Escherichia coli

Eur J Biochem, 1985 Nov 4, 152(3), 515 - 22
Molecular cloning and expression of human tumor necrosis factor and comparison with mouse tumor necrosis factor; Marmenout A et al.; U-937 cells, a monocytic line derived from a human histiocytic lymphoma, were induced for human tumor necrosis factor (TNF) secretion into the medium and were used for the preparation of TNF mRNA . Biological activity of the latter was quantified in a Xenopus laevis oocyte injection system . TNF mRNA was enriched by gradient centrifugation and this size-fractionated mRNA was used for synthesis of cDNA and inserted into the unique PstI site of pAT153 . A recombinant plasmid containing human TNF cDNA was selected by colony hybridization using an internal fragment of a mouse TNF cDNA clone {Fransen, L., Mueller, R., Marmenout, A., Tavernier, J., Van der Heyden, J., Kawashima, E., Chollet, A., Tizard, R., Van Heuverswyn, H., Van Vliet, A., Ruysschaert, M . R . & Fiers, W . (1985) Nucleic Acids Res . 13, 4417-4429} as a probe . The sequence of this human TNF cDNA is in agreement with the one published by Pennica et al . {Pennica, D., Nedwin, G . E., Hayflick, J . S., Seeburg, P . H., Derynck, R., Palladino, M . A., Kohr, W . J., Aggarwal, B . B . & Goeddel, D . V . (1984) Nature (Lond.) 312, 724-729} . The 157-amino-acid-long mature sequence is about 80% homologous to mouse TNF and its hydrophilicity plot is also very similar, in spite of the apparent species specificity of TNF . In contrast to mouse TNF, it contains no potential N-glycosylation site . When compared to other cytokines, like IFN-beta, IFN-gamma, or IL-2, there is a remarkably high preference for G X C pairs in the third-letter positions . Expression of the TNF cDNA in monkey COS cells or in Escherichia coli gives rise to a protein having similar biological and serological properties as natural human TNF . A human genomic clone was also identified and sequenced; it was found to be in good agreement with the one recently published by Shirai et al . {Shirai, T., Yamaguchi, H., Ito, H., Todd, C . W . & Wallace, R . B . (1985) Nature (Lond.) 313, 803-806}, except for some differences in the introns and 5'-untranslated region.

Eur J Biochem, 1985 Nov 4, 152(3), 645 - 9
Analysis of modification-dependent structural alterations in the anticodon loop of Escherichia coli tRNAArg and their effects on the translation of MS2 RNA; Baumann U et al.; The conformation of the anticodon loop of Escherichia coli tRNAArg was investigated . It is shown that the structure of the anticodon loop is influenced by the base composition of the anticodon stem, and the natural modification of the nucleoside residue 32 in the anticodon loop . The structural effects detected by analysis of the accessibility of the anticodon loop to nuclease S1 could be correlated with the ability of different Arg-tRNAArg species to suppress frame-shifting during translation of MS2 RNA.

Eur J Biochem, 1985 Nov 4, 152(3), 633 - 43
Analysis of secondary structures in M13mp8 (+) single-stranded DNA by the pausing of DNA polymerase alpha; Reckmann B et al.; The pausing of DNA replication has been used as a tool for analyzing secondary structures in a single-stranded DNA . M13mp8 (+) single-stranded DNA was replicated in vitro by the DNA polymerase alpha from calf thymus . The positions of pausing were determined from DNA sequencing gels . All experimentally observed pausing sites could be correlated with computer-predicted secondary structures of the M13 single-stranded DNA . In the computer calculations of the secondary structures, long-range base-pairing, G.T mispairs and loop-out of bases were allowed . By using six different primers, the pausing site pattern and the corresponding secondary structure map of a region comprising 1400 nucleotides of the M13 genome has been established . Our experiments indicate that the M13 DNA is highly structured . Most of the stable structures are clustered around the origin of replication . With fragments of the M13 DNA, we show that long-range base-pairing exists in the M13 single-stranded genome and we present evidence for tertiary structure interactions . Furthermore we observe structures that form newly during the course of replication . The Escherichia coli single-stranded DNA-binding protein facilitates replication through the barriers.

Vet Immunol Immunopathol, 1985 Nov, 10(2-3), 225 - 43
Cellular and humoral elements of the lower respiratory tract of sheep . Immunological examination of cells and fluid obtained by bronchoalveolar lavage of normal lungs; Burrells C; Bronchoalveolar washings were harvested from the excised lungs of 68 normal sheep of 3 different age groups (A: birth to 8 weeks; B: 6 months to 2 years; C: older than 2 years) . Cells and fluid obtained were examined quantitatively and functionally . Fewer cells were present in the lavage fluids of Group A sheep compared with those of Groups B and C . Macrophages were the predominant cell type (70-80%) in all sheep, with lymphocytes being second in number . The lower proportion of lymphocytes in young sheep (Group A) was attributable to lack of B-lymphocytes . As a proportion of the lymphocyte population T-cells were in the majority (60-80%) in all sheep . The proportion of null cells was higher in young sheep than in adults . Pulmonary lymphocytes from sheep of all ages responded poorly to stimulation by the non-specific mitogens phytohaemagglutinin, concanavalin A, pokeweed mitogen and lipopolysaccharide of Escherichia coli . The proportion of neutrophils was higher in sheep over 2 years old compared with younger animals . Eosinophils were present in all age groups but their proportion was greatly increased in animals over 2 years of age . Basophils were absent in young sheep and were present in only low numbers in the lungs of adult animals . In young sheep (Group A) IgG was the predominant immunoglobulin . With age, the percentage of IgG in lung fluid decreased while that of IgA increased so that IgA became the predominant immunoglobulin in older animals (Group C) . In sheep of all ages IgM was present in negligible amounts . The highest value of complement (C3) occurred in adult sheep (Group B) . The total white blood cell counts and differential blood counts of all the sheep were within accepted normal ranges . As in the lungs, there was an age-associated reduction in the proportions of blood T-lymphocytes and an increase in B-lymphocytes . In contrast to lung lymphocytes, those from the blood of sheep of all ages exhibited a wide range of responses to mitogens . The lowest stimulation indices were observed in the oldest sheep (Group C) . The results provide background data against which assessment can be made of changes consequent upon infection with respiratory pathogens.

J Med Chem, 1985 Nov, 28(11), 1668 - 73
Design and synthesis of new transition-state analogue inhibitors of aspartate transcarbamylase; Farrington GK et al.; Six transition-state or bisubstrate analogue inhibitors (6-11) have been designed, synthesized, and tested against aspartate transcarbamoylase (ATCase) . Several of these inhibitors, 7-9, were designed as analogues of N-(phosphonoacetyl)-L-aspartate (PALA, 5a) and incorporated a tetrahedral sulfur group (-S-, -SO-, -SO2-) alpha to a phosphonic acid moiety . Synthesis of 7-9 was accomplished with a new reagent, diethyl (mercaptomethyl)phosphonate (19) . Thiol addition of 19 to diethyl itaconate or other olefins proves a new general synthetic route to (thiomethyl)-phosphonate analogues of acyl phosphates or diphosphate anhydrides . Analysis of the observed inhibition kinetics with ATCase and structural modeling studies indicate that increased steric size of the sulfur moieties in the sulfide 7, sulfoxide 8, sulfone 9, and sulfonamide 10 may cause these compounds to be less potent inhibitors of Escherichia coli ATCase than N-(phosphonoacetyl)-L-aspartate (PALA, 5a) . The pKa of the carbonyl groups (or S-analogue thereof) may be a key factor in determining the affinity of ATCase for inhibitor . The distance from the alpha-carbon to the phosphorus atom was judged to be a less important factor in determining the tightness of inhibitor binding since no significant change in the inhibition constant (Ki) occurred upon elimination of the alpha-methylene group in sulfide 7 to give sulfide 11 . The ester analogue of PALA (5a), O-(phosphonoacetyl)-L-malic acid (6), exhibited a Ki of 2 X 10(-6) M.

Carcinogenesis, 1985 Nov, 6(11), 1611 - 4
Extent of formation of O4-methylthymidine in calf thymus DNA methylated by N-methyl-N-nitrosourea and lack of repair of this product by rat liver O6-alkylguanine-DNA-alkyltransferase; Dolan ME et al.; Calf thymus DNA was methylated by reaction with N-{3H}-methyl-N-nitrosourea and the content of O6-methyldeoxyguanosine, 3-methylthymidine and O4-methylthymidine was determined . It was found that O4-methylthymidine represented only 0.06 +/- 0.02% of the total methylation and that the ratio of O6-methyldeoxyguanosine:O4-methylthymidine was 126 +/- 31 . 3-Methylthymidine represented only 0.05 +/- 0.01% of the total radioactivity and the ratio of O6-methyldeoxyguanosine:3-methylthymidine was 171 +/- 16 . The ability of O6-alkylguanine-DNA-alkyltransferases from Escherichia coli and from rat liver to repair O4-methylthymidine was determined using this methylated DNA as a substrate . When the methylated DNA substrate was incubated with an excess of either of the O6-alkylguanine-DNA-alkyltransferases greater than 95% of the O6-methyldeoxyguanosine was removed . The E . coli O6-alkylguanine-DNA-alkyltransferase also removed 89% of the O4-methylthymidine but the rat liver alkyltransferase did not alter the content of O4-methylthymidine . These results indicate that the mammalian O6-alkylguanine-DNA-alkyltransferase is specific for O6-methylguanine and differs from the bacterial protein in that it does not demethylate O4-methylthymine at any significant rate . This shows that the rat O6-alkylguanine-DNA-alkyltransferase is not able to protect against the possible hazards of the promutagenic lesion, O4-methylthymidine, but the very low extent of formation of this product may limit its significance in carcinogenesis and mutagenesis.

Mol Biol Evol, 1985 Nov, 2(6), 478 - 83
Sequence of the ebgR gene of Escherichia coli: evidence that the EBG and LAC operons are descended from a common ancestor; Stokes HW et al.; The sequence of ebgR, the gene that encodes the EBG repressor, was determined . There is 44% DNA sequence identity between ebgR and lacI, the gene that encodes the LAC repressor . There is also 25% identity between the amino acid sequence of lacI and the deduced amino acid sequence of ebgR . The sequence of 596 bp distal to ebgA, the structural gene for EBG beta-galactosidase, was also determined . Within that region there were two sequences, 74 and 100 bp long, that showed 46% and 50% identity, respectively, to sequences in the first 600 bp of lacY, the structural gene for the lactose permease . The organization and direction of transcription of the repressor and structural genes of the two operons are identical . Taken together with the homology between ebgA and lacZ (as demonstrated in the companion article in this issue), this provides strong evidence that the EBG and LAC operons are descended from a common ancestor . The map position of these two operons supports the notion that these operons diverged following a genome duplication event in an ancestor of Escherichia coli.

