J Immunol Methods, 1984 Jul 6, 71(2), 203 - 10 Development of a sensitive solid-phase radioimmunoassay technique for quantification of class-specific Escherichia coli antibodies; Stephens S; A specific and sensitive, quantitative solid-phase radioimmunoassay (RIA) has been developed for the detection of Escherichia coli antibodies in serum and secretions . Preparation of affinity-purified anti-E . coli standards allows accurate quantification of G, M and A classes of antibody down to 10 ng/ml using only 20 microliters of sample . This technique has considerable advantages over indirect haemagglutination in sensitivity and accuracy of immunoglobulin class detection . RIA also compares favourably with ELISA in sensitivity and sample size required . Affinity-purified standards may also be used to quantify the ELISA test. J Mol Biol, 1984 Jul 5, 176(3), 431 - 42 Roles of the 5' leader region of the ompA mRNA; Green PJ et al.; OmpA protein, a major outer membrane protein of Escherichia coli, is synthesized from a messenger RNA containing a 134-nucleotide 5' leader region . The role of this leader region in efficient ompA expression was investigated using a series of ompA-lacZ fusion plasmids . These plasmids differ in the amount of DNA encoding the ompA leader region which is fused to the lacZ structural gene . The fusion containing all but six nucleotides of the ompA leader produced the highest beta-galactosidase activity, while the fusion containing the shortest leader synthesized only 4% as much beta-galactosidase . Fusions with leaders intermediate in length produced between 6% and 24% of the activity found in the most efficient fusion . Quantitation of lacZ mRNA synthesis by DNA-RNA hybridization revealed differences in lacZ mRNA production reflecting the observed differences in beta-galactosidase activity . The primary effect of the ompA leader in maintaining high levels of mRNA is discussed in terms of the roles of mRNA secondary structure. Biochemistry, 1984 Jul 3, 23(14), 3330 - 5 Probing the conformation of 26S rRNA in yeast 60S ribosomal subunits with kethoxal; Hogan JJ et al.; The conformation and accessibility of 26S rRNA in yeast 60S ribosomal subunits were probed with kethoxal . Oligonucleotides originating from reactive sites were isolated by diagonal electrophoresis and sequenced . From over 70 oligonucleotide sequences, 26 kethoxal-reactive sites could be placed in the 26S rRNA sequence . These are in close agreement with a proposed secondary structure model for the RNA that is based on comparative sequence analysis . At least seven kethoxal-reactive sites in yeast 26S rRNA are in positions that are exactly homologous to reactive positions in E . coli 23S rRNA; each of these sites has previously been implicated in some aspect of ribosomal function. Biochemistry, 1984 Jul 3, 23(14), 3136 - 43 Elementary steps in the reaction mechanism of the alpha-ketoglutarate dehydrogenase multienzyme complex from Escherichia coli: kinetics of succinylation and desuccinylation; Waskiewicz DE et al.; The kinetics of the succinylation and the desuccinylation of the alpha-ketoglutarate dehydrogenase complex from Escherichia coli have been studied at 4 degrees C in 2 mM thiamin pyrophosphate, 2 mM MgCl2, and 20 mM potassium phosphate (pH 7.0) by steady-state and quenched-flow techniques . The initial steady-state velocity for the reaction of the complex is inhibited by high concentrations of alpha-ketoglutarate . The data are consistent either with cooperative interactions between two catalytic sites or with the existence of an alpha-ketoglutarate regulatory site . The time course of the succinylation by alpha-ketoglutarate of the unmodified complex or the complex in which a fraction of the alpha-ketoglutarate decarboxylase subunits (E1) has been inhibited with N-ethylmaleimide reveals a complex kinetic process . A mechanism consistent with the kinetic data is proposed in which some E1 subunits succinylate one lipoic acid per E1 and other E1 subunits succinylate two lipoic acids per E1 . Furthermore, each succinylation reaction occurs via a two-step process with rate constants of 49 and 89 s-1 at saturating concentrations of alpha-ketoglutarate for the first and second steps, respectively . At long times, 13-16 mol of succinate binds per mol of unmodified complex . The stoichiometry of binding obtained with N-ethylmaleimide-treated complex is initially lower but approaches the same values as for the unmodified complex over the course of minutes . Coenzyme A removes the succinyl groups on the unmodified enzyme with a rate constant greater than or equal to 200 s-1 . The results obtained suggest a limited accessibility between sites on the complex. Biochemistry, 1984 Jul 3, 23(14), 3111 - 4 Exploring the conformational roles of signal sequences: synthesis and conformational analysis of lambda receptor protein wild-type and mutant signal peptides; Briggs MS et al.; Secretion of the Escherichia coli lambda receptor protein (LamB protein) appears from genetic evidence to be correlated with the predicted tendency of its signal sequence to adopt an alpha-helical conformation {Emr, S . D., & Silhavy, T . J . (1983) Proc . Natl . Acad . Sci . U . S . A . 80, 4599} . We have tested this hypothesis by synthesizing major portions of signal sequences from the wild-type and mutant LamB proteins and analyzing their conformations by circular dichroism . The wild-type signal sequence contains a seven-residue hydrophobic region flanked by a proline and a glycine . Chou-Fasman rules predict that this segment will adopt an alpha-helical conformation . An export-deficient mutant is missing four residues from this region; the helix-breaking glycine and proline are thus separated by only three residues, and an alpha helix is not predicted to form . In each of the export-restored revertants, either the glycine or the proline is replaced with a residue which promotes helix formation . The helix content of the synthetic signal sequence fragments on the basis of CD measurements supports the secondary structure hypothesis described above . The relative helicity in aqueous sodium dodecyl sulfate, lysolecithin, or trifluoroethanol is as follows: wild type greater than R2 (Pro----Leu) greater than R1 (Gly----Cys) much greater than deletion mutant. Biochemistry, 1984 Jul 3, 23(14), 3322 - 30 Probing the conformation of 18S rRNA in yeast 40S ribosomal subunits with kethoxal; Hogan JJ et al.; Yeast 40S ribosomal subunits have been reacted with kethoxal to probe the conformation of 18S rRNA . Over 130 oligonucleotides were isolated by diagonal electrophoresis and sequenced, allowing identification of 48 kethoxal-reactive sites in the 18S rRNA chain . These results generally support a secondary structure model for 18S rRNA derived from comparative sequence analysis . Significant reactivity at positions 1436 and 1439, in a region shown to be base paired by comparative analysis, lends support to the earlier suggestion {Chapman, N.M., & Noller, H.F . (1977) J . Mol . Biol 109, 131-149} that part of the 3'-major domain of 16S-like rRNAs may undergo a biologically significant conformational rearrangement . Modification of positions in 18S rRNA analogous to those previously found for Escherichia coli 16S rRNA argues for extensive structural homology between 30S and 40S ribosomal subunits, particularly in regions thought to be directly involved in translation. Eur J Biochem, 1984 Jul 2, 142(1), 161 - 4 Two different aldolase A mRNA species in rat tissues; Tsutsumi R et al.; Double-stranded DNA was synthesized with reverse transcriptase from size-fractionated poly(A)-containing RNA from rat ascites hepatoma cells . The cDNA was introduced into Escherichia coli HB101 using pBR322 DNA as a cloning vector . Several plasmids containing aldolase A cDNA were identified by colony hybridization with 32P-labeled cDNA prepared from immunologically purified aldolase A mRNA . The partial amino acid sequence of the cDNA sequence was determined, and found to coincide with that of rabbit aldolase A . Using aldolase A cDNA as a hybridization probe, the aldolase A mRNA concentrations in various rat tissues were analysed, and two aldolase A mRNA species differing in nucleotide length were found; the smaller mRNA (about 1550 nucleotides) in muscle, and the larger one (about 1650 nucleotides) in brain and hepatoma cells. Cell, 1984 Jul, 37(3), 1001 - 7 Heterogeneous mitochondrial DNA D-loop sequences in bovine tissue; Hauswirth WW et al.; Mitochondrial DNA from bovine tissue contains heterogeneous sequences located within an evolutionary conserved cytosine homopolymer sequence near the 5' end of the D-loop region . This part of the mammalian mitochondrial genome is known to contain the origin of heavy strand DNA synthesis and the major transcriptional promoter for each strand . Nucleotide sequence analysis of cloned DNA and electrophoretic analysis of appropriate small fragments from animal tissue reveal a population of length polymorphs containing from nine to 19 cytosine residues . No individual length species represents more than 40% of the population . These data imply a state of significant intraanimal mtDNA sequence heterogeneity, which most likely occurs intracellularly as well . The localization of variability to a homopolymer run suggests that replication slip-page generated the sequence population . We also report that when recombinant clones containing this region are repeatedly passaged in E . coli, they begin to regenerate length variation similar to that seen in animal mtDNA. Br J Surg, 1984 Jul, 71(7), 537 - 9 The effect of tetracycline lavage and trauma on visceral and parietal peritoneal ultrastructure and adhesion formation; Phillips RK et al.; The clinical efficacy of tetracycline lavage (1 mg/ml) in the management of abdominal sepsis has led to advocacy of its use in potentially contaminated cases . Yet at higher concentrations (6 mg/ml), tetracycline is a pleural sclerosant . The possibility of early ultrastructural peritoneal damage and later adhesion formation has been examined in syngeneic female Wag rats . At high concentration (10 mg/ml), tetracycline caused adhesions in the absence of peritoneal trauma and there was an associated loss of serosal microvilli . Lavage with low concentration tetracycline (1 mg/ml) or saline after clean abdominal surgery led to more adhesions than if no lavage was employed . There was an unexplained paradoxically low incidence of adhesions if prior mild contamination of the peritoneal cavity with 1 ml 10(5) E . coli had been performed. Cancer Res, 1984 Jul, 44(7), 2855 - 7 O6-alkylguanine-DNA alkyltransferase activity in normal human tissues and cells; Grafstrom RC et al.; Normal adult human tissues and cultured bronchial epithelial cells and fibroblasts exhibit O6-alkylguanine-DNA alkyltransferase activity in vitro by catalyzing the repair of the promutagenic alkylation lesion O6-methylguanine from DNA . The amount repaired by extracts of liver, peripheral lung, and colon extracts was proportional to the amount of extract protein . Repair of O6-methylguanine led to stoichiometric regeneration of guanine in the DNA and stoichiometric formation of S-methylcysteine in protein . Alkyltransferase activity varies in the different human tissues tested in the decreasing order of liver greater than colon greater than esophagus greater than peripheral lung greater than brain . Extracts of lung tissues, cultured human bronchial epithelial cells, and fibroblasts had similar alkyltransferase activities . Various human tissues exhibit 2- to 10-fold higher alkyltransferase activity than corresponding rat tissues . Whereas the interindividual variation of the activity was 4- to 5-fold in ten or more human lung and colon specimens, the interindividual variation in the inbred rat was less than 20% . The present results show that different human tissues and cells have a several-fold higher capacity to repair O6-methylguanine in DNA than do rat tissues and that the repair process occurs via a mechanism similar to that shown previously in other mammalian cells and Escherichia coli. Biokhimiia, 1984 Jul, 49(7), 1059 - 65 {Availability of DNP-modified 5'- and 3'-ends of the poly(U) fragment bound to the 30S ribosomal subunit for interaction with antibodies}; Evstaf'eva AG et al.; Modification of the 5'- or 3'-terminal nucleotide residues of poly(U) by dinitrophenyl haptens was carried out . The accessibility of the modified ends of short poly(U) (20-70 nucleotide residues) bound to the 30S subunit of E . coli ribosomes for the interaction with antibodies that are specific against the dinitrophenyl group, was investigated . Some peculiarities of the structural organization of the "entry" (3'-end) and "exit" (5'-end) sites of the template polynucleotide on the 30S subunit and the possibility of the template polynucleotide folding on the ribosome are discussed. Mol Pharmacol, 1984 Jul, 26(1), 135 - 40 Lethality associated with incorporation of 5-fluorouracil into preribosomal RNA; Herrick D et al.; We have previously demonstrated a highly significant relationship between incorporation of 5-fluorouracil (FUra) into total cellular RNA and loss of clonogenic survival . The present study extends these findings by demonstrating a similar relationship between incorporation of FUra into preribosomal nuclear RNA (45 S and 32 S) and lethal cellular events . The results also demonstrate that the extent of FUra incorporation into preribosomal RNA (prRNA) correlates significantly with inhibition of maturation to cytoplasmic 28 S and 18 S rRNA . These findings suggest that the incorporation of FUra into prRNA alters recognition sites involved in the processing of 45 S and 32 S RNA . These data are further supported by our finding of an enhanced degradation of (FUra)prRNA by RNase III, an enzyme implicated in the maturation of Escherichia coli prRNA and T7 mRNA . These observations suggest that the incorporation of FUra into prRNA is responsible for altered processing to cytoplasmic rRNA and cell lethality. J Clin Microbiol, 1984 Jul, 20(1), 59 - 64 Simple and reliable enzyme-linked immunosorbent assay with monoclonal antibodies for detection of Escherichia coli heat-stable enterotoxins; Thompson MR et al.; We have developed a sensitive and specific competitive enzyme-linked immunosorbent assay for Escherichia coli heat-stable enterotoxins consisting of methanol-soluble, suckling mouse active peptides with similar core sequences (STa) by using monoclonal antibodies prepared against STa purified from a human isolate . The assay can detect 3 to 20 pg of purified STa, depending on the monoclonal antibody used in the assay . The assay is rapid, requiring ca . 