: clinical protection induced by vaccination of the mothers; Duchet-Suchaux M; Protection of infant mice against experimental E . coli B41 diarrhoea by immunization of the mothers with homologous E . coli strain was studied . The influence of the number, dose, route and moment of vaccination(s) on protection, measured by the reduced mortality rate of infant mice duri