Mol Biol Evol, 1985 Nov, 2(6), 469 - 77
Sequence of the ebgA gene of Escherichia coli: comparison with the lacZ gene; Stokes HW et al.; We have sequenced the ebgA (evolved beta-galactosidase) gene of Escherichia coli K12 . The sequence shows 50% nucleotide identity with the E . coli lacZ gene, demonstrating that the two genes are related by descent from a common ancestral gene . Comparison of the two sequences suggests that the ebgA gene has recently been under selection . A significant excess of identical, rather than synonymous, codons used to encode identical amino acids at the same positions in the aligned sequences implies that some form of selection is operating directly at the DNA level . This selection is independent of, and in addition to, selection based on codon usage or on function of the gene products.

Mol Cell Biol, 1985 Nov, 5(11), 3208 - 13
Regulated expression of a Drosophila melanogaster heat shock locus after stable integration in a Drosophila hydei cell line; Sinclair JH et al.; DNA-mediated cotransformation has been used to transfer a Drosophila melanogaster heat shock locus into cultured Drosophila hydei cells by use of the copia-based selectable vector pCV2gpt and of pMH10A, a cloned 87A7 heat shock locus encoding a mutant heat shock protein (hsp) . Transformed lines contain between 50 and 200 copies of both plasmids, each separately organized as a head-to-tail concatemer which is stably maintained in the transformed lines . Exposure of the cotransformants to heat shock temperatures induces the regulated expression of the hsp RNA and the mutant hsp in all the lines analyzed.

Immunobiology, 1985 Nov, 170(4), 270 - 83
Contribution of immune interferon (IFN-gamma) in lymphokine-induced anti-toxoplasma activity: studies with recombinant murine IFN-gamma; Sethi KK et al.; Recombinant E . coli-derived murine interferon gamma (cDNA IFN-gamma) per se induced resident mouse peritoneal macrophages (MPM) and mouse embryo cells to exert marked antitoxoplasma activity . This capacity of cDNA IFN-gamma was abrogated by a specific antiserum to cDNA IFN-gamma which could only neutralize the antiviral activity mediated by this product, whereas a rabbit antiserum directed against murine IFN-alpha/beta proved ineffective in neutralizing these functions . It has been found that rabbit antiserum to cDNA IFN-gamma could also neutralize IFN-gamma-mediated antiviral activity present in crude lymphokine-enriched supernatants of antigen-stimulated toxoplasma-sensitized spleen cells (Toxo-LK) but proved ineffective in abolishing the capacity of Toxo-LK to trigger macrophage anti-toxoplasma activity . The data obtained suggest that macrophage anti-toxoplasma activity induced by Toxo-LK may be an interplay of multiple factor(s) and that Toxo-LK preparations contain soluble factor(s) other than IFN-gamma, which can induce macrophages to kill intracellular Toxoplasma . Experiments in which crude Toxo-LK preparations were incubated with lectin concanavalin A (Con A) showed that this treatment resulted in a block of anti-toxoplasma arming factor(s) activity, as well as a significant reduction of IFN-gamma-mediated antiviral activity present in Toxo-LK . By contrast, no significant difference was observed in the macrophage anti-toxoplasma activity mediated by Con A or untreated cDNA-IFN-gamma.

Arch Biochem Biophys, 1985 Nov 1, 242(2), 440 - 6
Thermodynamics of the reactions catalyzed by the multifunctional enzyme complex tryptophan synthase; Wiesinger H et al.; The intrinsic enthalpy changes (corrected for hydration of D-glyceraldehyde 3-phosphate) for the reactions catalyzed by the alpha and beta 2 subunits of tryptophan synthase from Escherichia coli have been determined calorimetrically . Cleavage of indoleglycerol phosphate (alpha reaction) was found to be associated with a delta H value of 54.0 +/- 2.5 kJ mol-1, while condensation of indole with L-serine (beta reaction) involved -80.3 +/- 4.6 kJ mol-1' . By direct determination of the enthalpy concomitant with the overall synthesis of tryptophan from indoleglycerol phosphate and L-serine an enthalpy value of -13.4 +/- 5.6 kJ mol-1 was observed . In view of the uncertainties of the literature data used for calculation of the hydration contribution, the agreement between the directly measured delta H value of the overall reaction and the sum of the enthalpies of the alpha and beta reactions is fair . Deamination of L-serine, a side reaction catalyzed preferentially by the isolated beta 2 pyridoxal 5'-phosphate2 subunit, was shown to be associated with an enthalpy change of -7.3 +/- 0.4 kJ mol-1.

J Bacteriol, 1985 Nov, 164(2), 836 - 44
Identification and genetic analysis of sbcC mutations in commonly used recBC sbcB strains of Escherichia coli K-12; Lloyd RG et al.; Evidence is presented to show that Escherichia coli JC7618, JC7621, and JC7623, previously regarded as having a recB recC sbcB genotype, carry an additional mutation in a new gene designated sbcC at minute 9 on the standard genetic map . In the absence of the sbcC mutation these strains are sensitive to mitomycin C and have a reduced efficiency of recombination . Cultures of recBC sbcB (sbcC+) strains grow slowly, contain many inviable cells, and rapidly accumulate fast-growing variants due to mutation of sbcC . sbcC has been identified on recombinant plasmids and tentatively located by Tn1000 mutagenesis to a 0.9-kilobase DNA section between proC and phoR.

Genetics, 1985 Nov, 111(3), 655 - 74
Limits of adaptation: the evolution of selective neutrality; Hartl DL et al.; Many enzymes in intermediary metabolism manifest saturation kinetics in which flux is a concave function of enzyme activity and often of the Michaelis-Menten form . The result is that, when natural selection favors increased enzyme activity so as to maximize flux, a point of diminishing returns will be attained in which any increase in flux results in a disproportionately small increase in fitness . Enzyme activity ultimately will reach a level at which the favorable effect of an increase in activity is of the order 1/(4Ne) or smaller, where Ne is the effective population number . At this point, many mutations that result in small changes in activity will result in negligible changes in fitness and will be selectively nearly neutral . We propose that this process is a mechanism whereby conditions for the occurrence of nearly neutral mutations and gene substitutions can be brought about by the long-continued action of natural selection . Evidence for the hypothesis derives from metabolic theory, direct studies of flux, studies of null and other types of alleles in Drosophila melanogaster and chemostat studies in Escherichia coli . Limitations and complications of the theory include changes in environment or genetic background, enzymes with sharply defined optima of activity, overdominance, pleiotropy, multifunctional enzymes and branched metabolic pathways . We conclude that the theory is a useful synthesis that unites many seemingly unrelated observations . The principal theoretical conclusion is that the conditions for the occurrence of neutral evolution can be brought about as an indirect result of the action of natural selection.

J Immunol, 1985 Nov, 135(5), 3505 - 11
Macrophage activation to kill Leishmania major: activation of macrophages for intracellular destruction of amastigotes can be induced by both recombinant interferon-gamma and non-interferon lymphokines; Nacy CA et al.; Macrophages treated with lymphokine (LK)-rich culture fluids from antigen- or mitogen-stimulated spleen cells or the hybridoma T cell 24/G1, or murine recombinant interferon-gamma (IFN-gamma) from either transfected monkey kidney cells (cos rIFN-gamma) or bacterial (E . coli) DNA (rIFN-gamma) developed the capacity to kill intracellular amastigotes of Leishmania major . Removal of IFN activity from LK by neutralizing fluid phase monoclonal anti-rIFN-gamma antibody, or by solid phase immunoadsorption, left residual macrophage activation factors that induced approximately 50% of the macrophage anti-leishmanial activity of untreated LK . In contrast, rIFN-gamma subjected to the same antibody treatments lost all capacity to induce this macrophage effector function . These results suggest that the intracellular destruction of amastigotes is regulated by several different factors . One of these factors is clearly IFN-gamma, which is pleiotropic in its effects on macrophage functions . The other non-IFN LK factors are immunochemically unrelated to IFN-gamma, and may regulate macrophage microbicidal activities in a more selective manner.

Plasmid, 1985 Nov, 14(3), 217 - 23
Conserved regions at the DNA primase locus of IncP alpha and IncP beta plasmids; Lanka E et al.; Genes specifying DNA primases (pri) are common in all IncP plasmids examined so far . These plasmids suppress the thermosensitive character of the Escherichia coli dnaG3 mutation . The mechanism of suppression appears to be identical to that known for RP4 and IncI alpha plasmids . The DNA primases of both these plasmid types can substitute for the dnaG protein in chromosomal DNA replication . The pri genes of the alpha and beta subgroup of IncP plasmids are related to each other as judged from Southern hybridization and immunological data . Extensive DNA and protein sequence homology has been detected although the gene products of the alpha and beta subgroups exhibit substantial differences in size . The arrangement of overlapping genes at the pri locus of IncP alpha plasmids also appears to be present in the IncP beta group.

J Gen Microbiol, 1985 Nov, 131 ( Pt 11), 3135 - 7
Detection of tellurite-resistance determinants in IncP plasmids; Bradley DE; Six IncP plasmids were tested for their ability to generate tellurite-resistant variants by plating bacterial strains harbouring them on medium containing potassium tellurite . Four plasmids, three of subgroup IncP alpha and one not allocated, formed variants that could transfer tellurite-resistance at the same frequency as plasmid-determined drug resistance . This property was not shared by two examples of subgroup IncP beta.