1 h to complete . With this competitive enzyme-linked immunosorbent assay, we measured STa production by enterotoxigenic E . coli directly in Casamino Acid-yeast extract culture supernatants . The assay was suitable for measuring STa in culture supernatants from human, bovine, and porcine E . coli isolates . No cross-reactivity was observed with heat-labile enterotoxin, cholera toxin, or heat-stable enterotoxin STb, which is a methanol-insoluble peptide(s) active in the ligated pig jejunal loop test . A 100% correlation of toxin production was found by comparing the enzyme-linked immunosorbent assay with the previously established radioimmunoassay for STa and with suckling mouse activity. Vet Microbiol, 1984 Jul, 9(3), 287 - 99 The effect of oral immunization on the population of lymphocytes migrating to the mammary gland of the sow; Kortbeek-Jacobs JM et al.; Sows were immunized orally with live Escherichia coli according to various immunization schedules . Six pregnant gilts were used; 4 immunized at various intervals during the last month of gestation, 1 control immunized after parturition following suppression of lactation by weaning and 1 non-immunized control . The effect of oral vaccination on cell populations from lymphoid organs was studied . The in vitro proliferative responses of the cell populations to K88 antigen, anti-Ig sera and mitogens were used to demonstrate the distribution of sensitized lymphocytes over different lymphoid organs . The capacity of these cells to produce antigen-specific Ig was determined by in ovo translation of their mRNA . Oral administration of antigen resulted in the appearance of K88-positive cells in lymphoid organs . In lactating sows, sensitized cells preferentially occurred in the mammary lymph nodes, whereas after suppression of lactation such a distribution was not seen . A possible route of migration of sensitized lymphocytes is discussed in relation to the local immune response . The antibody isotype produced by sensitized lymphocytes seemed to depend on the immunization schedule . The most effective schedule was one starting early in gestation and comprising frequent administration of antigen . This caused an optimal distribution of sensitized lymphocytes capable of IgA production. Rev Infect Dis, 1984 Jul-Aug, 6(4), 528 - 31 Lipid A-dependent lymphocyte proliferation in "endotoxin-nonresponder" mice; Vukajlovich SW et al.; Lipopolysaccharides (LPS) with a subunit composition reduced in heterogeneity were prepared by gel-filtration chromatography of detergent-dissociated LPS from the smooth strain of Escherichia coli 055:B5 . The splenocyte mitogenic activity of reassociated column-fraction pools was markedly dependent on the ratio of O-antigen polysaccharide to lipid A, where the activity of the fraction richest in lipid A (F5 LPS) was approximately three orders of magnitude greater than the least active fraction (F1 LPS) . The F5 LPS, which contains only a trace of O-antigen polysaccharide, induces a spleen-cell proliferative response in C3H/HeJ "LPS nonresponder" spleen cells . This mitogenic activity is not present in either unfractionated LPS or the other column fractions . These latter findings indicate that LPS macromolecular aggregates of the appropriate physico-chemical structure and/or compositions have the capacity to elicit C3H/HeJ spleen-cell proliferative responses. Rev Infect Dis, 1984 Jul-Aug, 6(4), 488 - 92 Physical interaction between lipid A and phospholipids: a study with spin-labeled phospholipids; Takeuchi Y et al.; When mixed bilayers containing spin-labeled phosphatidylethanolamine (or phosphatidylglycerol) and Escherichia coli B lipopolysaccharide were prepared, electron spin resonance signals indicated that the patches that were initially present and contained only phospholipids or only lipopolysaccharides were unusually stable and that little lateral diffusion of phospholipids into lipopolysaccharide domains, or vice versa, took place . These results explain how the outer layer of the outer membrane, which essentially contains only lipopolysaccharides in addition to proteins, can be generated and maintained stably in bacterial cells . Furthermore, the stability of pure lipopolysaccharide domains may have important implications in the mode of action of endotoxins in the body of the host . For example, lipopolysaccharide molecules may tend to form stable domains or patches spontaneously in the animal cell membrane, and special mechanisms (such as the binding to a special receptor) may be needed to disperse the lipopolysaccharide molecules within the host cell membranes. Rev Infect Dis, 1984 Jul-Aug, 6(4), 478 - 82 Recent developments in the organic synthesis of lipid A in relation to biologic activities; Shiba T et al.; For elucidation of a relationship between chemical structure and biologic activity of lipid A, syntheses of 13 derivatives of glucosamine beta (1-6) disaccharide, which were acylated at 3, 4, and 6'-hydroxyl groups and/or phosphorylated at positions 1 and 4', were carried out . The results showed the importance of the presence of the phosphate group at either position 1 or 4' and of the beta-hydroxyl group in acyl function, particularly in N-linked fatty acids, for the exhibition of biologic activities characteristic of lipid A . New evidence on the positions of fatty acids in natural lipid A of Escherichia coli species obtained by means of 2D-H nuclear magnetic resonance was also demonstrated; the findings indicate that the 3'-hydroxyl group is acylated and the 6'-hydroxyl group in the molecule must be free. Gene, 1984 Jul-Aug, 29(1-2), 27 - 31 One-step purification of hybrid proteins which have beta-galactosidase activity; Ullmann A; A one-step purification method of hybrid proteins exhibiting beta-galactosidase activity, based on affinity chromatography in the presence of high salt concentration, is described . Starting from crude bacterial extracts, several milligrams of near-homogeneous proteins can be obtained in a few hours with an overall yield of 85 to 95% . The purified hybrid proteins can be used to obtain antibodies against the foreign portion of the protein fusion. Mutat Res, 1984 Jul-Aug, 132(1-2), 15 - 20 Expression of the SOS response following simultaneous treatment with methyl-nitrosoguanidine and mitomycin C in Escherichia coli; Vericat JA et al.; Simultaneous treatment of Escherichia coli cultures with methyl-nitrosoguanidine and mitomycin C induces recA-dependent inhibition of respiration but not inhibition of cell division . This pattern of SOS functions expression is the same as that is found following treatment with methyl-nitrosoguanidine alone and contrary to the pattern induced after mitomycin C addition . The same result is obtained when a culture of E . coli RecA441 (formerly tif) is shifted to 42 degrees C and treated simultaneously with methyl-nitrosoguanidine . The suppressor effect of this compound over the pattern of SOS functions expression induced by mitomycin C or high temperature in recA441 mutants is directly related to the increase in its dose . Moreover, the division temperature-sensitive mutant ftsA treated with methyl-nitrosoguanidine and high temperature does not show any decrease in its normal filamentous growth when cultured at 42 degrees C . This indicates that the effect of methyl-nitrosoguanidine on the recA-independent inhibition of cell division is not due to any indiscriminate effect of this compound over the division process . These results suggest that the specific kind of lesion caused in DNA is very important in determining which SOS function is induced. Carbohydr Res, 1984 Jul 1, 129, 243 - 55 Chemical modification and serological properties of the 3-deoxy-alpha-D-manno-2-octulosonic acid-containing polysaccharide from Escherichia coli LP1092; Jennings HJ et al.; The alpha-D configuration of the 3-deoxy-D-manno-2-octulosylonic acid residues in the E . coli LP1092 polysaccharide was definitively assigned by a comparison of its c.d . spectrum with those of methyl 3-deoxy-alpha- and -beta-D-manno-2-octulopyranosidonic acid . The c.d . spectrum of the polysaccharide showed a negative n----pi transition at 211 nm, associated with carboxyl groups, which correlated well with the negative band at 218 nm exhibited in the c.d . spectrum of the methyl alpha-D-glycoside . In contrast, the methyl beta-D-glycoside gave a positive band in the same region of its c.d . spectrum, at 221 nm . Treatment of the E . coli LP1092 polysaccharide with 3-(3-dimethylaminopropyl)-1-ethylcarbodiimide hydrochloride yielded a modified polysaccharide containing O-acylisourea groups and intramolecular lactones, in addition to some unesterified carboxyl groups . Both forms of ester could be reduced to hydroxymethyl groups by sodium borohydride . Although immuno-precipitation of an antiserum specific for the E . coli LP1092 polysaccharide is not sensitive to the introduction of O-acylisourea groups into the polysaccharide, precipitation was completely eliminated when approximately half of the carboxyl groups of the polysaccharide were converted either into lactone or hydroxymethyl groups . Failure to precipitate the antiserum can be attributed to significant conformational changes in the polysaccharide brought about by the introduction of the latter two groups. EMBO J, 1984 Jul, 3(7), 1567 - 72 Antibodies against a fused 'lacZ-yeast mitochondrial intron' gene product allow identification of the mRNA maturase encoded by the fourth intron of the yeast cob-box gene; Jacq C et al.; Several missense or nonsense mutations have been localized in the fourth intron open reading frame (ORF) of the yeast mitochondrial cytochrome b gene . These results and the phenotypes of mutants strongly suggested that a mRNA maturase, controlling the expression of both cytochrome b and cytochrome oxidase subunit I (COXI) genes, is encoded in this ORF . To investigate more directly the biosynthesis of mRNA maturase we raised antibodies against a part of the putative ORF translation product . For that purpose we inserted a fragment of the ORF sequence, in phase, into the C-terminal EcoRI site of lacZ gene . The hybrid gene was then expressed in Escherichia coli under the control of either the wild-type lac promoter or the thermoregulated lambda system PR/cI857 . The hybrid protein was partially purified and antibodies were raised against it . These antibodies recognized a mitochondrially coded protein, p27, in intron mutants, whereas no such protein was detected in the wild-type cell . These results demonstrate that the p27 protein, previously shown to be associated with the mRNA maturase activity, is actually translated from the intron ORF . The autoregulated mRNA maturase synthesis model is discussed in relation to these results. Biochem Pharmacol, 1984 Jul 1, 33(13), 2041 - 6 N-glucuronide formation of carcinogenic aromatic amines in rat and human liver microsomes; Lilienblum W et al.; (1) Sensitive fluorimetric assays were developed for the determination of microsomal UDP-glucuronosyltransferase activities towards 1- and 2-naphthylamine and 4-aminobiphenyl . (2) In rat liver microsomes, enzyme activity towards 1-naphthylamine was orders of magnitude higher than the activities towards 2-naphthylamine, 4-aminobiphenyl or aniline . The differences were less marked with human liver microsomes . (3) Glucuronidation of aniline and 4-aminobiphenyl was not appreciably altered in rat liver microsomes from 3-methylcholanthrene- or phenobarbital-treated rats . UDP-glucuronosyltransferase activities towards 1- and 2-naphthylamine were selectively increased (about 2-fold) by 3-methylcholanthrene-treatment . However the increases were less marked than those observed with representative substrates of the 3-methylcholanthrene-inducible enzyme form . The results suggest that the arylamines investigated are predominantly conjugated by constitutive enzyme forms in rat liver . (4) Arylamine N-glucuronides were found to be susceptible to hydrolysis by E . coli beta-glucuronidase suggesting the release of carcinogenic arylamines in the gut and their enterohepatic circulation. J Urol, 1984 Jul, 132(1), 179 - 83 The ultrastructure of acute pyelonephritis in the monkey; Fussell EN et al.; P-fimbriated E . coli, which have specific receptors on urothelial cells for their fimbriae, were used to produce pyelonephritis . Using both scanning and electron microscopy, it was shown that the bacteria attached to ureteral epithelium by 12 hours after inoculation . They developed into a biolayer of bacteria covering the epithelium by 48 hours, but did not invade within that time . In the kidney, adherence was seen by 6 hours in collecting proximal and distal tubules . While many bacteria were seen, tubular epithelium remained normal through 24 hours . The inflammatory response was first seen at 48 hours . Phagocytosis was accompanied by mortal damage to both phagocytes and surrounding tubules . This was followed by bacterial invasion of the interstitium. Mol Biol Evol, 1984 Jul, 1(4), 317 - 34 Immunological similarities between specific chloroplast ribosomal proteins from Chlamydomonas reinhardtii and ribosomal proteins from Escherichia coli; Schmidt RJ et al.; Polyclonal antibodies were elicited against seven of the 33 different proteins of the large subunit