J Gen Microbiol, 1985 Nov, 131 ( Pt 11), 3037 - 45
F41 antigen as a virulence factor in the infant mouse model of Escherichia coli diarrhoea; Bertin A; The properties responsible for the virulence in infant mice of the bovine enterotoxigenic Escherichia coli strain B41 were investigated . A B41K99- variant previously found to be nearly as virulent as the original strain B41 (B41K99+) possessed F41 antigen and haemagglutinating properties . Two variants that did not haemagglutinate sheep and human erythrocytes were isolated from strain B41K99- . These variants simultaneously lost their ability to agglutinate with F41 antiserum and their haemagglutinating properties . They still produced heat-stable enterotoxin . The first B41K99-F41- variant was much less virulent than strains B41K99+ and B41K99-, the second was not virulent at all . F41 properties were not acquired by other E . coli strains by plasmid transfers . Non-haemagglutinating variants could not be obtained from the original strain B41K99+ . However, a B41K99+F41- strain was obtained by a four-step procedure: (i) spontaneous loss of the K99 plasmid, (ii) obtaining a nalidixic acid-resistant mutant, (iii) obtaining a non-haemagglutinating F41- variant, (iv) reacquisition of the K99 plasmid . This B41NalrK99+F41- strain, although producing heat-stable toxin, was not at all virulent, whereas reacquisition of the K99 plasmid by the strain B41NalrF41+ restored virulence . These results show that F41 antigen is an important virulence factor of strain B41 in the infant mouse model.

Can J Microbiol, 1985 Nov, 31(11), 988 - 93
Identification of a trans-dominant mutation affecting proline dehydrogenase in Escherichia coli; Deutch CE et al.; L-Proline dehydrogenase catalyzes the oxidation of L-proline to delta 1-pyrroline-5-carboxylate, a reaction that is an important step in the utilization of proline as a carbon or nitrogen source by bacteria . A mutant of Escherichia coli K-12 lacking L-leucyl-tRNA:protein transferase had been found previously to contain about five times as much proline dehydrogenase activity as its parent strain . This difference has now been shown to be due to the presence in the parent strain of a previously unrecognized mutation . This mutation, which has been designated put-4977, specifically affects proline dehydrogenase rather than proline uptake . Although proline dehydrogenase remains inducible by L-proline in strains carrying the mutation, there is a premature cessation of differential synthesis during induction that results in a lower specific activity . The mutation shows about 50% P1-mediated cotransduction with pyrC and is therefore located at about 22 min on the E . coli chromosome . Merodiploids containing a normal F' factor still exhibit decreased enzyme activity, indicating that the put-4977 mutation is trans-dominant . The mutation cannot be detected in present stocks of the transferase-deficient mutant, suggesting that this mutant is a revertant for put-4977.

Biochem Int, 1985 Nov, 11(5), 709 - 18
Purification of ribosomal proteins from Escherichia coli by cation exchange and reversed phase FPLC; Tam MF et al.; A complex mixture of 21 proteins from the 30S ribosomal subunit of Escherichia coli was fractionated on a cation-exchanger, then further separated on a C8 reversed-phase column . A set of 14 proteins were purified to homogeneity . The same protein mixture was also analysed on a C8 RPC column using a triethylamine phosphate (TEAP, pH2.2)/acetonitrile or a trifluoroacetic acid/acetonitrile solvent system which gave 11 and 8 purified proteins, respectively . Altogether, 16 out of 21 proteins from the 30S ribosomal subunit were purified.

Biochem Int, 1985 Nov, 11(5), 661 - 8
Serine-specific tRNAs in Escherichia coli: relative abundance and sequence; Fischer W et al.; Serine isoaccepting tRNAs were isolated from bulk tRNA of Escherichia coli by affinity chromatography on immobilized bacterial elongation factor Tu and their relative abundance was determined . The three major species, which are sufficient to read all six serine codons, were identified by sequencing . The sequence of a novel tRNASer with the anticodon GGA was elucidated.

Appl Environ Microbiol, 1985 Nov, 50(5), 1187 - 91
Synthetic oligodeoxyribonucleotide probes for detecting heat-stable enterotoxin-producing Escherichia coli by DNA colony hybridization; Hill WE et al.; DNA colony hybridization was used to identify and enumerate enterotoxigenic Escherichia coli strains in foods . The cells were identified and enumerated by using synthetic polynucleotide probes for the heat-stable enterotoxin genes . These 22-base oligonucleotides, made from known nucleotide sequences of the genes for the heat-stable enterotoxins of human and porcine strains of E . coli, contain two mismatches between the two heat-stable enterotoxins . Colonies were replicated from agar medium onto paper filters and lysed with alkali followed by steam; probes were end labeled . After overnight hybridization at 40 degrees C and washing at 50 degrees C, autoradiograms were exposed at -70 degrees C . Results were consistent with suckling-mouse tests for heat-stable enterotoxins . A stronger signal was obtained on paper filters than on nitrocellulose filters . Enterotoxigenic E . coli cells were detected when mixed with a 1,000-fold excess of nonenterotoxigenic E . coli cells . This procedure appears to be more acceptable for routine testing than the use of cloned DNA fragments, labeling by nick translation, and lysing colonies on nitrocellulose filters.

Appl Environ Microbiol, 1985 Nov, 50(5), 1162 - 4
Disinfecting capabilities of oxychlorine compounds; Noss CI et al.; The bacterial virus f2 was inactivated by chlorine dioxide at acidic, neutral, and alkaline pH values . The rate of inactivation increased with increasing pH . Chlorine dioxide disproportionation products, chlorite and chlorate, were not active disinfectants . As chlorine dioxide solutions were degraded under alkaline conditions, they displayed reduced viricidal effectiveness, thereby confirming the chlorine dioxide free radical as the active disinfecting species.

Zentralbl Bakteriol Mikrobiol Hyg {A}, 1985 Nov, 260(3), 293 - 9
The use of the coagglutination test to determine whether Australian and New Zealand isolates of Escherichia coli produce the heat-labile enterotoxin; Bettelheim KA et al.; A rapid method for identifying strains of enterotoxigenic Escherichia coli (ETEC) which produce the heat-labile enterotoxin (LT) was evaluated . It was shown generally to be more sensitive in identifying LT producing ETEC than the traditional methods . It is considered particularly useful for the small laboratory, because no complex equipment is required.

Tohoku J Exp Med, 1985 Nov, 147(3), 281 - 93
Effects of pH on the deconjugation of conjugated bilirubin in human bile; Shinya F et al.; The enzymatic activity of bacterial beta-glucuronidase plays an essential role in the formation of calcium bilirubinate in bile . There are, however, many unsettled problems such as methodology of the assay for its enzymatic activity . In the present study (1) the azopigments from monoconjugated bilirubin (MCB) and unconjugated bilirubin (UCB) in native bile were semiquantitatively determined, (2) the deconjugation of conjugated bilirubin (CB) in bile was estimated with azopigment analysis and (3) factors affecting the deconjugation of CB in bile, especially for pH value, were investigated . CB in bile was stable at physiologic pH during 6-hr incubation at 37 degrees C, but was hydrolyzed at alkaline pH . At physiologic pH, addition of beta-glucuronidase from E . coli hydrolyzed CB in bile and increased MCB and UCB in bile . Based upon the results mentioned above, it is suggested that alkaline pH and enzymatic activity of beta-glucuronidase should cause the increase of UCB in bile . It can be said that beta-glucuronidase is essential for the formation of calcium bilirubinate gallstone at physiologic pH.

J Biochem (Tokyo), 1985 Nov, 98(5), 1387 - 94
A micro-scale method for the conjugation of affinity-purified Fab' to beta-D-galactosidase from Escherichia coli; Inoue S et al.; A micro-scale method for the conjugation of affinity-purified Fab' to beta-D-galactosidase from Escherichia coli is described . Rabbit anti-human chorionic gonadotropin serum (0.2 ml) was digested with pepsin to convert IgG to F(ab')2 and applied to a column of human chorionic gonadotropin-Sepharose 4B, followed by elution at pH 2.5 . The affinity-purified anti-human chorionic gonadotropin F(ab')2 was mixed with non-specific goat F(ab')2 (0.5 mg) as a carrier, reduced with 2-mercaptoethylamine to split F(ab')2 to Fab' and conjugated to beta-D-galactosidase using N,N'-o-phenylenedimaleimide . The affinity-purified rabbit anti-human chorionic gonadotropin Fab'-beta-D-galactosidase conjugate was separated from non-specific goat Fab'-beta-D-galactosidase conjugate and unconjugated beta-D-galactosidase by affinity chromatography on a column of goat (anti-rabbit IgG) IgG-Sepharose 4B using 4 M urea . The amount of the affinity-purified conjugate obtained was 56-69 micrograms . The detection limit of human chorionic gonadotropin by a sandwich enzyme immunoassay technique was improved 30-fold by using the affinity-purified conjugate as compared with that before affinity-purification . This method is applicable to the conjugation with alkaline phosphatase from calf intestine and probably also other enzymes which are stable in 4 M urea.

J Appl Bacteriol, 1985 Nov, 59(5), 443 - 9
Improvements in the microtitre GM1 ganglioside enzyme-linked immunosorbent assay for Escherichia coli heat-labile enterotoxin; Bongaerts GP et al.; A variant of the microtitre GM1-ELISA for Escherichia coli heat-labile enterotoxin was studied . The test was improved by both reducing the assay time from 2 1/2 d to 8 h and by determining the most appropriate GM1 coating concentration . Coating the plates with greater than or equal to 3 micrograms of GM1/ml yielded a maximal sensitivity and ensured a linear relationship between the enterotoxin concentration and the extinction observed when using the final assay-procedure . Thus an optimal accuracy was obtained . This ELISA was 4- to 8-times more sensitive than the Vero cell monolayer assay . The sensitivity of this ELISA and of the chinese hamster ovary cell monolayer assay were identical.

Acta Anaesthesiol Scand, 1985 Nov, 29(8), 831 - 45
Prophylactic and delayed treatment with high-dose methylprednisolone in a porcine model of early ARDS induced by endotoxaemia; Borg T et al.; The effects of prophylactic and delayed treatment with high-dose methylprednisolone were evaluated in a porcine model of early adult respiratory distress syndrome induced by endotoxaemia . Spontaneously breathing pigs under ketamine anaesthesia were infused i.v . with E . coli endotoxin (10 micrograms . h-1 . kg-1) over 6h . Twenty animals received endotoxin without treatment . Eight animals were pretreated with methylprednisolone i.v., 60 mg . kg-1, followed by an i.v . infusion at a rate of 10 mg . h-1 . kg-1 . Ten animals received the same dosage of methylprednisolone beginning 2 h after the start of endotoxin infusion . Pretreatment with methylprednisolone prevented the endotoxin-induced impairment in pulmonary gas exchange and the development of pulmonary oedema . The pulmonary hypertension was counteracted . Cardiac output (Qt) and O2 delivery were improved . Mean arterial blood pressure (MAP) increased and was higher than in the untreated endotoxin group . The profound fall in PMN count was inhibited, while the accumulation of these cells in the lung was still substantial . Survival was improved . Delayed methylprednisolone treatment prevented further deterioration in pulmonary gas exchange and tended to restore it towards baseline . The pulmonary oedema and pulmonary hypertension were reduced . Qt and O2 delivery did not improve . MAP was higher than in the untreated endotoxin group towards the end of the observation period . The decline in PMN count and the pulmonary accumulation of these cells were not significantly influenced . Survival was improved . These results indicate that high-dose methylprednisolone, when given early in the course of sepsis, might be of clinical value in prevention of the devastating pulmonary and circulatory complications of this disease.

Acta Anaesthesiol Scand, 1985 Nov, 29(8), 814 - 30
A porcine model of early adult respiratory distress syndrome induced by endotoxaemia; Borg T et al.; To study the pathophysiology of early adult respiratory distress syndrome (ARDS) induced by sepsis, spontaneously breathing pigs under ketamine anaesthesia were investigated . Twenty animals were infused i.v . with E . coli endotoxin (10 micrograms . h-1 . kg-1) over 6 h, and ten control animals received physiological saline . In the controls, cardiac output (Qt) and O2 delivery decreased slightly . There were no changes in pulmonary gas exchange, pulmonary haemodynamics or extravascular lung water (EVLW) . The polymorphonuclear (PMN) leucocyte count gradually increased, while the platelet count decreased slightly . Endotoxin infusion caused profound deterioration of pulmonary gas exchange, a marked rise in pulmonary vascular resistance (PVR) and a moderate increase in EVLW . The pulmonary dysfunction was not attributable to the pulmonary oedema per se, whereas a "dry" ventilation/perfusion inequality played an important role . The "responders" (peak venous admixture greater than 20%; n = 14) were characterized by higher Qt and lower PVR than the "non-responders" . Qt declined progressively, especially in non-survivors . O2 delivery decreased considerably . Metabolic acidosis probably indicated oxygen deficit . Eleven of 20 animals died during the observation period . Mortality was related more to the imbalance between O2 delivery and oxygen demand than to the deterioration in pulmonary gas exchange . The PMN count decreased markedly while the gradual decline in platelet count was similar to that in the controls . Lung microscopy revealed PMN accumulation in the microvasculature, moderate interstitial oedema and microvascular blood stasis . Our porcine model, which closely mimics early ARDS in man, will be useful in further studies of the pathophysiological pathways and the treatment of this syndrome.

Vet Immunol Immunopathol, 1985 Nov, 10(2-3), 155 - 65
Assessment of attachment, ingestion, and killing of Escherichia coli by bovine polymorphonuclear cells with combined micromethods; Rainard P; A set of microassays separately measuring attachment, ingestion, and overall killing of Escherichia coli by bovine granulocytes was devised and its analytical potential used to test the effect of drugs which block intracellular killing: sodium azide, phenylbutazone, chloroquine phosphate were all inactive, suggesting that O2-dependent systems were not the sole pathway involved in the killing of E.coli by granulocytes . The microtechniques were also used to investigate the opsonic requirements for phagocytosis of two E.coli strains . Absorption of normal bovine serum with the homologous and the heterologous strains showed that specific antibodies were necessary to induce attachment of bacteria to phagocytes . Once bound to granulocytes, the unencapsulated strain P4 was engulfed, whereas for the encapsulated strain B117, complement was required for the internalization step of phagocytosis . With immune serum the need for complement was not absolute.

Res Commun Chem Pathol Pharmacol, 1985 Nov, 50(2), 233 - 43
Conformational analysis of methotrexate; Takle H et al.; Using classical potential functions, the conformation of methotrexate is obtained . Calculations show that glutamate side chain will not have much folding and the plane of pteridine ring would be tilted approximately with an angle of 30 degrees with respect to the plane of phenyl group.

Radiobiologiia, 1985 Nov-Dec, 25(6), 795 - 8
{Radiosensitizing activity of maleic acid derivatives . Effect of maleic acid o-nitroanilide, anilide and diethyl ester on the survival of gamma-irradiated cells of Escherichia coli B/r}; Riabchenko NI et al.; Among three substances under study, only o-nitroanilide of maleic acid (oNAM) possessed a radiosensitizing activity . Radiosensitivity of E . coli B/r cells irradiated in the presence of Ar and oNAM was higher by 4.8 times than that of irradiated controls . The survival rate of E . coli B/r cells irradiated in the presence of oxygen was changed by 1.4 times by the effect of oNAM.

Radiobiologiia, 1985 Nov-Dec, 25(6), 744 - 7
{Radiosensitizing and toxic effect of metronidazole and its ortho-isomer 1-(2-hydroxyethyl)-2-methyl-4-nitroimidazole on Escherichia coli B/r cells}; Riabchenko NI et al.; A study was made of the effect of metronidazole and isometronidazole on the survival rate of irradiated and nonirradiated E . coli B/r cells . These substances had similar radiosensitizing activity with regard to anoxic cells and did not sensitize cells irradiated in the air . At the same time, isometronidazole was found to be less toxic than metronidazole.

Vet Clin North Am Food Anim Pract, 1985 Nov, 1(3), 495 - 508
Bovine enteric colibacillosis; Haggard DL; In this article, the authors discusses procedures used to determine enteropathogenic strains of Escherichia coli, the history of the development of prophylactic procedures, including cow vaccination and specific monoclonal antibody, and other preventative measures with as proper management, nutrition, and sanitation.

Vet Clin North Am Food Anim Pract, 1985 Nov, 1(3), 461 - 9
Pathophysiology of neonatal calf diarrhea; Argenzio RA; Neonatal calf diarrhea caused by bacterial enterotoxins, bacterial or parasitic-induced inflammation, or virus-induced villous atrophy leads to intestinal hypersecretion, malabsorption, or both . Mechanisms of secretion and malabsorption differ depending on the agent, suggesting that different modes of treatment must be employed to be effective . Currently, oral rehydration solutions and the pharmacologic blockade of secretory processes are being evaluated in these various diseases.

Vet Clin North Am Food Anim Pract, 1985 Nov, 1(3), 445 - 59
Septicemic colibacillosis and failure of passive transfer of colostral immunoglobulin in calves; Besser TE et al.; Septicemic colibacillosis is a highly fatal disease that occurs in calves less than 2 weeks of age . The disease occurs when a calf that fails to absorb protective levels of immunoglobulin from colostrum is exposed to an invasive serotype of E . coli . Management to ensure good passive transfer of colostral immunoglobulin will prevent this disease and reduce calf mortality caused by other infectious diseases as well.

Am J Vet Res, 1985 Nov, 46(11), 2288 - 93
Effects of endotoxin on lung water, hemodynamics, and gas exchange in anesthetized ponies; Olson NC; Effects of endotoxemia on lung water, hemodynamics, and gas exchange were determined in ponies breathing a mixture of halothane and 100% O2 . Escherichia coli endotoxin was infused IV at 20 micrograms/kg of body weight for 1 hour followed by 10 micrograms/kg/hr the subsequent 4 hours . By 0.25 hour, endotoxin increased mean pulmonary artery pressure and pulmonary vascular resistance; this was followed by a return to base-line values by 0.5 and 1 hour, respectively . A 2nd increase in pulmonary vascular resistance occurred by 5 hours of endotoxemia . During the last 2 hours of endotoxin infusion, cardiac index was significantly (P less than 0.05) decreased . Hematocrit was increased from 1 to 5 hours of endotoxemia, whereas, the plasma protein concentration was increased from 2 to 4 hours, indicating a loss of plasma volume . The PaO2 and PaCO2 were unchanged . After 5 hours of endotoxemia, lung extravascular thermal volume, postmortem bronchoalveolar lavage albumin content, and extravascular lung water/extravascular dry weight ratio of bloodless lungs were not increased, indicating no increase in alveolar-capillary permeability or pulmonary edema.

Proc Natl Acad Sci U S A, 1985 Nov, 82(22), 7599 - 603
Transient fluorescence in synchronously dividing Escherichia coli; Layne SP et al.; Using a spectrometer equipped with an optical multichannel analyzer as the detector, we observed the Stokes laser-Raman spectra of metabolically synchronous Escherichia coli from 100 to 2100 cm-1 . After more than 400 separate recordings, at cell concentrations of 10(7)-10(8) per ml, no Raman lines attributable to the metabolic process nor to the cells themselves were found . However, we did find that synchronous E . coli cultures become more fluorescent during a limited phase of the division cycle . This transient increase in fluorescence may be ascribed to a variation in the redox state of a chemical species within the bacteria or to a variation of the intracellular optical field . The effect is reproducible in synchronous cultures and it is not seen in asynchronous ones . The results suggest that spectral features seen in previous laser-Raman spectra of synchronous bacteria (taken with scanning monochromators) are due to a time-dependent variation in bacterial fluorescence.

Proc Natl Acad Sci U S A, 1985 Nov, 82(22), 7480 - 4
Mapping of functional domains in adenovirus E1A proteins; Krippl B et al.; We have modified the E1A gene of human subgroup C adenovirus by introducing deletions in its coding sequence . Various truncated E1A proteins were expressed in Escherichia coli, purified, and microinjected via glass capillaries into Vero cells . We monitored their movement from the cell cytoplasm to the nucleus and their ability to induce expression of H5dl312, an adenovirus E1A deletion mutant . Our results show that the carboxyl terminus of E1A contains sequences essential for rapid and efficient nuclear localization . Essential information for efficient H5dl312 complementation is contained in an internal region, comprising sequences of both exons of the E1A gene . A first exon-encoded region, however, is sufficient to induce low levels of adenovirus gene expression . Information for nuclear localization and for H5dl312 complementation are therefore encoded by distinct domains of the E1A gene . In addition, we determined that the human c-myc product was unable to complement H5dl312.