of the chloroplast ribosome from Chlamydomonas reinhardtii . Three of these proteins are synthesized in the chloroplast and four are made in the cytoplasm and imported . In western blots, six of the seven antisera are monospecific for their respective large subunit ribosomal proteins, and none of these antisera cross-reacted with any chloroplast small subunit proteins from C . reinhardtii . Antisera to the three chloroplast-synthesized ribosomal proteins cross-reacted with specific Escherichia coli large subunit proteins of comparable charge and molecular weight . Only one of the four antisera to the chloroplast ribosomal proteins synthesized in the cytoplasm cross-reacted with an E . coli large subunit protein . None of the antisera cross-reacted with any E . coli small subunit proteins . On the assumption of a procaryotic, endosymbiotic origin for the chloroplast, those chloroplast ribosomal proteins still synthesized within the organelle appear to have retained more antigenic sites in common with E . coli ribosomal proteins than have those which are now the products of cytoplasmic protein synthesis . Antisera to this cytoplasmically synthesized group of chloroplast ribosomal proteins did not recognize any antigenic sites among C . reinhardtii cytoplasmic ribosomal proteins, suggesting that the genes for the cytoplasmically synthesized chloroplast ribosomal proteins either are not derived from the cytoplasmic ribosomal protein genes or have evolved to a point where no antigenic similarities remain. Vopr Med Khim, 1984 Jul-Aug, 30(4), 127 - 32 {Method of determining low-molecular weight oligoamines in various biological materials}; Chudinov AA et al.; A modified procedure is described for thin-layer chromatography of dansyl-spermine, -spermidine, -cadaverine and -putrescine . Distinct separation of the dansyl derivatives studied from dansyl-ammonium, which usually causes difficulties, was achieved . This procedure enabled to estimate the pyckomolar quantities of biogenic oligoamines . The method does not require radioactive compounds, enables to carry out the measurements without the use of spectrofluorimeter or fluorescent densitometer . The densitometry of the chromatogram photo-prints was successfully used for quantitative estimation of the oligoamines. Mol Biol (Mosk), 1984 Jul-Aug, 18(4), 1042 - 52 {Study of the regulation of colicin E1 and K synthesis}; Rekesh AN et al.; Synthesis of colicins E1 and K and other plasmids was studied using DNA templates of corresponding plasmids in a cell-free system of coupled transcription and translation . Up to 15 plasmid-specific polypeptides differing by molecular weight were detected electrophoretically in conditions of complete denaturation . Samples of polypeptides related to plasmids ColE1 and ColK were similar in size . A complete or partial substitution of plasmid-free cell extracts by plasmid-containing cell extracts in a cell-free system leads to repression of the synthesis of colicins and some other proteins which is indicated by diminishing or complete disappearance of electrophoretic bands of corresponding polypeptides . The repression is relieved by addition of the recA gene protein into a cell-free system . Based on the obtained results, a conclusion is made that the repressors of colicin genes are not strictly specific and are, probably, coded by plasmids. Stain Technol, 1984 Jul, 59(4), 221 - 4 A method to identify microinjected nephrons of rat; Shimamura T et al.; This paper describes a technique for identifying individual nephrons that have been subjected to micropuncture . The general location of the nephron is marked on the surface of the kidney by implanting two micropipette tips on opposite sides of it two or three tubule diameters away . The tubule itself is marked by the injection into the lumen of a tracer material, for purposes of this account, a suspension of E . coli . After perfusion fixation the kidneys are removed and a block of tissue containing the extrapapillary portion of the nephron is excised . This block is cut into thin slices parallel to the surface of the kidney; these are embedded in plastic for subsequent sectioning . On sectioning, the marker material makes the nephron in question readily discernible under the microscope . A major advantage of this technique is that it allows the tubule of interest to be located as much as 48 hours later. Plasmid, 1984 Jul, 12(1), 37 - 40 A mutational hot spot in the incompatibility gene incC of mini-F plasmid; Seelke RW et al.; The incC incompatibility locus of the mini-F plasmid is also a replication control locus . We have selected and sequenced six independent incC mutations . Each mutation causes a partial loss of incompatibility . All mutations are GC----AT transitions . Five of six mutations occur in the same base pair located at coordinate 46.070 kb; the sixth mutation occurs in an adjacent base pair at 46.069 kb . Both mutational sites are in a hexanucleotide sequence common to several origins of replication. Plasmid, 1984 Jul, 12(1), 19 - 36 Factors affecting the isolation of CCC DNA from Streptomyces lividans and Escherichia coli; Kieser T; Based on the results of a systematic study of factors affecting plasmid yield and purity, a procedure suitable for the rapid screening for and isolation of covalently closed circular DNA from Streptomyces lividans and Escherichia coli was developed . The method consists of lysis of lysozyme-treated bacteria combined with alkaline denaturation of DNA at high temperature . Renaturation of CCC DNA and precipitation of single-stranded DNA together with protein is achieved by the addition of a minimal amount of phenol/chloroform . The screening procedure uses only a single tube and the samples can be analyzed by agarose gel electrophoresis about 30 min after lysis . Removal of phenol and further purification of the plasmid preparation is achieved by consecutive precipitations with isopropanol and spermine, followed by extraction with ethanol, producing samples suitable for restriction endonuclease digestion, ligation, and transformation of S . lividans protoplasts or competent E . coli cells in about 2 h . All steps of the procedure are explained in detail with information about the effects of changing parameters . This should help the experimenter to obtain reproducible results and may be useful if the method has to be adapted to new strains or plasmids. Infection, 1984 Jul-Aug, 12(4), 309 - 12 Studies on immunity against Escherichia coli K13 with monoclonal anti-K13 and anti-anti-K13; Soderstrom T et al.; The structural basis for the cross-reactivity between the Escherichia coli K13, K20 and K23 capsular polysaccharides is the ----)-beta-ribofuranosyl-(1----7)-beta-2-keto-3-deoxyoctonate polymer . Monoclonal antibodies against E . coli K13 which require O-acetyl-2-keto-3-deoxyoctonate for binding were further investigated . Such antibodies, of both the IgG and the IgM isotype, opsonized E . coli K13 in vitro and protected against intraperitoneal infection in mice as well as ascending pyelonephritis in rats . A monoclonal IgG1 anti-idiotype, specific for the K13 polysaccharide combining site of a protective IgM idiotype, primed for protection against intraperitoneal infection with live E . coli K13 following K13 injections at four as well as 12 weeks of age . the K13 polysaccharide alone did not immunize and protect . The monoclonal anti-K13 idiotype only primed for protection at four weeks of age . These findings suggest a strong effect of a single idiotype on the outcome of a bacterial infection. Gene, 1984 Jul-Aug, 29(1-2), 125 - 34 Overproduction of a gastrointestinal hormone, secretin, in Escherichia coli cells and its chemical characterization; Sumi S et al.; A cloned synthetic DNA fragment coding for 27-desamidosecretin (DAS), which differs from natural porcine secretin by the lack of the amide group at the carboxyl-terminal valine residue, was fused to the beta-galactosidase gene (lacZ) at the EcoRI site near the 3'-terminus of the gene . The fusion gene was efficiently expressed in Escherichia coli, yielding 1.15 X 10(6) fused-protein molecules per cell . After the treatment of the fused protein with cyanogen bromide, DAS was purified by a combination of ion-exchange column chromatography and reverse-phase high-performance liquid chromatography (HPLC) . The structure of bacterial DAS was confirmed by tryptic mapping and amino acid composition analyses . The bacterial DAS was not amidated at its carboxyl-terminal valine residue . Nevertheless, it stimulated pancreatic secretion in rats as does authentic porcine secretin, indicating that the carboxyl-terminal amide is not essential to secretin activity. Vet Q, 1984 Jul, 6(3), 134 - 40 Some pharmacokinetic aspects of four sulphonamides and trimethoprim, and their therapeutic efficacy in experimental Escherichia coli infection in poultry; Goren E et al.; Pharmacokinetic studies in broilers and layers of different sulphonamides indicate a good absorption and a long elimination half-life (of sulphaquinoxaline, sulphadimidine and to a lesser degree sulphadiazine) resulting in high plasma concentrations during drinking water medication in the recommended therapeutic doses . In contrast drinking water medication with high concentrations of trimethoprim (up to 1,320 mg/liter) resulted in a maximal mean plasma concentration of 1.2 micrograms/ml . Very good therapeutic effects were demonstrated in broilers experimentally infected with a sulphonamide-susceptible E . coli strain when treated with sulphaquinoxaline (200 mg/liter), sulphadimidine sodium (2 gram/liter), sulphachloropyridazine 30 per cent (1 gram/liter) and to a lesser degree sulphadiazine sodium (250 mg/liter) . Synergism was demonstrated between trimethoprim and sulphadiazine (1:5) . The combination of trimethoprim with sulphaquinoxaline (1:3) did not induce better therapeutic effects than sulphaquinoxaline in proportional doses . However, significant synergism was demonstrated between trimethoprim and both sulphonamides in treatment of experimental infection with sulphonamide-resistant E . coli . No signs resembling sulphonamide intoxication were observed during these studies. Kardiologiia, 1984 Jul, 24(7), 14 - 9 {Septic endocarditis in cardiosurgical clinical practice}; Solov'ev GM et al.; The authors analyze the experience of the treatment of eight patients with primary acute septic endocarditis (SE), 35 patients with rheumatic heart disease complicated by secondary subacute SE, 68 patients with early postsurgery SE including 25 who had had closed mitral comissurotomy, and 43 with prosthetic SE . Surgical treatment consisting in the removal of the infection source (the impaired valve or prosthesis) and the implantation of a new prosthesis should be carried out before the development of irreversible alterations of the internal organs. Biosci Rep, 1984 Jul, 4(7), 565 - 72 The immunity genes of colicins E2 and E8 are closely related; Lau PC et al.; We have determined the nucleotide sequence of the newly characterized colicin E8 imm gene which exists in tandem with the colicin E3 imm gene in the ColE3-CA38 plasmid . Comparison of these immunity structures reveals considerable sequence divergence, but the ColE8 imm gene is markedly homologous to the colicin E2 imm gene from the ColE2-P9 plasmid. Biochem Int, 1984 Jul, 9(1), 105 - 13 Purification and characterisation of a 12 K dalton peptide with an affinity for oriC containing DNA fragments from Escherichia coli K12 membrane fractions; Chen YQ et al.; A DNA binding protein capable of interacting specifically with oriC containing DNA was purified to near homogeneity from E . coli membrane fractions . It has a molecular weight of 12 KD in denaturing conditions . The specific binding to oriC DNA is more resistant to the presence of salt than the non specific binding . Neither the DNA binding activity nor the specificity for oriC DNA was destroyed by heating at 100 degrees C for 2 min. Arch Microbiol, 1984 Jul, 138(3), 191 - 4 Expression of surface hydrophobicity encoded by R-plasmids in Escherichia coli laboratory strains; Ferreiros CM et al.; The inclusion of drug-resistance plasmids (R-plasmids) in Escherichia coli strains has been shown to determine the formation of specific surface structures which could modify bacterial surface characteristics relevant for pathogenic processes . Thirtyone R-plasmids (from different incompatibility groups) have been transferred to three E . coli laboratory strains, and surface hydrophobicity modifications have been measured by three methods: "salting-out", adsorption to hexadecane and adsorption to xylene . The results obtained show that the presence of R-plasmids produced variations which are dependent on the receptor strains and measuring method employed . Also, it has been found that the plasmids behave differently depending on the strain in which they are included . The results obtained by the "salting-out" method are not correlative with those obtained by adsorption to hydrocarbons, probably due to the implication of different hydrophobic molecules in the interaction with salt or hydrocarbons . Concluding, the choice of receptor strain and measuring method are of great importance for the investigation of surface hydrophobicity (and probably other characteristics) encoded by R-plasmids. Antimicrob Agents Chemother, 1984 Jul, 26(1), 10 - 2 Identification of aminoglycoside-acetylating enzymes by high-pressure liquid chromatographic determination of their reaction products; Lovering AM et al.; A method to identify the aminoglycoside-acetyltransferase (AAC) enzymes AAC(3), AAC(2') and AAC(6') by high-pressure liquid chromatographic characterization of their products of reaction with tobramycin or sisomicin is described . Conditions are given for the chromatography of kanamycin A, netilmicin, neomycin, and apramycin, and their products of reaction, if any, with the three AAC enzymes are listed. Tsitol Genet, 1984 Jul-Aug, 18(4), 268 - 71 {Cytogenetic