J Appl Physiol, 1985 Nov, 59(5), 1464 - 71
Effects of flunixin meglumine on cardiopulmonary responses to endotoxin in ponies; Olson NC et al.; The effects of endotoxemia on cardiopulmonary parameters, before and after cyclooxygenase blockade, were determined in anesthetized ponies spontaneously breathing a mixture of halothane and 100% O2 . Escherichia coli endotoxin was infused intravenously at 20 micrograms/kg for 1 h followed by 10 micrograms X kg-1 X h-1 the subsequent 4 h . By 15 min endotoxin increased mean pulmonary arterial pressure (Ppa), pulmonary vascular resistance (PVR), and alveolar dead space ventilation (VDA/VT), and these were followed by a return to base-line values by 30 min . A second increase in PVR occurred by 5 h of endotoxemia . The early increases in Ppa, PVR, and VDA/VT were blocked by flunixin meglumine (FM), a cyclooxygenase inhibitor . Endotoxin decreased central plasma volume by 1 h and cardiac index by 3 h; hematocrit and plasma protein concentration were increased by 0.5 and 1.5 h, respectively, indicating a loss of plasma volume . These changes were also blocked or attenuated by FM . Moreover, in ponies treated with endotoxin + FM, cardiac index increased, indicating the presence of a cardiac-stimulating factor . We conclude that endotoxemia in ponies causes cardiopulmonary dysfunction that is mediated by cyclooxygenase-dependent and possibly cyclooxygenase-independent metabolites.

EMBO J, 1985 Nov, 4(11), 2731 - 7
Liposome-mediated transformation of tobacco mesophyll protoplasts by an Escherichia coli plasmid; Deshayes A et al.; An Escherichia coli plasmid, pLGV23neo, carrying a kanamycin resistance gene expressed in plant cells, was encapsulated into negatively charged liposomes prepared by the reverse phase evaporation technique . These liposomes were induced to fuse with tobacco mesophyll protoplasts by polyethyleneglycol treatment . Kanamycin-resistant clones were reproducibly isolated from transfected cultures at an average frequency of 4 X 10(-5) . Plants regenerated from these resistant colonies were confirmed to be transformed according to three criteria . Protoplasts isolated from their leaves were resistant to 100 micrograms/ml kanamycin . The enzyme aminoglycoside 3'-phosphotransferase II encoded by the plasmid pLGV23neo was detected in leaf extracts . Approximately 3-5 copies of the gene encoding for kanamycin resistance were inserted in the genome of at least one of the studied transformants . The restriction pattern of inserted DNA was best explained by assuming a tandem integration of the pPLGV23neo sequences, implying an homologous recombination event between these sequences during transformation . Kanamycin resistance was transmitted as a single dominant nuclear marker to the progeny of resistant plants after selfing or cross-pollination with the wild-type.

Endoscopy, 1985 Nov, 17(6), 228 - 30
Unusual long-term complication after endoscopic sphincterotomy: stone formation in the gallbladder; Matsumoto S et al.; Long-term complications of endoscopic sphincterotomy hitherto reported consist of recurrent common bile duct stones, cholangitis, cholecystitis and stenosis of the sphincterotomy opening . A case of unusual late complication - formation of new stones in the gallbladder - is described and consideration is given to the etiology.

Brain Res Bull, 1985 Nov, 15(5), 459 - 63
Effects of endotoxin and sodium salicylate on the preoptic thermosensitive neurons in tissue slices; Nakashima T et al.; Effects of E . coli endotoxin and sodium salicylate (Sal) on single-unit activity of thermosensitive neurons recorded in slices of preoptic and anterior hypothalamic area (PO/AH) were studied in vitro . Perfusion of endotoxin-containing Krebs-Ringer's solution or local application of endotoxin in the immediate vicinity of recording neurons decreased and increased the firing rate of 31 of 34 warm-sensitive neurons and a cold-sensitive neuron, but had no effect on the majority of thermally insensitive neurons . In about half of warm-sensitive neurons the inhibitory response to endotoxin was preceded by a transient increase in firing rate . The pyrogen-induced decrease in firing rate in warm-sensitive neurons was reversibly blocked or attenuated by local application of Sal in a dose-dependent manner . The results are consistent with the view that pyrogen and Sal act in the PO/AH to produce fever and antipyresis, respectively, by appropriately offsetting the activity of thermosensitive neurons.

Proc Natl Acad Sci U S A, 1985 Nov, 82(21), 7374 - 8
korA function of promiscuous plasmid RK2: an autorepressor that inhibits expression of host-lethal gene kilA and replication gene trfA; Young C et al.; In broad host-range plasmid RK2, korA function prevents the lethal effect of kilA on Escherichia coli host cells and inhibits expression of trfA, the essential replication gene . From gene fusion and promoter replacement studies, we determined that control of kilA is also mediated at the level of gene expression and that the target resides in the kilA promoter region . The nucleotide sequence of this region shows the same two operator-like palindromes present in the previously sequenced promoters of trfA and korA . One of the palindromes (5'-GTTTAGCTAAAC-3') at the -10 position is sufficient to confer sensitivity to korA function . The presence of the same sequences in the korA promoter region suggested that korA might also regulate its own expression . Using the structural gene for chloramphenicol acetyltransferase (cat) fused to the korA promoter, we found that korA gene expression is indeed autoregulated . The results show that korA gene product is very likely a repressor that negatively regulates expression of at least three different genes by interacting with an operator-like sequence in their promoter regions . Coordinate regulation of host-lethal gene kilA and essential replication gene trfA by a common mechanism also supports our hypothesis that these genes are functionally related.

Proc Natl Acad Sci U S A, 1985 Nov, 82(21), 7252 - 5
Oxygen binding properties of human mutant hemoglobins synthesized in Escherichia coli; Nagai K et al.; Human beta-globin was synthesized in Escherichia coli as a cleavable fusion protein, using the expression vector pLcIIFX beta-globin {Nagai, K . & Thogersen, H . C . (1984) Nature (London) 309, 810-812} . The fusion protein cIIFX beta-globin was purified to homogeneity and cleaved at the junction by blood coagulation factor Xa; the authentic beta-globin was liberated . Beta-globin was folded in vitro and reconstituted with heme and alpha subunits to form alpha 2 beta 2 tetramers . The oxygen binding properties of reconstituted Hb are essentially the same as those of human native Hb . Two mutant Hbs (Hb Nympheas {Cys-93 beta----Ser} and Hb Daphne {Cys-93 beta----Ser, His-143 beta----Arg}) were constructed by site-directed mutagenesis using synthetic oligonucleotides . Hb Nympheas showed a slightly increased oxygen affinity and diminished cooperativity with normal 2,3-diphosphoglyceric acid and slightly reduced alkaline Bohr effects . Hb Daphne showed low cooperativity with high oxygen affinity . The alkaline Bohr effect was slightly reduced but the diphosphoglycerate effect was enhanced by 50% by the His-143 beta----Arg mutation . As arginine is fully charged at physiological pH and has a long flexible side chain, diphosphoglycerate binds more strongly to Hb Daphne.

Proc Natl Acad Sci U S A, 1985 Nov, 82(21), 7212 - 6
Synthesis and secretion of human epidermal growth factor by Escherichia coli; Oka T et al.; A synthetic gene for human epidermal growth factor (hEGF) was joined to a sequence encoding the signal peptide of Escherichia coli alkaline phosphatase . This hybrid gene was placed under the control of the alkaline phosphatase gene (phoA) promoter in a recombinant plasmid, which was used to transfect E . coli . The hybrid protein that was expressed in host cells under conditions of phosphate limitation was processed accurately during the secretion process, and mature hEGF was recovered in the periplasmic fraction . On the other hand, no EGF was detected in the periplasmic space when the synthetic hEGF gene was not accompanied by the phoA signal sequence.

Mutat Res, 1985 Nov, 144(3), 145 - 9
Competition between the dam mutator and the isogenic wild-type of Escherichia coli; Trobner W et al.; Previous studies have shown that the mutT, mutH, mutL and mutS mutators of Escherichia coli confer a marked selective advantage on their respective hosts in competition with otherwise isogenic wild-type strains . We have conducted competition experiments between dam- and dam+ strains of Escherichia coli and have found that dam mutator strains are negatively selected . Although dam- is the first mutator to have a lower fitness than wild-type under chemostat conditions our result does not contradict the hypothesis that increased mutation rates are of evolutionary advantage under environmental stress conditions . Only in the special case of dam- does the advantage of higher mutation rates not outweigh the disadvantage due to the dam- -caused heavy pleiotropic effects.

J Clin Microbiol, 1985 Nov, 22(5), 705 - 7
Enterotoxigenic Escherichia coli strains belonging to a new serogroup, Escherichia coli O166; Gross RJ et al.; Thirty-two strains of Escherichia coli belonging to a new O group, O166, were examined . Twenty-one strains had the flagella antigen H27, five had the H15 antigen, five had the H7 antigen, and one was nonmotile . All the H27 strains and the nonmotile strain produced heat-stable enterotoxin but not heat-labile enterotoxin . All the H7 strains produced heat-labile enterotoxin but not heat-stable enterotoxin . The remaining strains were nonenterotoxigenic . None of the strains possessed colonization factor antigens CFA/I, CFA/II, or PCF8775.

J Bacteriol, 1985 Nov, 164(2), 861 - 5
Involvement of the relA gene in the autolysis of Escherichia coli induced by inhibitors of peptidoglycan biosynthesis; Kusser W et al.; It is generally assumed that inhibitors of peptidoglycan biosynthesis do not kill nongrowing bacteria . An exceptional case is reported here . The addition of chloramphenicol to amino acid-deprived cultures of relA+ strains of Escherichia coli which were treated with beta-lactam antibiotics, D-cycloserine, or moenomycin resulted in lysis . This phenomenon is termed chloramphenicol-dependent lysis . To be effective, chloramphenicol had to be present at its minimum growth-inhibitory concentration (or higher) . Analogs of chloramphenicol which did not bind to ribosomes were completely ineffective . Amino acid deprivation was actually not required to demonstrate chloramphenicol-dependent lysis, and cultures treated with growth-inhibitory levels of chloramphenicol alone were lysed when challenged with inhibitors of peptidoglycan synthesis . Peptidoglycan synthesis has been shown previously to be under stringent (relA+) control, and chloramphenicol is known to be an antagonist of stringent control . Thus, it is proposed that the mechanism of chloramphenicol-dependent lysis is based on the ability of chloramphenicol to relax peptidoglycan synthesis in nongrowing relA+ bacteria . This is also consistent with the observation that treatment of amino acid-deprived relA mutants with inhibitors of peptidoglycan synthesis resulted in lysis, i.e., without the mediation of chloramphenicol.

J Bacteriol, 1985 Nov, 164(2), 854 - 60
Regulation of sialic acid metabolism in Escherichia coli: role of N-acylneuraminate pyruvate-lyase; Vimr ER et al.; In Escherichia coli, synthesis of sialic acid is not regulated by allosteric inhibition mediated by cytidine 5'-monophospho-N-acetylneuraminic acid (CMP-NeuNAc) . Evidence for the lack of metabolic control by feedback inhibition was demonstrated by measuring the intracellular level of sialic acid and CMP-NeuNAc in mutants defective in sialic acid polymerization and in CMP-NeuNAc synthesis . Polymerization-defective mutants could not synthesize the polysialic acid capsule and accumulated ca . 25-fold more CMP-NeuNAc than the wild type . Mutants unable to activate sialic acid because of a defect in CMP-NeuNAc synthetase accumulated ca . sevenfold more sialic acid than the wild type . An additional threefold increase in sialic acid levels occurred when a mutation resulting in loss of N-acylneuraminate pyruvate-lysase (sialic acid aldolase) was introduced into the CMP-NeuNAc synthetase-deficient mutant . The aldolase mutation could not be introduced into the polymerization-defective mutant, suggesting that any further increase in the intracellular CMP-NeuNAc concentration was toxic . These results show that sialic acid aldolase can regulate the intracellular concentration of sialic acid and therefore the concentration of CMP-NeuNAc . We conclude that regulation of aldolase, mediated by sialic acid induction, is necessary not only for dissimilating sialic acid (E.R . Vimr and F . A . Troy, J . Bacteriol . 164:845-853, 1985) but also for modulating the level of metabolic intermediates in the sialic acid pathway . In agreement with this conclusion, an increase in the intracellular sialic acid concentration was correlated with an increase in aldolase activity . Direct evidence for the central role of aldolase in regulating the metabolic flux of sialic adid in E . coli was provided by the finding that exogenous radiolabeled sialic acid was specifically incorporated into sialyl polymer in aldolase-negative strain but not in the wild type.

J Bacteriol, 1985 Nov, 164(2), 845 - 53
Identification of an inducible catabolic system for sialic acids (nan) in Escherichia coli; Vimr ER et al.; Escherichia coli K-12 and K-12 hybrid strains constructed to express a polysialic acid capsule, the K1 antigen, were able to efficiently use sialic acid as a sole carbon source . This ability was dependent on induction of at least two activities: a sialic acid-specific transport activity, and an aldolase activity specific for cleaving sialic acids . Induction over basal levels required sialic acid as the apparent inducer, and induction of both activities was repressed by glucose . Induction also required the intracellular accumulation of sialic acid, which could be either added exogenously to the medium or accumulated intracellularly through biosynthesis . Exogenous sialic acid appeared to be transported by an active mechanism that did not involve covalent modification of the sugar . Mutations affecting either the transport or degradation of sialic acid prevented its use as a carbon source and have been designated nanT and nanA, respectively . These mutations were located by transduction near min 69 on the E . coli K-12 genetic map, between argG and glnF . In addition to being unable to use sialic acid as a carbon source, aldolase-negative mutants were growth-inhibited by this sugar . Therefore, the intracellularly accumulated sialic acid was toxic in aldolase-deficient E . coli strains . The dual role of aldolase in dissimilating and detoxifying sialic acids is consistent with the apparent multiple controls on expression of this enzyme.

J Bacteriol, 1985 Nov, 164(2), 665 - 73
In vivo and in vitro synthesis of Escherichia coli maltose-binding protein under regulatory control of the lacUV5 promoter-operator; Rasmussen BA et al.; It has not been possible to obtain in vitro expression of the positively regulated malE gene encoding the periplasmic maltose-binding protein (MBP) of Escherichia coli . To facilitate in vitro malE expression, we constructed plasmids that place the malE gene under transcriptional control of the lacUV5 promoter-operator . These plasmids could be grouped into three classes, based upon their ability to complement in vivo a chromosomal malE deletion in the presence or absence of isopropyl thiogalactoside . In the one class I plasmid analyzed, the lacUV5-malE junction was just 3' to the malE ATG initiation codon, and this plasmid did not complement the malE deletion . Class II and class III plasmids retained various amounts of the malE promoter . MBP synthesis was solely under control of the lacUV5 promoter in the class II plasmids, and MBP synthesis was under control of both the lacUV5 and malE promoters in the class III plasmids . A malE mutation that renders the MBP signal peptide export defective was genetically recombined onto one of the class II plasmids . The in vivo synthesis and export of plasmid-encoded MBP were studied in the presence and absence of isopropyl thiogalactoside and maltose and in a strain harboring a prlA mutation that suppresses the malE signal sequence mutation and is thought to alter the export machinery of cells . In addition, both class II and class III plasmids programmed the synthesis of precursor MBP in an in vitro-coupled transcription-translation system . When precursor MBP was synthesized in vitro in the presence of E . coli membrane vesicles, a significant portion of wild-type precursor MBP, but not export-defective precursor MBP, was converted to a form that migrated on sodium dodecyl sulfate-polyacrylamide gels identically to mature MBP synthesized in vivo.

J Bacteriol, 1985 Nov, 164(2), 646 - 52
Properties of an Escherichia coli mutant deficient in phosphoenolpyruvate carboxylase catalytic activity; Coomes MW et al.; A mutant Escherichia coli (Ppcc-) which was unable to grow on glucose as a sole carbon source was isolated . This mutant had very low levels of phosphoenolpyruvate carboxylase activity (approximately 5% of the wild type) . Goat immunoglobulin G prepared against wild-type phosphoenolypyruvate carboxylase cross-reacted with the Ppcc- enzyme . The amount of enzyme protein in the mutant cells was similar to that found in wild-type cells, but it had greatly diminished specific activity . The catalytically less active mutant enzyme retained the ability to interact with fructose 1,6-bisphosphate, but did not exhibit stabilization of the tetrameric form by aspartate . The pI of the mutant protein was lower (4.9) than that of the wild-type protein (5.1) . After electrophoresis and immunoblotting of the partially purified protein, several immunostaining bands were seen in addition to the main enzyme band . A novel method for showing that these bands represented proteolytic fragments of phosphoenolpyruvate carboxylase was developed.

J Bacteriol, 1985 Nov, 164(2), 585 - 90
Domains involved in osmoregulation of the ompF gene in Escherichia coli; Inokuchi K et al.; Expression of the ompF gene, which is under the control of the OmpR protein, is regulated by the osmolarity of the medium . To study the mechanism of osmoregulation, plasmids carrying two different types of chimeric genes were constructed . In one type, the coding region of the ompF gene was linked to the trp promoter (trpPO) preceding ompF, and in the other type the ompF upstream region, mostly composed of the region for regulation by OmpR and the promoter region, was linked to the lacZ gene by protein fusion . Expression of beta-galactosidase by the lacZ chimeric gene was OmpR dependent and osmoregulated as sensitively as that of the intact ompF gene . In the ompR20 background the direction of osmoregulation was opposite that of normal osmoregulation, as was the direction of osmoregulation of the intact ompF gene . Osmoregulation was also observed with trpPO-ompF chimeric genes . However, the regulation was not as sensitive to the osmolarity of the medium as was regulation of the intact ompF gene and was independent of OmpR . These results suggest that OmpR-dependent osmoregulation played a primary role in the osmoregulation of ompF expression and that ompR-independent osmoregulation most likely did not play a crucial role . Studies with a series of trpPO-ompF chimeric genes also suggest that the untranslated leader region, about 100 base pairs in length, between the transcription initiation site and the initiation codon was not required for osmoregulation.

Infect Immun, 1985 Nov, 50(2), 506 - 9
In vitro adherence of Escherichia coli to endometrial epithelial cells of rats and influence of estradiol; Nishikawa Y et al.; The influence of ovarian hormones on the adhesion of Escherichia coli to endometrial epithelial cells was investigated in an in vitro system . Endometrial cells liberated by collagenase from rat uteri were used . Optimal test conditions were obtained when 5 X 10(8) E . coli bacteria were added to 10(5) epithelial cells and incubated for 60 min . The adhesion of the organisms was inhibited by the addition of either mannose or alpha-methyl-D-mannopyranoside . When epithelial cells collected from uteri of estradiol-treated rats were used, the number of E . coli adhering to the cells was markedly lower than that adhering to epithelial cells collected from control rats . These results suggest that E . coli adheres to endometrial epithelial cells with so-called type 1 pili and that estradiol alters the nature of the endometrial epithelium and prevents the adherence of the organisms to the cells.

Proc Natl Acad Sci U S A, 1985 Nov, 82(21), 7355 - 9
Mutagenesis of the lac promoter region in M13 mp10 phage DNA by 4'-hydroxymethyl-4,5',8-trimethylpsoralen; Piette J et al.; Double-stranded M13 phage DNA (M13 mp10 replicative form) was photoreacted with 4'-hydroxymethyl-4,5',8-trimethylpsoralen, using light of wavelength greater than 320 nm or greater than 390 nm to generate predominantly crosslinks or monoadducts, respectively . The damaged DNAs were scored for inactivation and mutagenesis after transfection into Escherichia coli . The appearance of light-blue or colorless plaques on indicator medium showed that mutation had occurred in the lac insert of the viral DNA . A comparison of the consequences of the two phototreatments with psoralen supports the idea that crosslinks are both more lethal and more mutagenic than monoadducts . Numerous mutant clones partially or totally deficient in beta-galactosidase were plaque-purified and amplified . The viral DNA of each clone was sequenced by the dideoxy chain-terminating procedure . All of the observed base-pair changes were mapped to the lac promoter region and consisted of 3 transition, 14 transversion, and 6 single base-pair frame-shift mutations . The predominant mutation was a T.A----G.C transversion.

J Biochem (Tokyo), 1985 Nov, 98(5), 1275 - 84
Large scale isolation and some properties of AGY-specific serine tRNA from bovine heart mitochondria; Ueda T et al.; A method was developed for large scale isolation of AGY-specific serine tRNA (tRNASerAGY) from bovine heart mitochondria . By this method, 5 A260 units of tRNASerAGY were recovered from 6.3 kg of bovine hearts . The nucleotide sequence was identical to that reported previously . tRNASerAGY showed abnormal melting profiles, as was predicted from its unique primary sequence . Its secondary and/or tertiary structure was analyzed by nuclease digestion method . It was suggested that three extra base pairs could occur in the anticodon stem region, with one adenosine residue protruding . The T loop was quite sensitive to nuclease S1, suggesting that the T loop doesn't interact with other regions . This finding is consistent with the model proposed by Sundaralingam (1980) . tRNASerAGY was aminoacylated in vitro with only mitochondrial enzyme but not with the enzymes from E . coli and yeast . The aminoacylation rate of tRNASerAGY with mitochondrial enzyme was much faster than that of cytosolic tRNASerUCN, perhaps reflecting differences due to the presence and absence of the D arm of the tRNAs.