characteristics of 5 experimental mammalian pathology models}; Zolotareva GN et al.; A cytogenetic study of bone marrow cells was carried out first under conditions of toxic hepatopathy, experimental toxic nephropathy, alloxan diabetes in rats and under conditions of colibacillar and pyocyanic sepsis in mice in different periods of pathology . These experimental models may be used in research on medicinal mutagenesis. Rev Infect Dis, 1984 Jul-Aug, 6(4), 463 - 71 The enzymatic synthesis of lipid A: molecular structure and biologic function of monosaccharide precursors; Raetz CR; Certain Escherichia coli mutants, deficient in phosphatidylglycerol, accumulate novel, glucosamine-derived phospholipids that appear to be very early precursors of lipid A . The simplest of these, lipid X, is a derivative of glucosamine-1-phosphate substituted with beta-hydroxymyristoyl moieties at positions 2 and 3 . The discovery of lipid X makes it possible to predict a biosynthetic pathway and a unified structure for lipid A . In addition, the minimal molecular requirements for the mitogenic and endotoxic properties of lipopolysaccharides can be elucidated with compounds of this kind. J Gen Microbiol, 1984 Jul, 130 ( Pt 7), 1839 - 44 The effect of plasmid R391 and other IncJ plasmids on the survival of Escherichia coli after UV irradiation; Pembroke JT et al.; The presence of the IncJ plasmids R391, R997, R705, R706, R748 and R749 was shown to sensitize Escherichia coli AB1157 and both its uvrA and lexA derivatives to UV irradiation . No alteration in post-irradiation survival was observed in a recA mutant containing these plasmids, compared with the non-plasmid-containing recA strain . Analysis of recombination frequency in Hfr crosses to recA+ cells containing plasmid R391 indicated a reduction in recombination frequency compared with that obtained in similar crosses to a non-plasmid-containing strain . This effect was not due to plasmid-encoded restriction or entry exclusion systems and therefore must be considered as a real block in recombination . When cells containing plasmid R391 were irradiated and allowed to photoreactivate, an increase in survival was observed which was comparable to that observed in the non-plasmid-containing derivative . This indicated that post-irradiation processing of UV-induced damage, or lack of such processing, by mechanisms other than photoreactivation was responsible for the UV sensitivity associated with plasmid R391. J Gen Microbiol, 1984 Jul, 130 ( Pt 7), 1651 - 63 Molecular gentic analysis of the trfB and korB region of broad host range plasmid RK2; Smith CA et al.; A 3.2 kb region of the broad host range IncP plasmid RK2 (indistinguishable from RP1, RP4, R68 and R18) anticlockwise from the EcoRI site may be separated phenotypically into three loci . The trfB/korA/korD locus both complements a temperature-sensitive maintenance mutation and suppresses the deleterious effects of the loci kilA and kilD; the incC locus expresses incompatibility towards complete RK2-like replicons, and the korB locus suppresses the host lethal effect the kilB locus . Transcriptional fusions of the galK gene to various segments of this region revealed that all three loci are transcribed anticlockwise from a common promoter . A weak secondary promoter may also contribute to the expression of korB . Analysis of the sizes of the polypeptides produced from these segments led to the identification of two cistrons, the first encoding a polypeptide of 38 kDal associated with incC function and the second a polypeptide of 49 kDal associated with korB activity . The trfB/korA/korD activities are associated with a polypeptide of 14kDal which may be an N-terminal fragment of the incC-associated polypeptide. J Gen Microbiol, 1984 Jul, 130 ( Pt 7), 1641 - 50 Genetic evidence for the direction of transcription of the trfA gene of broad host range plasmid RK2; Thomas CM; For replication of broad host range plasmid RK2 in Escherichia coli two regions of the plasmid genome are essential, oriV RK2 between 12.0 and 12.7 kb on the genome (defined clockwise from the unique EcoRI site) and trfA, located between 16.0 and 17.4 kb, which provides a trans-acting product necessary for oriV RK2 function . The properties of an insertion mutant of a mini-RK2/ColE1 hybrid plasmid suggest that the trfA promoter lies clockwise from the 17.4 kb RK2 coordinate . Fusion of the trfA gene lacking its normal promoter to the E . coli trpE gene in a hybrid plasmid confirmed that trfA is transcribed anticlockwise, towards the Tcr gene of RK2 . Repression of trpE promoter activity in these fusions showed that replication of an RK2 replicon can be regulated by varying the level of trfA gene expression . The results also indicate the presence of a transcription unit running anticlockwise through the trfB region located between 54.0 and 56.0 kb on the RK2 genome. Biull Eksp Biol Med, 1984 Jul, 98(7), 117 - 9 {Determination of the nucleotide and isoplith composition of DNA by scanning thin-layer chromatograms in ultraviolet light}; Vasil'ev VK; The author studied the UV spectra of the bases and pyrimidine sequences of DNA after separation in thin layers of cellulose, and calculated the coefficients permitting the determination of nucleotide and isoplith composition of DNA from UV light reflection during thin-layer chromatogram scanning. Proc Natl Acad Sci U S A, 1984 Jul, 81(14), 4290 - 3 Ribonuclease T: new exoribonuclease possibly involved in end-turnover of tRNA; Deutscher MP et al.; Examination of double mutants lacking one of the exoribonucleases, RNase II, RNase D, RNase BN, or RNase R, and also devoid of tRNA nucleotidyltransferase has suggested that none of these RNases participates in the end-turnover of tRNA . This prompted a search for and identification of a new exoribonuclease, termed RNase T . RNase T could be detected in mutant Escherichia coli strains lacking as many as three of the known exoribonucleases, and it could be separated from each of the four previously described RNases . RNase T is optimally active at pH 8-9 and requires a divalent cation for activity . The enzyme is sensitive to ionic strengths greater than 50 mM and is rapidly inactivated by heating at 45 degrees C . Its preferred substrate is tRNA-C-C-{14C}A, with much less activity shown against tRNA-C-C . RNase T is an exoribonuclease that initiates attack at the 3' hydroxyl terminus of tRNA and releases AMP in a random mode of hydrolysis . The possible involvement of RNase T in end-turnover of tRNA and in RNA metabolism in general are discussed. Mol Immunol, 1984 Jul, 21(7), 609 - 20 Interaction of human complement proteins with serum-sensitive and serum-resistant strains of Escherichia coli; Taylor PW et al.; Exposure of serum-susceptible Escherichia coli strains to lethal concns of lysozyme-free human serum resulted in stable binding of complement components to the outer membrane (OM), but not to the cytoplasmic membrane (CM) . The short prekilling phase of the reaction was accompanied by binding of C3b; loss of viability was immediately preceeded by stable deposition onto the OM of the component proteins of the membrane attack complex . During the early stages of the active killing phase, bound monomeric C9 could be resolved into two distinct bands on SDS-polyacrylamide gels . Serum exposure lead to a progressive loss of CM recoverability, which appeared to result from partial degradation of CM phospholipids . In contrast, exposure of a resistant E, coli strain to human serum resulted in little change in the membrane profile and very little stable deposition of terminal complement components onto the OM. JPEN J Parenter Enteral Nutr, 1984 Jul-Aug, 8(4), 440 - 2 Clearance rate of intravenously administered lipid emulsion in canine endotoxemia; Iriyama K et al.; The effect of endotoxemia on the initial catabolism of intravenously given lipid emulsion was investigated in dogs . Two types of endotoxemia were prepared . One was produced by peritonitis which was established by ligation of the artery and vein of an isolated intestine (group 1, n = 6) . The other was made by an intravenous injection of Escherichia coli lipopolysaccharide in a dose of 1.5 mg/kg of body weight (group 2, n = 6) . Group 1 showed evident peritonitis with a positive limulus test 48 hr after the procedure, but no significant changes of blood sugar level and lactate/pyruvate ratio, while group 2 demonstrated profound hypoglycemia, significant elevation of lactate/pyruvate ratio, and low arterial pressure 3 to 5 hr after the injection of lipopolysaccharide . The clearance rate of intravenously administered lipid emulsion (K value) of group 1 before the peritonitis was 0.0105 +/- 0.0017 and after the peritonitis it was 0.0105 +/- 0.0019 . The difference was not significant, while the K value of group 2 which was 0.0133 +/- 0.0056 before the injection of lipopolysaccharide decreased significantly to 0.0069 +/- 0.0024 after the injection of lipopolysaccharide . These results suggest that, in case of endotoxemia with normally maintained oxidation-reduction potential, the initial catabolism of intravenously given lipid emulsion is kept in a normal level, while oxidation-reduction potential is impaired, it is inhibited. EMBO J, 1984 Jul, 3(7), 1609 - 12 RNA secondary structure and translation inhibition: analysis of mutants in the rplJ leader; Christensen T et al.; We have carried out measurements of the stable binding of the ribosomal protein (r-protein) complex L10-L7/L12 to mutant forms of the mRNA leader of the rplJ operon of Escherichia coli . One of the point mutations, base 1548, which lies within the L10-L7/L12-protected region, almost completely abolishes in vitro formation of a stable complex of L10-L7/L12 with rplJ mRNA leader, and a second point mutation, base 1634, strongly reduces it . These observations constitute strong support for the proposition that L10-L7/L12 binds to the rplJ leader in bringing about translational feedback . To account for the action of these and other mutations, and to explain the mechanism of translation feedback inhibition, we suggest a secondary structure model involving alternate forms of the rplJ mRNA leader. EMBO J, 1984 Jul, 3(7), 1513 - 9 The korB gene of broad host range plasmid RK2 is a major copy number control element which may act together with trfB by limiting trfA expression; Thomas CM et al.; For replication, plasmid RK2 encodes a vegetative replication origin, oriV RK2, and a gene, trfA, whose polypeptide product(s) is essential for oriV RK2 activity . The trfA gene is transcribed as part of a polycistronic operon which also includes kilD . Transcription of this operon is negatively regulated by the products of the trfB/korD/korA and korB loci . Mini replicons previously studied in detail lack the korB locus and have copy numbers significantly higher than RK2 itself . Here we report that korB in trans expresses incompatibility towards RK2 replicons either when the korB gene dosage is high or when it is expressed from a strong foreign promoter . This incompatibility can be largely overcome if a trfA gene which is expressed from a foreign promoter, and is therefore not regulated by korB, is supplied in trans . When korB is introduced in cis to mini RK2 replicons the copy number is reduced to within the range estimated for parental RK2 . Deletions in the oriV RK2 region which otherwise cause quite large increases in plasmid copy number have only a small effect when korB is present in cis . These results suggest that korB in combination with trfB may be the overriding copy number control element in RK2 reducing trfA expression to levels limiting for replication. Am J Surg, 1984 Jul, 148(1), 117 - 24 Oriental cholangitis; Carmona RH et al.; Oriental cholangitis is a poorly understood syndrome consisting of intrahepatic pigment stone formation with chronically recurrent exacerbations and remissions . Endemic to Asia, it is being encountered more frequently in the United States due to increased immigration of asians . Twenty-one patients with oriental cholangitis (9 men and 12 women), 19 to 84 years of age, all of whom immigrated from asian countries, were treated between 1970 and 1983 . All had histories of episodic abdominal pain, most with jaundice, chills, and fever . Laboratory results were nonspecific but frequently included leukocytosis and hyperbilirubinemia . All patients were operated on with 15 having cholecystectomy, common duct exploration, and a bilioenteric anastomosis . E . coli was cultured from specimens obtained from the biliary tracts of all patients, and 13 patients had more than one organism . Four patients had a previous history of parasitic infection, and four different patients had parasites identified in the biliary tract intraoperatively . Early recognition and appropriate operation will decrease morbidity and mortality. Proc Natl Acad Sci U S A, 1984 Jul, 81(13), 4139 - 43 Multiple regulatory signals in the control region of the Escherichia coli carAB operon; Bouvier J et al.; The first reaction in pyrimidine and arginine biosynthesis in Escherichia coli is catalyzed by a single enzyme, carbamoyl-phosphate synthetase (EC 6.3.5.5), the product of the carAB operon . Expression of this operon is cumulatively repressed by arginine and pyrimidines . The nucleotide sequence of the carAB control region was determined and transcriptional starts were localized . Two adjacent promoters, 70 base pairs apart, appear to be used in vivo, the downstream one overlapping a typical arginine operator . The absence of any attenuation-like sequence excludes such a mechanism for pyrimidine-mediated repression . Various fragments of the carA promoter-proximal region were fused in vitro with the lacZ gene . Results obtained with these fusions indicate that (i) translation of the carA gene can be initiated in vivo without an AUG codon but very likely with an UUG or an AUU codon; (ii) the carAB downstream promoter is repressed by arginine; and (iii) the carAB upstream promoter is repressed by pyrimidines and subject to stringent control . When carried by a multicopy plasmid the carAB control region escapes repression by arginine and pyrimidines . The existence of a pyrimidine repressor, present in limiting amounts in the cell, is therefore postulated. Proc Natl Acad Sci U S A, 1984 Jul, 81(13), 4120 - 4 Regulation of the araC gene of Escherichia coli: catabolite repression, autoregulation, and effect on araBAD expression; Miyada CG et al.; The araC gene encodes a positive regulatory protein required for L-arabinose utilization in Escherichia coli . Transcription from the araC promoter has been shown to be under positive control by cAMP receptor protein and under negative control by its protein product (autoregulation) . This work describes the identification of the region of the araC promoter that interacts with the cAMP receptor protein to mediate catabolite repression . A 3-base-pair deletion centered 60 base pairs from the transcriptional initiation site results in a mutant araC promoter that, in the absence of araC protein, reduces transcriptional activity when compared with the wild-type promoter and is unresponsive to various concentrations of intracellular cAMP in vivo . The same deletion results in a lowered affinity of the araC promoter for cAMP receptor protein in vitro . However, this lowered affinity for the mutant araC promoter does not result in substantial reduction of intracellular araC protein because autoregulation of the araC gene dominates catabolite repression . The 3-base-pair deletion in the cAMP receptor protein binding site of the araC promoter does not affect catabolite repression of the adjacent araBAD operon . The implications of these results on current models for expression of the araBAD operon and the araC gene are discussed. Proc Natl Acad Sci U S A, 1984 Jul, 81(13), 4037 - 40 Structure of unligated aspartate carbamoyltransferase of Escherichia coli at 2.6-A resolution; Ke HM et al.; The three-dimensional structure of the allosteric enzyme aspartate carbamoyltransferase (EC 2.1.3.2) has been refined to a crystallographic R-factor of 0.24 at 2.6-A resolution in the space group P321, where a and b are 122.1 A and c is 142.2 A . This structure is isomorphous to the form of the enzyme complexed to the allosteric inhibitor cytidine triphosphate . All sources of sequence information have been evaluated against the electron density . The corrected amino acid sequences of the catalytic and regulatory proteins have been incorporated in the model, and three regions in the active site are described: (i) near arginine-105, histidine-134, and arginine-167, (ii) near lysine-232 and arginine-229, and (iii) near lysine-83 and lysine-84. Proc Natl Acad Sci U S A, 1984 Jul, 81(13), 3973 - 7 Model of specific complex between catabolite gene activator protein and B-DNA suggested by electrostatic complementarity; Weber IT et al.; Calculation of the electrostatic potential energy surfaces of Escherichia coli catabolite gene activator protein (CAP) dimer suggests a model for the complex between CAP and a specific DNA sequence . The positive electrostatic charge density of CAP lies on the two COOH-terminal domains and about 20-30 A from the molecular 2-fold axis . Assuming that the 2-fold axes of the CAP dimer and the DNA to which it binds are coincident, the positions of the positive electrostatic potential surfaces strongly suggest the rotational orientation of the DNA relative to the protein . A specific complex between CAP and its DNA binding site in the lac operon has been built with the DNA in this orientation . The amino ends of the two protruding F alpha-helices interact in successive major grooves of the DNA . Four side chains emanating from each F helix can form hydrogen bonds with the exposed edges of four bases in the major groove . Electrostatic considerations as well as the necessity to make interactions between CAP and a DNA site as much as 20 base pairs long require us to bend or kink the DNA . In our model of CAP complexed with B-DNA, as with those proposed for Cro and lambda cI repressors, the protruding second helices of the two-helix motif from both subunits interact in successive major grooves of B-DNA . However, unlike Cro and similar to lambda cI, the protruding alpha-helices are nearly parallel to the bases rather than the groove. Proc Natl Acad Sci U S A, 1984 Jul, 81(13), 3969 - 72 Cross-linking of tRNA at two different sites of the elongation factor Tu; Van Noort JM et al.; Recently, we reported on the induction by kirromycin of two tRNA binding sites on elongation factor Tu . To obtain independent information on the existence of these two sites and to characterize them further, 3' oxidized tRNA was cross-linked to elongation factor Tu by {3H}borohydride reduction . Specific cross-linking occurred exclusively in the presence of kirromycin . In the case of elongation factor Tu X GDP X kirromycin, cross-linking was found at lysine-208; in elongation factor Tu X GTP X kirromycin, cross-linking was at lysine-208 and lysine-237 . In both elongation factor Tu complexes, kirromycin itself was found cross-linked to lysine-357 . The tRNA cross-linking sites are in agreement with the idea of two different binding sites of tRNA on elongation factor Tu. J Bacteriol, 1984 Jul, 159(1), 421 - 3 Kinetics of methylation in Escherichia coli K-12; Lyons SM et al.; Newly synthesized DNA is undermethylated in E . coli K-12 . The amount of N6-methyl deoxyadenylic acid in labeled DNA varied from 0.3 mol% of total adenine for a 2-min pulse to 1.7 mol% for DNA that was labeled for more than two generations. J Bacteriol, 1984 Jul, 159(1), 368 - 74 Chemosensory and thermosensory excitation in adaptation-deficient mutants of Escherichia coli; Imae Y et al.; Methyl-accepting chemotaxis protein-methyltransferase-deficient mutants, cheR mutants, of Escherichia coli showed a tumble response to repellents only at low temperatures, and the resultant tumbling lasted unless the condition was changed . The swimming pattern of the repellent-treated cells was different at different temperatures, indicating that the absolute temperature is a determinant of the tumbling frequency of those cells . The tumbling of those cells was also suppressed by the addition of attractants . Under a suitable repellent concentration, the tumbling frequency of the cells was found to be simply determined by the ligand occupancy of chemoreceptors for many attractants . In a methyl-accepting chemotaxis protein-methylesterase-deficient mutant, a cheB deletion mutant, the tumbling frequency was also determined by receptor occupancy of some attractants . These results indicate that in the adaptation-deficient mutants, sensory signals are produced in proportion to the amount of ligand-bound or of thermally altered receptors and transmitted to the flagellar motors without any modification . Thus, it is concluded that the adaptation system, namely, the methylation-demethylation system of methyl-accepting chemotaxis proteins, is not concerned with the step of chemosensory or thermosensory excitation . A simple model is proposed to explain how the swimming pattern of the adaptation-deficient mutants is determined. J Bacteriol, 1984 Jul, 159(1), 360 - 7 Conditional inversion of the thermoresponse in Escherichia coli; Mizuno T et al.; Mutants in Escherichia coli having defects in one of the methyl-accepting chemotaxis proteins, Tsr protein, which is the chemoreceptor and transducer for L-serine, showed a reduced but similar type of thermoresponse compared with wild-type strains; the cells showed smooth swimming upon temperature increase and tumbling upon temperature decrease . However, when the mutant cells were adapted to attractants such as L-aspartate and maltose, which are specific to another methyl-accepting chemotaxis protein, Tar protein, the direction of the thermoresponse was found to be inverted; a temperature increase induced tumbling and a temperature decrease induced smooth swimming . Consistent with this, the mutant cells showed inverted changes in the methylation level of Tar protein upon temperature changes . Wild-type strains but not Tar protein-deficient mutants exhibited the inverted thermoresponse when the cells were simultaneously adapted to L-aspartate and L-serine, indicating that Tar protein has a key role in the inversion of the thermoresponse . Thus, besides Tsr protein, Tar protein has a certain role in thermoreception . A simple model for thermoreception and inversion of the thermoresponse is also discussed. J Bacteriol, 1984 Jul, 159(1), 329 - 34 Acquisition of Escherichia coli outer membrane proteins by Bdellovibrio sp . strain 109D; Diedrich DL et al.; The ability of Bdellovibrio sp . to acquire the OmpF major outer membrane protein from its Escherichia coli prey was examined to determine if there were other outer membrane proteins which could or could not be acquired . Growth of bdellovibrios on mutant prey which were defective in the expression of outer membrane proteins revealed that Bdellovibrio sp . could acquire the OmpC protein in the absence of the OmpF protein . However, the OmpA, LamB, and protein 2 proteins could not be found in the Bdellovibrio Triton-insoluble outer membrane . The disappearance of the OmpF and OmpC proteins from the bdelloplast surface was measured, and it was determined that Bdellovibrio sp . exhibited a kinetic and temporal preference for the OmpF protein . Bdellovibrios could be grown on porin-deficient prey, and the progeny bdellovibrios possessed outer membranes with a protein mass deficiency. Infect Immun, 1984 Jul, 45(1), 278 - 80 Species differences in Kupffer cells and endotoxin sensitivity; McCuskey RS et al.; The relative species sensitivity to Escherichia coli O111:B4 endotoxin was found to be guinea pig greater than hamster greater than mouse greater than rat . The 50% lethal dose of this endotoxin correlated with both the rate at which single latex particles were phagocytosed by individual Kupffer cells and the number of Kupffer cells in hepatic lobules that phagocytosed latex . The results suggests that the intrahepatic density and the level of activation of Kupffer cells participate in determining endotoxin sensitivity. Infect Immun, 1984 Jul, 45(1), 242 - 7 Molecular homogeneity of heat-stable enterotoxins produced by bovine enterotoxigenic Escherichia coli; Saeed AM et al.; Heat-stable enterotoxins (STs) from four strains of bovine enterotoxigenic Escherichia coli representing four serogroups were purified to homogeneity by utilizing previously published purification schemata . Biochemical characterization of the purified STs showed that they met the basic criteria for the heat-stable enterotoxins of E . coli . Amino acid analysis of the purified STs revealed that they were peptides of identical amino acid composition . This composition consisted of 18 residues of 10 different amino acids, 6 of which were cysteine . The amino acid composition of the four ST peptides was identical to that reported for the STs of human and porcine E . coli . In addition, complete sequence analysis of two of the ST peptides and partial sequencing of several others revealed strong homology to the sequences of STs from human and porcine E . coli and to the sequence predicted from the last 18 codons of the transposon Tn1681 . There was also substantial homology to the sequence predicted from the ST-coding genetic element of human E . coli, which may indicate the existence of identical bioactive configuration among ST peptides of E . coli strains of various host origins . These data support the hypothesis that STs produced by human, bovine, and porcine E . coli are coded by a closely related genetic element which may have originated from a single, widely disseminated transposon. Proc Soc Exp Biol Med, 1984 Jul, 176(3), 292 - 6 Changes in protein degradation in chickens due to an inflammatory challenge; Klasing KC et al.; Tissue-specific changes in protein catabolism were examined in chicks 16 hr following an inflammatory challenge . It was determined that tyrosine was not catabolized or converted to phenylalanine in muscle, thymus, bursa, or spleen . Therefore, rates of tyrosine release from protein were used to estimate rates of protein catabolism in these tissues . Arginine was not catabolized to urea by chick liver; consequently, arginine release from liver protein was used to measure protein catabolism in this tissue . An injection of sheep red blood cells (SRBC) or Escherichia coli did not change rates of protein catabolism in liver or bursa as compared to saline-injected controls . SRBC significantly increased protein catabolism in muscle and spleen by 29 and 15%, respectively . E . coli resulted in significant increases in muscle, spleen, and thymus of 43, 30, and 34%, respectively . These changes in protein catabolism, together with known changes in protein synthesis, suggest that an inflammatory response to SRBC and E . coli result in increased protein accretion in the bursa and liver, and net protein loss from muscle. Proc Soc Exp Biol Med, 1984 Jul, 176(3), 285 - 91 Changes in protein synthesis due to an inflammatory challenge; Klasing KC et al.; Rates of protein synthesis in various chick tissues were examined 16 hr after an inflammatory challenge . Protein synthetic rates were calculated from the rate at which {14C}leucine was incorporated into protein and the specific activity of {14C}leucine in the precursor pool . An injection of either Escherichia coli or sheep red blood cells (SRBC) decreased the rate of protein synthesis in the gastrocnemius muscle, and increased the rate in liver, bursa, spleen, and thymus . E . coli, but not SRBC, decreased protein synthesis in the pectoralis muscle . E . coli significantly decreased the aggregation of pectoralis muscle polysomes and increased the aggregation of polysomes in the thymus, bursa, and spleen . E . coli increased the aggregation of free, but not bound, polysomes in liver, suggesting