Bioorg Khim, 1985 Nov, 11(11), 1574 - 6
{Aminooxypropylamine--an effective inhibitor of ornithine decarboxylase in vitro and in vivo}; Khomutov RM et al.; Hydroxylamine-containing analogues of putrescine and cadaverine have been found effective in inhibiting the mouse liver ornithine decarboxylase, the best among synthesized were 1-aminooxy-3-aminopropane (I50 2.10(-8) M) and 1-aminooxy-4-aminobutane (I50 2.10(-7) M) . The inhibitory effect of these substances on the mouse liver ornithine-transaminase and S-adenosylmethionine decarboxylase from E . coli was displayed at concentrations higher by several orders of magnitude, that demonstrated the specificity of the compounds of this type . 1-Aminooxy-3-aminopropane in experiments in vivo suppressed the ornithine decarboxylase activity in mouse liver at 16 mg/kg by 75%, the toxic effect being insignificant.

Mol Gen Mikrobiol Virusol, 1985 Nov, (11), 20 - 4
{Recombinant plasmids capable of integration into the chromosome of the cyanobacterium Anacystis nidulans R2}; Elanskaia IV et al.; Recombinant plasmids, series pIAB and pIAH, have been constructed by insertion of BamHI or HindIII chromosomal fragments from Anacystis nidulans R2 into the tet gene of plasmid pACYC184 . Plasmids pIAB and pIAH are stably maintained in Escherichia coli cells and transfer the CmR marker in transformation of Anacystis nidulans . Blot hybridization technique has shown the formation of CmR clones in transformation to result from integration of plasmid pACYC184 with the chromosome of cyanobacterium.

Mol Gen Mikrobiol Virusol, 1985 Nov, (11), 16 - 20
{Identification of the plasmid R1drd-19 region complementing the mutant phenotype recB- in Escherichia coli K12 strain}; Ovadis MI et al.; Plasmid R1-19 and its copy number mutants markedly increase the recombinational efficiency of a recB- strain of E . coli K12 and its resistance to the lethal action of UV and mitomycin C . These effects are associated with the appearance of a new ATP-dependent exonuclease activity in recB- cells known to be deficient in the ATP-dependent exonuclease V . Using hybrid plasmids carrying different EcoRI fragments of R1-19 (in the pSF124 vector), the gene(s) responsible for effect of R1-19 in recB-cells were localized in the EcoRI-C fragment (8.5 MD) belonging to the RTF portion of R1-19 . Expression of the gene(s) in hybrid plasmids depends on the orientation of EcoRI-C fragment in the vector . The copy number of the EcoRI-C fragment was not strictly correlated with the degree of expression of the effects in the recB- mutant.

Mol Cell Biol, 1985 Nov, 5(11), 3241 - 50
Extrachromosomal replication of shuttle vectors in Dictyostelium discoideum; Firtel RA et al.; We cloned a 12.3-kilobase (kb) endogenous plasmid, Ddp1, found in several wild-type and laboratory strains of Dictyostelium discoideum into pBR322 . The cloned plasmids have been used to cotransform D . discoideum cells with B10S, a transformation vector carrying a gene fusion conferring resistance to G418 . Whereas B10S DNA alone appears to integrate in a tandem array, the cloned Ddp1 plasmids replicate extrachromosomally and are stably maintained in the absence of selection with an average copy number of 50 to 100 copies per cell . The Ddp1-derived plasmids can be directly recovered by transforming Escherichia coli with bulk nuclear DNA from these cells . Preliminary deletion analysis indicates that not all regions of Ddp1 are necessary for stable replication in D . discoideum . Several recombinant vectors which replicate extrachromosomally in D . discoideum were also isolated . One contains the Act6-neor gene fusion from B10S recombined into one of the cloned derivatives of Ddp1 and can be used to directly transform D . discoideum amoebae, selecting for G418 resistance . Another recombinant is only 5.6 kb and resulted from a deletion of a 16.6-kb cloned Ddp1 hybrid plasmid . An analysis of the vector DNAs present in clones derived from single D . discoideum transformants is also described.

Mol Cell Biol, 1985 Nov, 5(11), 2993 - 3000
The proto-oncogene c-ets is preferentially expressed in lymphoid cells; Chen JH; The transforming sequences of the avian acute leukemia virus, E26, contain two distinct oncogenes, v-mybE and v-ets, fused together . By using a probe containing v-ets sequences, polyadenylated transcripts of the c-ets proto-oncogene were detected in avian tissues; they included a major 7.0-kilobase and a minor 2.0-kilobase species . These c-ets mRNAs were detected at high levels only in lymphoid organs and in avian T and B lymphoid cell lines . A similar pattern of c-ets transcription was observed in human hematopoietic cell lines, with transcripts detected in lymphoid B and T cells but not in erythroid or myeloid cells . The E26 oncogene was inserted into an inducible expression vector, and a 90-kilodalton protein (bp90) was produced in bacteria . Rabbit antisera raised to purified bp90 precipitated P135gag-mybE-ets, the v-mybE-ets polyprotein expressed in E26-transformed cells, and also reacted with p50v-mybA, the transforming protein of the avian myeloblastosis virus . Antiserum to bp90 was absorbed with a bacterially synthesized v-mybA protein to remove anti-myb activity . The absorbed anti-bp90 serum retained the ability to immunoprecipitate P135gag-mybE-ets from E26-transformed cells and specifically reacted with a 56-kilodalton polypeptide (p56) detected in chicken lymphoid organs and in T and B lymphocytes of both avian and human origin . The data suggest that p56 is a translational product of the c-ets proto-oncogene and imply that p56 may be involved in regulating the growth of lymphoid cells.

Ann Inst Pasteur Immunol, 1985 Nov-Dec, 136D(3), 231 - 43
Monoclonal antibody detection of 2-acetyl-aminofluorene-modified DNA probes for the specific detection of nucleic acids in hybridization procedures; Masse MJ et al.; We describe the use of acetoxy-acetyl-aminofluorene-modified DNA probes in several hybridization techniques . Hybrids were detected with the help of a monoclonal antibody raised against AAF-guanosine and a second antibody coupled to an enzyme . The sensitivity achieved with AAF-DNA probes routinely detected 0.25 pg DNA bound to a filter . AAF-DNA probes were highly stable and were prepared by simple chemical modification of DNA . Their use as a possible diagnostic tool is discussed.

Plasmid, 1985 Nov, 14(3), 245 - 54
DNA segments of the IncX plasmid R485 determining replication and incompatibility with plasmid R6K; Stalker DM et al.; The expression of incompatibility properties between the IncX plasmids R6K and R485 of Escherichia coli was examined . For small autonomously replicating derivatives of both plasmid elements, the requirements for incompatibility expression include a functional R485 replicon and an active R6K beta-origin region . Functional R6K alpha and gamma origins are not directly involved in incompatibility expression between R6K and R485 . A trans-acting replication system was constructed for plasmid R485 . It consists of a 3.2-(kb) DNA fragment of R485 that specifies a product(s) in trans which supports replication from an R485 origin plasmid . A minimal R485 origin region of 591 bp was derived utilizing this trans-acting replication system and the nucleotide sequence of this origin region determined . The most striking feature of the sequence is the presence of six tandem 22-bp nucleotide sequence direct repeats.

Plasmid, 1985 Nov, 14(3), 224 - 34
The EcoR124 and EcoR124/3 restriction and modification systems: cloning the genes; Firman K et al.; The Escherichia coli plasmid R124 codes for a type I restriction and modification system EcoR124 and carries genetic information, most probably in the form of a "silent copy," for the expression of a different R-M specificity R124/3 . Characteristic DNA rearrangements have been shown to accompany the switch in specificity from R124 to R124/3 and vice versa . We have cloned a 14.2-kb HindIII fragment from R124 and shown that it contains the hsdR, hsdM, and hsdS genes which code for the EcoR124 R-M system . An equivalent fragment from the plasmid R124/3 following the switch in R-M specificity has also been cloned and shown to contain the genes coding for the EcoR124/3 R-M system . These fragments, however, lack a component present on the wild-type plasmid essential for the switch in specificity . Restriction fragment maps and preliminary heteroduplex analysis indicate the near identity of the genes that encode the two different DNA recognition specificities . Transposon mutagenesis was used to locate the positions of the hsdR, hsdM, and hsdS genes on the cloned fragments in conjunction with complementation tests for gene function . Indirect evidence indicates that hsdR is expressed from its own promoter and that hsdM and hsdS are expressed from a single promoter, unidirectionally.

Plasmid, 1985 Nov, 14(3), 192 - 9
The nucleotide sequences of the replication origins of plasmids ColA and ColD; Zverev VV et al.; The nucleotide sequences of the replication origin regions of plasmids ColA-CA31 and ColD-CA23 were obtained . Analysis of the nucleotide sequences showed a high degree of homology of these regions with the plasmid ColE1 region responsible for its autonomous replication . In the ColA and ColD regions involved in the regulation of replication, sites have been revealed identical to those participating in the transcription initiation of the ColE1 plasmid RNA I and primer RNA . The presumed RNA polymerase binding sites and the RNA polymerase recognition sequences are identical in ColA, ColD, and ColE1 plasmids . In spite of the differences in the nucleotide sequences, RNA I and the preprimer RNA of ColA and ColD may form structures analogous to the respective structures of ColE1.

Mikrobiologiia, 1985 Nov-Dec, 54(6), 889 - 92
{Effect of cyclic 3',5'-adenosine monophosphate on the growth rate of Escherichia coli}; Mikheeva GA; Cyclic 3',5'-adenosine monophosphate (cAMP) inhibits the rate of Escherichia coli growth in media with glucose . When the exogenous nucleotide is added, the generation time and the lag phase become longer . These parameters decrease if cAMP is entirely absent from the cya- mutant as compared to the parent cya+ strain . The nucleotide exerts a low activity in media with glycerol . The action of cAMP is highly specific.