an increase in synthesis of export proteins . SRBC significantly increased polysomal aggregation in bursa and spleen only . A crude preparation of leukocyte endogenous mediator, isolated from peritoneal macrophages, decreased muscle-polysomal aggregation . These studies indicate that tissue-specific changes in protein synthesis occur after a noninfectious inflammatory challenge . These changes may be part of a homeostatic mechanism which supports the immune response. Proc Natl Acad Sci U S A, 1984 Jul, 81(13), 4149 - 53 Construction of mutants of Moloney murine leukemia virus by suppressor-linker insertional mutagenesis: positions of viable insertion mutations; Lobel LI et al.; A highly efficient method for the generation of insertion mutations is described . The procedure involves the use of a 220-base-pair (bp) EcoRI fragment bearing the SuIII+ suppressor tRNA gene as an insertional mutagen . The plasmid DNA to be mutagenized is linearized by a variety of means, and the suppressor fragment is ligated into the site of cleavage . Successful insertion mutants can be readily detected in Escherichia coli carrying lac- amber mutations on MacConkey lactose plates; virtually 100% of the red colonies contain insertions of the fragment . Subsequent removal of the SuIII+ gene and recyclization leaves a 12-bp insertion if the original cleavage was blunt-ended and a 9-bp insertion if the original cleavage generated 3-bp cohesive termini . This technique, as well as conventional linker mutagenesis with decamer and dodecamer linkers, was used to generate a large library of insertion mutations in cloned DNA copies of the genome of Moloney murine leukemia virus . A number of viable mutants were isolated bearing 9-, 10-, and 12-bp insertions in various domains of the genome . The map positions of the viable mutations suggest that the viral long terminal repeats and portions of the gag and env genes are quite insensitive to alteration . Although most of the mutations were stable for many passages, some of the mutants lost the inserted DNA; we presume that the insertion was somewhat deleterious in these mutants and that continued passage of the virus selected for overgrowth by a revertant. Proc Natl Acad Sci U S A, 1984 Jul, 81(13), 4134 - 8 DNA sequence of the carA gene and the control region of carAB: tandem promoters, respectively controlled by arginine and the pyrimidines, regulate the synthesis of carbamoyl-phosphate synthetase in Escherichia coli K-12; Piette J et al.; The carAB operon of Escherichia coli K-12, which encodes the two subunits of carbamoyl-phosphate synthetase (glutamine hydrolyzing) {carbon-dioxide: L-glutamine amido-ligase (ADP-forming, carbamate-phosphorylating); EC 6.3.5.5}, is cumulatively repressed by arginine and the pyrimidines . We describe the structure of the control region of carAB and the sequence of the carA gene . Nuclease S1 mapping experiments show that two adjacent tandem promoters within the carAB control region serve as initiation sites . The upstream promoter P1 is controlled by pyrimidines; the downstream promoter P2 is regulated by arginine . Attenuation control does not appear to be involved in the expression of carAB . A possible mechanism by which control at these promoters concurs to produce a cumulative pattern of repression is discussed . The translational start of carA is atypical; it consists of a UUG or AUU codon. Proc Natl Acad Sci U S A, 1984 Jul, 81(13), 4110 - 4 Induction of specific Escherichia coli genes by sublethal treatments with alkylating agents; Volkert MR et al.; Fusions of the lac operon to genes induced by treatment with sublethal levels of alkylating agents have been selected from random insertions of the Mu-dl(ApRlac) phage by screening for induction of beta-galactosidase activity in the presence of methyl methanesulfonate . Genetic analysis reveals that these fusions resulted from insertion of Mu-dl(ApRlac) into two regions of the chromosome . One region (aidA) is near his and, based on phenotypic effects, appears to represent insertion into the alkA gene . The other region (aidB) is in the 92.3- to 98-min region, which harbors no previously identified genes involved in repair of alkylation damage . The aidB fusions caused increased resistance to alkylating agents and caused little or no change in the biological effects of adaptation to alkylating agents . Unlike the aidA fusions, aidB fusions showed increased beta-galactosidase activity in untreated cells in a growth phase-dependent fashion . The ada-5 mutation, which blocks expression of the adaptive response, decreased induction of beta-galactosidase activity in both aidA and aidB fusions after alkylation treatments . Thus, both aidA and aidB share with adaptive response a common regulatory mechanism involving the ada gene . The growth phase-dependent control of the aidB fusions, however, is unaffected by ada, suggesting that a second regulatory mechanism exists that controls only aidB. Proc Natl Acad Sci U S A, 1984 Jul, 81(13), 4051 - 4 Accurate initiation of human epsilon-globin RNA synthesis by Escherichia coli RNA polymerase in isolated nuclei of K562 erythroleukemia cells; Gilmour RS et al.; The human epsilon-globin gene was transcribed in vitro in isolated K562 cell nuclei by using exogenous Escherichia coli RNA polymerase (EC 2.7.7.6) . Newly formed RNA transcripts were distinguished from nuclear RNA molecules by (i) incorporating mercurated UTP into RNA under conditions in which the endogenous polymerase II is inactive and (ii) subsequently isolating the mercurated RNA by affinity chromatography on thiolated Sepharose . A specific 5'-end-labeled probe spanning the epsilon-globin gene cap site was used in nuclease S1 mapping studies to examine the in vitro initiation site of the isolated transcripts . It was found that transcription occurred from the coding strand only and originated almost entirely from a point that was identical to that of the major cap site for epsilon-globin mRNA in vivo. Proc Natl Acad Sci U S A, 1984 Jul, 81(13), 3983 - 7 Intracellular targeting and import of an F1-ATPase beta-subunit-beta-galactosidase hybrid protein into yeast mitochondria; Douglas MG et al.; The gene coding for the yeast mitochondrial F1-ATPase beta subunit (ATP2) has been fused to the Escherichia coli lacZ gene . The chimeric ATP2-lacZ gene codes for a hybrid protein consisting of some 350 amino acids of the F1-ATPase beta subunit at its amino terminus and a large enzymatically active portion of the lacZ gene product, beta-galactosidase (beta-D-galactoside galactohydrolase, EC 3.2.1.23), at its carboxyl terminus . The beta-subunit-beta-galactosidase hybrid protein is expressed in both E . coli and yeast . In yeast, this hybrid molecule is targeted to the mitochondrion and is protected in isolated mitochondria from added protease under conditions in which an outer membrane enzymatic marker is digested . Yeast cells carrying the ATP2-lacZ gene fusion on plasmid p beta Z1 are unable to grow on a nonfermentable carbon source . Upon loss of the p beta Z1 plasmid, growth of the cured host strain on the nonfermentable substrate is restored . In the presence of the beta-subunit-beta-galactosidase hybrid protein, the energy-transducing capacity of the mitochondrial membrane as measured by the 32Pi-ATP exchange reaction is only 9% of that measured in the absence of the gene fusion product . The results indicate that it is the presence of the beta-subunit-beta-galactosidase hybrid protein within mitochondria that interferes with function(s) essential for respiratory growth . These observations open up the prospect of genetic characterization of the signals and cellular machinery responsible for mitochondrial protein delivery. J Invest Dermatol, 1984 Jul, 83(1 Suppl), 82s - 87s Transforming functions associated with Epstein-Barr virus; Sugden B et al.; Epstein-Barr virus (EBV) transforms the human B-lymphocytes it infects into lymphoblasts that are competent to proliferate indefinitely in tissue culture {1} . The dividing transformed lymphoblasts carry multiple and complete copies of EBV DNA as plasmids {1} . No viral functions that are necessary for EBV-induced transformation have been identified and characterized to date . We have developed an assay that aids in the identification of viral functions needed to maintain the viral plasmid in transformed cells . The assay employs a plasmid vector that encodes resistance to ampicillin and can replicate in E . coli . It also encodes resistance to the neomycin derivative G418, so that its presence can be selected in mammalian cells {2} . Fragments of viral DNA that span the genome have been molecularly cloned into this vector . These recombinant molecules have been transfected into four cell types, and survivors to G418 have been scored . The DNA from one region of EBV gives a 10- to 100-fold higher rate of survival per microgram of added DNA than does the DNA from all other recombinants, as tested in cells that already contain EBV genomes . This recombinant fragment of EBV is maintained in an apparently unrearranged state as a plasmid and therefore carries at least those cis-acting viral elements necessary for plasmid replication. J Bacteriol, 1984 Jul, 159(1), 93 - 9 Transposon Tn10-dependent expression of the lamB gene in Escherichia coli; Brass JM et al.; Among Tn10 insertions isolated in or near the malB region of Escherichia coli, one (zjb-729::Tn10) mapped between malK and lamB or late in malK and allowed MalT-independent expression of lamB . Tn10-dependent expression of a lamB-lacZ protein fusion was 25% of the expression of the fusion from the malK-lamB operon promoter in malTc constitutive strains . The maltoporin content of a strain carrying this Tn10 was about 20% that of a malTc malB+ strain . Transport of maltose at concentrations of below 10(-6) M was reduced about threefold . When maltoporin was present at about 50% of the level of malTc malB+ strains, maltose transport was largely restored . We conclude that maltoporin is not rate limiting for maltose transport in wild-type cells but becomes rate limiting when the ratio of maltoporin to other maltose transport components is reduced more than twofold. J Bacteriol, 1984 Jul, 159(1), 57 - 62 Nucleotide sequence of Escherichia coli pabB indicates a common evolutionary origin of p-aminobenzoate synthetase and anthranilate synthetase; Goncharoff P et al.; Biochemical and immunological experiments have suggested that the Escherichia coli enzyme p-aminobenzoate synthetase and anthranilate synthetase are structurally related . Both enzymes are composed of two nonidentical subunits . Anthranilate synthetase is composed of proteins encoded by the genes trp(G)D and trpE, whereas p-aminobenzoate synthetase is composed of proteins encoded by pabA and pabB . These two enzymes catalyze similar reactions and produce similar products . The nucleotide sequences of pabA and trp(G)D have been determined and indicate a common evolutionary origin of these two genes . Here we present the nucleotide sequence of pabB and compare it with that of trpE . Similarities are 26% at the amino acid level and 40% at the nucleotide level . We propose that pabB and trpE arose from a common ancestor and hence that there is a common ancestry of genes encoding p-aminobenzoate synthetase and anthranilate synthetase. J Bacteriol, 1984 Jul, 159(1), 424 - 6 Physical mapping of hemolysin plasmid pCW2, which codes for virulence of a nephropathogenic Escherichia coli strain; Waalwijk C et al.; The hemolysin plasmid pCW2, which enhances the virulence of Escherichia coli, has been mapped . Comparison of the region coding for hemolysin with other hemolysin determinants reveals differences that could explain differences in the ability to confer virulence. J Bacteriol, 1984 Jul, 159(1), 418 - 20 Cloning of the iron superoxide dismutase gene (sodB) in Escherichia coli K-12; Sakamoto H et al.; A clone overproducing iron superoxide dismutase has been isolated from an Escherichia coli cosmid bank . Subcloning located the gene responsible for iron superoxide dismutase overproduction on a 6.6-kilobase PstI restriction endonuclease fragment . Maxicell analysis, followed by immunological identification of iron superoxide dismutase protein, demonstrated that the structural gene, sodB, of iron superoxide dismutase has been cloned. J Bacteriol, 1984 Jul, 159(1), 404 - 6 Isolation and analysis of inhibitors of transposon Tn3 site-specific recombination; Fennewald MA et al.; We have constructed a genetic bioassay for inhibitors of site-specific recombination by transposon Tn3 resolvase . Of 6,000 compounds tested, 26 inhibited in vivo, and 5 of these 26 inhibited in vitro . At least two inhibitors also inhibit the topoisomerase of resolvase . We have also identified analogs of A1062 which inhibit. J Bacteriol, 1984 Jul, 159(1), 283 - 7 lon gene product of Escherichia coli is a heat-shock protein; Phillips TA et al.; The product of the pleiotropic gene lon is a protein with protease activity and has been tentatively identified as protein H94.0 on the reference two-dimensional gel of Escherichia coli proteins . Purified Lon protease migrated with the prominent cellular protein H94.0 in E . coli K-12 strains . Peptide map patterns of Lon protease and H94.0 were identical . A mutant form of the protease had altered mobility during gel electrophoresis . An E . coli B/r strain that is known to be defective in Lon function contained no detectable H94.0 protein under normal growth conditions . Upon a shift to 42 degrees C, however, the Lon protease was induced to high levels in K-12 strains and a small amount of protein became detectable at the H94.0 location in strain B/r . Heat induction of Lon protease was dependent on the normal allele of the regulatory gene, htpR, establishing lon as a member of the high-temperature-production regulon of E . coli. J Bacteriol, 1984 Jul, 159(1), 265 - 70 Purine metabolism in Acholeplasma laidlawii B: novel PPi-dependent nucleoside kinase activity; Tryon VV et al.; Acholeplasma laidlawii B-PG9 was examined for 16 cytoplasmic enzymes with activity for purine salvage and interconversion . Phosphoribosyltransferase activities for adenine, guanine, xanthine, and hypoxanthine were shown . Adenine, guanine, xanthine, and hypoxanthine were ribosylated to their nucleoside . Adenosine, inosine, xanthosine, and guanosine were converted to their base . No ATP-dependent phosphorylation of nucleosides to mononucleotides was found . However, PPi-dependent phosphorylation of adenosine, inosine, and guanosine to AMP, inosine monophosphate, and GMP, respectively, was detected . Nucleotidase activity for AMP, inosine monophosphate, xanthosine monophosphate, and GMP was also found . Interconversion of GMP to AMP was detected . Enzyme activities for the interconversion of AMP to GMP were not detected . Therefore, A . laidlawii B-PG9 cannot synthesize guanylates from adenylates or inosinates . De novo synthesis of purines was not detected . This study demonstrates that A . laidlawii B-PG9 has the enzyme activities for the salvage and limited interconversion of purines and, except for purine nucleoside kinase activity, is similar to Mycoplasma mycoides subsp . mycoides . This is the first report of a PPi-dependent nucleoside kinase activity in any organism. J Bacteriol, 1984 Jul, 159(1), 260 - 4 Evidence for antitermination in Escherichia coli RRNA transcription; Aksoy S et al.; The stable RNA operons of Escherichia coli do not exhibit polarity, even though they make an RNA product that is not translated . By contrast, most E . coli operons that specify proteins exhibit polarity if their translation is interrupted . The transcriptional component of this polarity depends on the action of Rho protein on the exposed mRNA, which results in premature transcription termination . Here we examine how a stable RNA operon (rrnG) transcript is protected from the Rho protein-mediated polarity response . We compared transcription from the ara and the rrnG promoters through a 16S DNA segment . In each case, the promoter-16S sequences were joined to a trp-lac fusion, and lacZ mRNA was examined in rho+ and rho-115 strains . We found significant Rho protein-dependent termination of transcripts from the ara promoter but little or no Rho protein effect on transcription from the rrnG promoter . We concluded that the transcript of the 16S ribosomal DNA segment does contain Rho protein-dependent transcription terminators, but there is an antitermination system in the rrnG control region that allows it to transcribe through those terminators. J Bacteriol, 1984 Jul, 159(1), 19 - 25 Identification of the phoM gene product and its regulation in Escherichia coli K-12; Ludtke D et al.; Plasmids containing the chromosome region of Escherichia coli encoding phoM, whose product is a positive regulator of alkaline phosphatase expression, were isolated from the Clarke and Carbon plasmid bank . A 9.9-kilobase EcoRI fragment of plasmid pLC17-39 (subcloned into pBR322) was able to complement both phoM and thrB mutations . Restriction endonuclease analysis and in vitro mutagenesis of the hybird plasmids enabled the localization of the phoM gene locus to 3 kilobases of the cloned chromosomal fragment . The phoM gene product was identified, with maxicell techniques, as a protein with an approximate molecular weight of 55,000 . A phoM-lacZ protein fusion was constructed by using a plasmid carrying the phoM gene and a derivative of phage lambda, lambda plac Mu2 . Restriction endonuclease analysis of the plasmid carrying the fusion indicated that phoM is transcribed in a clockwise direction on the circular E . coli chromosome . Analysis of strains bearing the fusion on a multiple-copy plasmid or integrated at the lambda attachment site of the chromosome indicated that the synthesis of the phoM gene product was unaffected by phosphate limitation of growth . The expression of the phoM gene was studied in strains with mutations in genes encoding effectors of the pho regulon . A threefold increase in phoM expression was seen in a phoU strain in comparison with the wild-type strain. J Bacteriol, 1984 Jul, 159(1), 184 - 90 Regulatory changes in the formation of chromosomal dihydrofolate reductase causing resistance to trimethoprim; Flensburg J et al.; High resistance to trimethoprim mediated by the several hundredfold overproduction of the drug target enzyme, dihyrofolate reductase, in a clinically isolated Escherichia coli strain, 1810, was cloned onto several vector plasmids and seemed to be comprised of a single dihydrofolate reductase gene, which by DNA-DNA hybridization and restriction enzyme digestion mapping was very similar to the corresponding gene of E . coli K-12 . Determination of mRNA formation in the originally isolated resistant strain and strains with cloned trimethoprim resistance determinant demonstrated an about 15-fold increase in production of dihydrofolate reductase mRNA compared with that in E . coli K-12 . This was explained by the occurrence of a promoter up mutation in the resistant isolate accompanied by changes in the restriction enzyme digestion pattern found by comparison with the corresponding pattern from E . coli K-12. J Bacteriol, 1984 Jul, 159(1), 159 - 66 Construction in vitro of a cloned nar operon from Escherichia coli; Rondeau SS et al.; To clone the nar operon of Escherichia coli without an effective selection procedure for the nar+ phenotype, a strategy utilizing nar::Tn5 mutants was employed . Partial segments of the nar operon containing Tn5 insertions were cloned into plasmid pBR322 by using the transposon resistance character for selection . A hybrid plasmid was constructed in vitro from two of these plasmids and isolated by a procedure that involved screening a population of transformed nar(Ts) mutant TS9A for expression of thermal stable nitrate reductase activity . A detailed restriction site map of the resulting plasmid, pSR95, corresponded closely to the composite restriction endonuclease map deduced for the nar region from maps of the cloned nar::Tn5 fragments . When transformed with pSR95, wild-type strain PK27 overproduced the alpha, beta, and gamma subunits of nitrate reductase, although nitrate reductase activity was only slightly increased . The alpha and beta subunits were overproduced about 5- to 10-fold and accumulated mostly as an inactive aggregate in the cytoplasm; the gamma subunit overproduction was detected as a threefold increase in the specific content of cytochrome b555 in the membrane fraction . Functional nitrate reductase and the cytochrome spectrum associated with functional nitrate reductase were restored in the nar::Tn5 mutant EE1 after transformation with pSR95 . Although the specific activity of nitrate reductase in this case was less than that of the wild type, both the alpha and beta subunits appeared to be overproduced in an inactive form . In both strains PK27(pSR95) and EE1(pSR95), the formation of nitrate reductase activity and the accumulation of inactive subunits were repressed during aerobic growth . From these observations and the accumulation of inactive subunits were repressed during aerobic growth . From these observations and the demonstration that pSR95 contains a functional nor operon that encodes the alpha, beta, gamma subunits of nitrate reductase. Biochem J, 1984 Jul 1, 221(1), 43 - 51 An additional acidic residue in the membrane portion of the b-subunit of the energy-transducing adenosine triphosphatase of Escherichia coli affects both assembly and function; Jans DA et al.; Glycine at position 9 is replaced by aspartic acid in the mutant b-subunit of Escherichia coli F1F0-ATPase coded for by the uncF476 allele . The mutant b-subunit is not assembled into the membrane in haploid strains carrying the uncF476 allele, but, if the mutant allele is incorporated into a multicopy plasmid, then some assembly of the mutant b-subunit occurs . Two revertant strains were characterized, one of which (AN2030) was a full revertant, the other (AN1953) a partial revertant . DNA sequencing indicated that in strain AN2030 the uncF476 mutation had reverted to give the sequence found in the normal uncF gene . The partial-revertant strain AN1953, however, retained the DNA sequence of the uncF476 allele, and complementation analysis indicated that the second mutation may be in the uncA gene . Membranes prepared from the partial-revertant strain carried out oxidative phosphorylation, although the membranes appeared to be impermeable to protons, and the ATPase activity was sensitive to the inhibitor dicyclohexylcarbodi-imide. Mol Biol (Mosk), 1984 Jul-Aug, 18(4), 907 - 18 {Affinity modification of Escherichia coli ribosomes with photoactivated analogs of mRNA}; Gimautdinova OI et al.; Oligoribonucleotide derivatives containing the photoactivated arylazidogroup at 5'-end of the oligonucleotide fragment {2-(N-2,4-dinitro-5-azidophenyl) aminoethyl} phosphamides of the oligoribonucleotides, azido-NH (CH2)2NHpN (pN) n-1, were prepared . It was demonstrated that azido-NH(CH2)2NHpA(pA)4 and azido-NH (CH2)2NHpU (pU)3 stimulate the binding of the codonspecific aminoacyl-tRNA with ribosome . After irradiation of the ternary complex ribosome-azido-NH (CH2)2NHpU (pU) n-1 X tRNA with UV-light (lambda greater than 350 nm) covalent binding of the reagent to ribosome occurs . Up to 10% of the reagent, bound in the ternary complex with ribosome, is cross-linked with the ribosomal proteins of 30S and 50S subunits . The ribosomal RNA are not modified by azido-NH (CH2)2NHpU (pU) n-1 . The proteins of 30S and 50S subunits, modified with azido-NH (CH2)2NHpU (pU) n-1 with n = 4,7 and 8, were identified . It is shown that proteins of 30S subunits S3, S4, S9, S11, S12, S14, S17, S19, S20 undergo modification . The proteins of 50S subunits L2, L13, L16, L27, L32, L33 are modified . The set of the modified proteins essentially depends on the length of the oligonucleotide part of the reagent and on occupancy of ribosome A-site by a molecule of tRNA. Ann Clin Biochem, 1984 Jul, 21 ( Pt 4), 310 - 7 Evaluation of beta-D-galactosidase from Escherichia coli and horseradish peroxidase as labels by sandwich enzyme immunoassay technique; Imagawa M et al.; beta-D-galactosidase from Escherichia coli and horseradish peroxidase were evaluated as labels of Fab' in dose-response curves for human alpha-fetoprotein and human chorionic gonadotropin by sandwich enzyme immunoassay technique using fluorogenic substrates for enzyme assay . The non-specific binding of Fab'-peroxidase conjugates to IgG-coated polystyrene balls was less than that of Fab'-beta-D-galactosidase conjugates, and the affinity-purified Fab'-peroxidase conjugates gave more sensitive dose-response curves for these antigens than the corresponding beta-D-galactosidase conjugates . However, a large quantity of Fab'-peroxidase conjugates was required and a longer incubation was necessary for the peroxidase assay, since the peroxidase assay was much less sensitive than the beta-D-galactosidase assay . Other advantages and disadvantages of the two enzymes are discussed. Acta Paediatr Scand, 1984 Jul, 73(4), 426 - 32 In-vivo immune responses of breast- and bottle-fed infants to tetanus toxoid antigen and to normal gut flora; Stephens S et al.; The effects of breast- and bottle-feeding on serum immunoglobulin levels and specific antibody responses have been examined in 30 infants on five occasions from 6 days until 9 months of age . No significant differences were found on any sample occasion between the two feeding groups in total immunoglobulin levels of G, M and A classes or in class-specific antibody responses to tetanus toxoid vaccine . This suggests that the capacity of the two groups to make serum antibodies develops similarly . Concentrations of antibodies to commensal Escherichia coli 'O' lipopolysaccharide antigens, however, were significantly greater in the bottle-fed group, and it is suggested that this difference is due to an increase in the exposure of the systemic immune system to these gut antigens in the bottle-fed infants . There are several possible explanations for this increased exposure and the resulting effects on the infants' immune system . These experiments also illustrate a possible role of breast milk in stimulating the immune system. Proc Natl Acad Sci U S A, 1984 Jul, 81(14), 4465 - 9 cis-acting mutations that affect rop protein control of plasmid copy number; Moser DR et al.; A number of pMB1 derivatives provide a trans-acting function that can suppress lethal runaway replication of a temperature-sensitive copy-number mutant of NTP1 . Deletion analysis indicates that the region of the pMB1 genome that contains the rop gene is required for this suppression . Mutant derivatives of the temperature-sensitive copy-number mutant plasmid whose conditional lethal phenotype is not suppressed in trans by the region encoding the rop gene have been isolated . These rop-insensitive derivatives contain single nucleotide changes within the RNA I coding region. Mol Immunol, 1984 Jul, 21(7), 673 - 7 Some monoclonal antibodies raised with a native protein bind preferentially to the denatured antigen; Friguet B et al.; Recent studies with monoclonal antibodies directed against different epitopes of the beta 2-subunit of Escherichia coli tryptophan synthase have shown that some of these antibodies bind rapidly in solution to the native protein; others bind very slowly to native beta 2 in solution while they recognize quite rapidly this antigen adsorbed on a microtitration plate . In the present work, an enzyme-linked immunosorbent assay competition test with either the native or a denatured form of the antigen has been developed . It allowed us to show that the rapidly binding antibodies recognize epitopes present on the native protein while those which react very slowly in solution bind preferentially to the denatured form of the protein . These results prompted us to emphasize how important it is, in the characterization of antibodies, to ascertain that the antigen they recognize remains native in the specificity test. Proc Natl Acad Sci U S A, 1984 Jul, 81(13), 3959 - 63 Tandem promoters preceding the gene for the M1 RNA component of Escherichia coli ribonuclease P; Motamedi H et al.; The nucleotide sequence of a cloned gene for the RNA component of Escherichia coli ribonuclease P, M1 RNA, is presented . The sequence determined extends 320 nucleotides upstream of the 377-base-pair (bp) structural gene and includes three sequences homologous to the consensus E . coli promoter sequence . Two nucleotides found in the M1 RNA structural gene sequence were not found in a previously determined gene sequence of another M1 RNA clone {Reed, R . E., Baer, M . F., Guerrier-Takeda, C., Donis-Keller, H . & Altman, S . (1982) Cell 30, 627-636} . In vitro transcription of supercoiled plasmid DNA containing the M1 RNA gene resulted in a major transcript arising from the strong promoter nearest to the mature M1 RNA . RNAs encoded by the M1 RNA clone in vivo were examined by S1 nuclease mapping . The results indicated that in vivo transcripts originate from all three promoters preceding the M1 RNA gene . These transcripts are apparently processed in a multistep pathway to generate the 5' end of mature M1 RNA. J Cell Physiol, 1984 Jul, 120(1), 75 - 82 Induction of ornithine decarboxylase, RNA, and protein synthesis in macrophage cell lines stimulated by immunoadjuvants; Prosser FH et al.; Early biochemical changes associated with adjuvant stimulation of macrophage protein synthesis were studied using two murine macrophage cell lines, PU5-1.8 and J774.1 . An induction of ornithine decarboxylase (ODC) was detected 2 hours after exposure of PU5-1.8 and J774.1 cells to two crude immunoadjuvants, BCG cell walls (BCGcw) and lipopolysaccharides from Escherichia coli (LPS) . The chemically defined immunoadjuvant glycopeptide, N-acetyl-muramyl-L-alanyl-D-isoglutamine (MDPL) also promoted an increase in ODC activity at 2 hours that was maximal after 4 hours, while little or no effect was observed with the D-alanyl analog (MDPD) that is devoid of adjuvant activity . The increase in ODC activity promoted by BCGcw in PU5-1.8 and J774.1 cells returned toward control levels by 6 to 8 hours . BCGcw also stimulated RNA and protein synthesis which remained elevated for at least 24 hours and was associated with a decrease in DNA synthesis and cell proliferation . ODC induction by BCGcw and MDPL was enhanced by the addition of PGE2 in both cell lines . Indomethacin slightly depressed the magnitude of ODC stimulation by BCGcw in J774.1 cells but failed to alter the response of PU5-1.8 cells . Additional observations indicated that the induction of ODC by BCGcw in both cell lines was preceded by an activation of cyclic AMP-dependent protein kinase . These observations suggest that a cyclic AMP-mediated induction of ODC may be an early biochemical marker of adjuvant stimulation in macrophages. J Bacteriol, 1984 Jul, 159(1), 63 - 70 Energy-requiring translocation of the OmpA protein and alkaline phosphatase of Escherichia coli into inner membrane vesicles; Rhoads DB et al.; In developing a reliable in vitro system for translocating bacterial proteins, we found that the least dense subfraction of the membrane of Escherichia coli was superior to the total inner membrane, both for a secreted protein (alkaline phosphatase) and for an outer membrane protein (OmpA) . Compounds that eliminated the proton motive force inhibited translocation, as already observed in cells; since protein synthesis continued, the energy for translocation appears to be derived from the energized membrane and not simply from ATP . Treatment of the vesicles with protease, under conditions that did not interfere with subsequent protein synthesis, also inactivated them for subsequent translocation . We conclude that export of some proteins requires protein-containing machinery in the cytoplasmic membrane that derives energy from the proton motive force. Infect Immun, 1984 Jul, 45(1), 203 - 9 Escherichia coli O18ac antigen: structure of the O-specific polysaccharide moiety; Gupta DS et al.; The O-specific polysaccharide moiety (O18ac polysaccharide) of the O18ac antigen (lipopolysaccharide) from Escherichia coli 2980 (O18ac:K5:Fim+:H-) was isolated in pure form by degradation of the lipopolysaccharide and chromatography on Sephadex G-50 . The primary structure of the O18ac polysaccharide was elucidated by composition, fragmentation procedures, methylation analysis, and nuclear magnetic resonance spectroscopy . The polysaccharide consists of repeating units of the pentasaccharide: (formula; see text) which are joined in the polymer by alpha-1,2 linkages. J Pharm Pharmacol, 1984 Jul, 36(7), 467 - 70 Rifampicin curing of plasmids in Escherichia coli K12-rifampicin resistant host; Obaseiki-Ebor EE; The in-vitro rifampicin curing of R-plasmids JR225, BN102, BN106 and F'-plasmid F'lac+/R386 in Escherichia coli K12 rifampicin-resistant host J62-2 is described . The minimum rifampicin curing concentration and the frequency of curing of plasmids from the rifampicin-resistant host cell J62-2 and the isogenic rifampicin-sensitive J62-1 host cell were similar . However, rifampicin did not cure some test R-plasmids such as R222 and RP4 from rifampicin-resistant and rifampicin-sensitive host cells, indicating that it is the R-plasmid itself and not the host strain that determines rifampicin curing. Infect Immun, 1984 Jul, 45(1), 299 - 301 Pilus-mediated adherence of Escherichia coli K1 to human oral epithelial cells; Clegg H et al.; We examined the effect of host age and health status on the adherence of mannose-sensitive piliated Escherichia coli K1 to human oral epithelial cells . Mannose-sensitive piliated bacteria adhered in comparable numbers to newborn, older infant, and adult cells (125 +/- 61, 198 +/- 54, and 139 +/- 69 bacteria per cell, respectively) . Prematurity and serious illness did not alter adherence in newborns . The increased susceptibility of premature newborns to E . coli K1 cannot be explained by enhanced epithelial cell adherence. Mol Cell Biol, 1984 Jul, 4(7), 1411 - 5 Detection of deletion mutations in pSV2gpt-transformed cells; Tindall KR et al.; We have developed a system to study mutations that affect xanthine-guanine phosphoribosyltransferase gene (gpt) expression in hypoxanthine-guanine phosphoribosyltransferase-deficient CHO cells that have been transformed by the plasmid vector pSV2gpt . One isolated transformant, designated AS52, carries a single copy of the Escherichia coli gpt gene stably integrated into the high-molecular-weight DNA and expresses the bacterial gene for the enzyme xanthine-guanine phosphoribosyltransferase . Mutants deficient in this enzyme can be induced in the AS52 cell line by a variety of mutagens, and spontaneous or induced mutants can be selected for resistance to 6-thioguanine (Tgr) . Two Tgr clones derived from the AS52 line were analyzed by Southern blot hybridization and were found to contain deletions involving at least a portion of the gpt gene . Because of the small size and stability of the integrated pSV2gpt plasmid, and the well-defined selection protocol for mutant isolation, the AS52 line offers promise as a system suitable for the study of mutation at the molecular level in CHO cells. Mol Biol (Mosk), 1984 Jul-Aug, 18(4), 1081 - 9 {Directed modification of the Tcr gene region of the plasmid pBR322 using complementary single-stranded DNA fragments carrying alkylating groups}; Mazin AV et al.; A method is suggested for chemical modification of preselected regions of plasmid DNA by complementary single-stranded restriction fragments of DNA (srf DNA), carrying alkylating reagents . The gene coding for tetracycline resistance of plasmid pBR322 was used as a target . Srf DNA was prepared by a partial digestion of a double-stranded EcoRI-BamHI restriction fragment (377 base pairs) from Tcr by E . coli exonuclease III . The residues of an alkylating reagent N,N,N'-tri(beta-chlorethyl)-N'-(p-formylphenyl) propylenediamine 1,3 (TFP) were attached covalently to 4-5% of sfr DNA bases . The alkylating derivative of the sfr DNA was hybridized with supercoiled pBR322 plasmid DNA . The hybridization conditions (37 degrees C, 40% formamide, 0,2 M NaCl, 0,1 M Tris-HCl pH 7,5, 0,001 M EDTA) under which the bases carrying TFP residues are not eliminated from the sfr DNA, and transforming activity of pBR322 DNA does not decrease were established . It was shown that about 20% of plasmid pBR322 molecules form D-loops with alkylating sfr DNA under these conditions . It was shown that sfr DNA, carrying TFP can alkylate the complementary region of plasmid DNA, forming cross-linked D-loops . A method for the site-directed mutagenesis of switching off the preselected genes or non-transcribed DNA functional regions (promotors, introns etc) integrated into plasmids of other vectors is suggested. Gene, 1984 Jul-Aug, 29(1-2), 41 - 9 Expression of cloned calf prochymosin cDNA under control of the tryptophan promoter; Nishimori K et al.; To increase yields of calf prochymosin (PC) produced in Escherichia coli, PC cDNA was cloned in a plasmid vector under control of the trp promoter . The hybrid plasmid pCR501 constructed for this purpose contains cDNA coding for PC (from the 5th Arg to the C-terminal Ile) fused to the N-terminal fragment of the trpE gene preceded by the trp promoter and attenuator region . E . coli C600 harboring this plasmid produces approx . 300 000 molecules of PC per cell . This is about a tenfold increase above the amount obtained using lacUV5 promoter {Nishimori et al., Gene 19 (1982) 337-344} . A similar plasmid, pCR601, which contains the same coding sequence fused to the trp promoter and N-terminal fragment of the trpL gene, directs the production of PC at the same rate as pCR501 . In pCR601 the trp attenuator is deleted . Another plasmid, pCR701, in which construction of a sequence coding for fMet-PC cDNA that was aided by chemical synthesis, was placed under direct control of the trp promoter, produced PC at a much lower rate . Extracts prepared from all these bacterial transformants in the presence of urea showed distinct milk-clotting activity after renaturation and processing. Gene, 1984 Jul-Aug, 29(1-2), 251 - 4 Synthesis of human insulin gene . VIII . Construction of expression vectors for fused proinsulin production in Escherichia coli; Guo LH et al.; We have constructed two families of plasmids suitable for the cloning of genes and for directing the synthesis of large amounts of fused proteins in Escherichia coli . The plasmids include the E . coli lac promoter and a portion of the coding sequence for beta-galactosidase, which can code for approx . 590 or 450 amino acids . The truncated beta-galactosidase gene ends with a poly-linker region at the 3' end, which can be cleaved by any one of the eight common restriction enzymes and joined to the gene coding for any desired protein . Each family includes three plasmids that enable fusion to be made in all three of the translational reading frames . We have cloned a synthetic human proinsulin gene into these plasmids, and 30% of the total E . coli protein was represented by the 590 amino acid-long truncated beta-galactosidase fused to proinsulin . The yield of proinsulin in this system is more than twice the amount produced by using a 1007 amino acid-long beta-galactosidase gene for fusion. Gene, 1984 Jul-Aug, 29(1-2), 243 - 6 Molecular cloning of the hamster papovavirus genome in Escherichia coli plasmid vector pBR322; Zimmermann W et al.; The complete genome of the hamster papovavirus (HaPV) which was isolated from virions found in multiple skin tumors of Syrian hamsters was cloned in Escherichia coli using the plasmid vector pBR322 . The cloned viral DNAs were identified by digestion of the recombinant DNAs with various restriction enzymes followed by comparison of their electrophoretic mobilities in agarose gels with that of similarly digested uncloned DNAs . The cloned HaPV DNAs showed the same migration pattern as the corresponding fragments from the restricted uncloned DNAs, indicating that no major insertions or deletions occurred during cloning and plasmid propagation . The electrophoretic data were confirmed by Southern blot hybridization. Gene, 1984 Jul-Aug, 29(1-2), 231 - 41 A technique for integrating any DNA fragment into the chromosome of Escherichia coli; Raibaud O et al.; We describe a technique that allows the insertion of any DNA fragment into the EcoRI-site-containing malPpa, the promoter of malPQ, one of the three maltose operons of Escherichia coli . DNA fragments were cloned into the unique EcoRI site of the pBR322-derived plasmid pOM40, which carries malPpa . In the next step these fragments were transposed into the chromosome by homologous recombination events occurring on both sides of malPp . Cells in which such insertion of the entire recombinant plasmid have occurred can be conveniently selected . Excision and curing of the vector plasmid could then occur spontaneously at a high frequency, leaving behind the inserted fragment that can be manipulated as any chromosomal marker . When the inserted fragment contains a properly positioned promoter, its promoting activity can be estimated by assaying amylomaltase, the product of malQ . When required, the inserted fragment can be easily transferred back onto pOM40 . As examples of application we have transferred two different fragments into the chromosome of E . coli: one contained the ceaC-ceiC operon, which encodes colicin E3 and its immunity protein, and the other contained the lac promoter of E . coli. Gene, 1984 Jul-Aug, 29(1-2), 211 - 9 The replication origin of pSC101: the nucleotide sequence and replication functions of the ori region; Yamaguchi K et al.; The nucleotide sequence of a 770-bp ori region of plasmid pSC101 is presented . The sequence shows homologies to some parts of Escherichia coli