J Gen Microbiol, 1985 Nov, 131 ( Pt 11), 3047 - 53
Cloning of genes determining the production of vero cytotoxin by Escherichia coli; Willshaw GA et al.; Sequences encoding the production of a cytotoxin (VT) active on Vero cells were cloned in Escherichia coli K12 from a VT-determining phage that originated in E . coli strain H19 of serotype O26.H11 . Subcloning resulted in the identification of a 2.5 kb fragment that still coded for VT production . Mutagenesis with transposon Tn1000 was used to map VT sequences and a 0.75 kb probe was developed . In colony hybridization tests with strains isolated from patients with haemolytic uraemic syndrome or diarrhoea, this probe derived from the H19 VT genes detected only some of the VT+ strains belonging to serogroup 0157 . A VT+ strain, E32511, serotype 0157.H-, which was negative in colony hybridization was the source of another VT-determining phage from which VT sequences were cloned . Southern hybridization of the VT genes from E32511 with the H19 probe was negative under stringent conditions but there was weak homology under conditions of low stringency . These results indicate that there are differences in the VT genes of pathogenic E . coli.

Bioorg Khim, 1985 Nov, 11(11), 1572 - 3
{Substrate specificity of restriction endonuclease Eco781}; Butkus V et al.; The recognition sequence and cleavage point of restriction endonuclease Eco781 have been determined as 5'-GGCGCC- . There are several known enzymes recognizing the same sequence, although the prototype NarI and isoschizomers NdaI and NunII cleave the substrate to produce 5'-protruding ends, whereas cleavage with isoschizomer BbeI results in 3'-protruding ends . Therefore, restrictase Eco78I, generating flush ends, may be regarded as an enzyme with new specificity among the restriction endonucleases recognizing the 5'-GGCGCC-sequence.

Bioorg Khim, 1985 Nov, 11(11), 1533 - 46
{General approach to the engineering of synthetic DNA}; Chakhmakhcheva OG et al.; A useful and efficient approach to the synthesis of DNA duplexes of practically unlimited length has been developed . The proposed methodology is based on the use of temporary restriction sites for subcloning and assembling the segments of the desired DNA . It allows the utilization of chemically synthesized oligonucleotides of various length (from 10- to 100-mers) for the duplex construction . The application of this approach to the synthesis of a gene for the functionally active bacteriorhodopsin fragment is described.

Mol Biol (Mosk), 1985 Nov-Dec, 19(6), 1457 - 65
{Deletion of late genes of the prophage lambda}; Matvienko NI et al.; The deletions in tandem prophage lambda appear with high frequency (to 10%) in rec A- strain of Escherichia coli . The deletions were shown by marker rescue and hybridization of fragments of DNA on nitrocellulose filters with nick-translated phage lambda DNA localized only in prophage area . Right and left att sites are not involved . The majority of defective lysogens had all regulatory regions and deletions of late structural genes . These strains may be used for construction of the host-vector systems with the strongest promoter p'R of phage lambda.

Cell, 1985 Nov, 43(1), 47 - 57
Localization of the fushi tarazu protein during Drosophila embryogenesis; Carroll SB et al.; The fushi tarazu (ftz) gene of Drosophila acts early in embryogenesis to regulate body segmentation . The localization of the ftz protein product in embryos was examined using indirect immunofluorescence microscopy . Antibodies were prepared against a beta-galactosidase-ftz hybrid protein made in E . coli . The ftz protein was first detectable in blastoderm-stage embryos as seven stripes of nuclei encircling the embryos transversely . The stripes persist through the early events of gastrulation, but disappear before overt segmentation is visible . The ftz protein is expressed a second time in some nuclei of the developing nervous system . In contrast to the early pattern, at the later stage, ftz is expressed in each of fifteen metameric subunits of the embryo.

Cell, 1985 Nov, 43(1), 117 - 30
IS10 transposition is regulated by DNA adenine methylation; Roberts D et al.; We show that dam- mutants are a major class of E . coli mutants with increased IS10 activity . IS10 has two dam methylation sites, one within the transposase promoter and one within the inner terminus where transposase presumably binds . Absence of methylation results in increased activity of both promoter and terminus, and completely accounts for increased transposition in dam- strains . Transposition of Tn903 and Tn5 are also increased in dam- strains, probably for analogous reasons . Transposition is also increased when IS10 is hemimethylated . One hemimethylated species is much more active than the other and is estimated to be at least 1000 times more active than a fully methylated element . Evidence is presented that the promoter and inner terminus of IS10 are coordinately activated in a dam-dependent fashion, presumably because they are hemimethylated at the same time . Thus, in dam+ strains, IS10 will transpose preferentially when DNA is hemimethylated . We suggest specifically that IS10 transposition may preferentially occur immediately after passage of a chromosomal replication fork.

Somat Cell Mol Genet, 1985 Nov, 11(6), 617 - 24
Studies on gene transfer and reversion to UV resistance in xeroderma pigmentosum cells; Schultz RA et al.; We have examined several parameters which address the feasibility of complementing the UV-sensitive phenotype of xeroderma pigmentosum (XP) fibroblasts by gene transfer . We present a comparative study which demonstrates that, relative to immortalized cells, human diploid cells are poor recipients for gene transfer . As measured by both transient and stable expression assays, diploid fibroblasts were completely refractory to DNA transfer by calcium phosphate coprecipitation and exhibited substantially reduced levels of expression following gene transfer by fusion with E . coli protoplasts . We also examined the significance of reversion of the phenotype of UV sensitivity in SV40-immortalized XP-A cell lines . In addition to confirming a previous report of reversion to wild-type levels of UV resistance at a frequency of approximately 10(-7), we have attempted to facilitate the identification of XP-A cells complemented with genomic DNA by employing less stringent selection schemes and cotransfection of a selectable marker . Under these conditions, we observed an increased frequency of reversion and were unable to identify true transfectants.

Proc Natl Acad Sci U S A, 1985 Nov, 82(22), 7626 - 30
Tumor necrosis factor: specific binding and internalization in sensitive and resistant cells; Tsujimoto M et al.; Highly purified, Escherichia coli-derived recombinant human tumor necrosis factor (TNF) was labeled with 125I and employed to determine receptor binding, internalization, and intracellular degradation in murine L929 cells (highly sensitive to the cytotoxic action of TNF) and in diploid human FS-4 cells (resistant to TNF cytotoxicity) . 125I-labeled TNF bound specifically to high-affinity receptors on both L929 and FS-4 cells . Scatchard analysis of the binding data indicated the presence of 2200 binding sites per L929 cell and 7500 binding sites per FS-4 cell . The calculated dissociation constants are 6.1 X 10(-10) M and 3.2 X 10(-10) M for L929 and FS-4 cells, respectively . In both L929 and FS-4 cells, incubation at 37 degrees C resulted in a rapid internalization of the bulk of the cell-bound TNF, followed by the appearance of trichloroacetic acid-soluble 125I radioactivity in the tissue culture medium, due to degradation of TNF . Degradation but not cellular uptake of TNF was inhibited in the presence of chloroquine (an inhibitor of lysosomal proteases) in both L929 and FS-4 cells, suggesting that degradation occurs intracellularly, probably within lysosomes . These results show that resistance of FS-4 cells to TNF cytotoxicity is not due to a lack of receptors or their inability to internalize and degrade TNF.

Proc Natl Acad Sci U S A, 1985 Nov, 82(22), 7530 - 4
Genetic manipulation of membrane phospholipid composition in Escherichia coli: pgsA mutants defective in phosphatidylglycerol synthesis; Miyazaki C et al.; Unique mutants of Escherichia coli K-12, defective in phosphatidylglycerol synthesis, have been isolated from a temperature-sensitive strain incubated at its nonpermissive temperature . The parent strain had excess phosphatidylglycerol by harboring both the pss-1 allele {coding for a temperature-sensitive phosphatidylserine synthase (EC 2.7.8.8)} and the cls- allele (responsible for a defective cardiolipin synthase) . The newly acquired mutations caused better growth at higher temperatures . One of the mutations (pgsA3) has been identified in the structural gene for phosphatidylglycerophosphate synthase {glycerophosphate phosphatidyltransferase (EC 2.7.8.5)} . Phospholipid compositions of these mutants were remarkable; phosphatidylethanolamine was the sole major lipid . In media with low osmotic pressures, these cells grew more slowly than the wild-type cells . They grew normally without recovering from the phospholipid abnormality in media appropriately supplemented with sucrose and MgCl2 . Formation of cardiolipin and phosphoglycerol derivatives of membrane-derived oligosaccharides was reduced in a pgsA3 mutant . E . coli strains having the pgsA3, pss-1, and cls- mutations, either individually or in combination, constitute an empirical system in which the molar ratio of three major membrane phospholipids can be variously altered.

Proc Natl Acad Sci U S A, 1985 Nov, 82(22), 7500 - 4
GTP-binding membrane protein of Escherichia coli with sequence homology to initiation factor 2 and elongation factors Tu and G; March PE et al.; The amino acid sequence of LepA protein, which has been shown to be cotranscribed with signal peptidase I in Escherichia coli, was compared with greater than 2000 known protein sequences . It was revealed that, of the 598 amino acid residues contained in LepA, an amino-terminal domain of 112 residues is homologous to a domain of similar size found in initiation factor 2, elongation factor Tu, and elongation factor G (IF2, EF-Tu, and EF-G), factors required for translation in E . coli . In this domain, 46 and 34 residues align perfectly with the corresponding regions of EF-G and EF-Tu, respectively . If functionally conserved residues within this domain (19 for EF-G and 17 for EF-Tu) are included, the overall resemblance is 58% and 46%, respectively, for EF-G and EF-Tu . A similar domain exists internally in IF2, where there is 42% overall resemblance with the domain of LepA . Immediately adjacent to this region is a small sequence of limited similarity that exists not only in EF-G, EF-Tu, and IF2 but also in the protooncogene c-Ha-ras-1 (from human bladder) and other GTP-binding proteins . Given these homologies, GTP-photoaffinity labeling and subcellular fractionation experiments were undertaken, and it was found that LepA is indeed a membrane-bound GTP-binding protein.

EMBO J, 1985 Nov, 4(11), 2991 - 5
Identification of a telomeric DNA sequence in Plasmodium berghei; Ponzi M et al.; A fragment of Plasmodium berghei DNA was cloned using a technique designed to select for telomeric sequences . The cloned fragment recognizes Bal31-sensitive bands in P . berghei genomic digests . It contains at its distal end at least 70 tandem repeats of the heptanucleotide sequence CCCTGAAA . The presence of natural single strand discontinuities in the telomeric regions of P . berghei DNA is demonstrated by the selective incorporation of deoxyribonucleoside triphosphates in the absence of DNase . The number of copies of the cloned sequence present in each genome agrees with an estimate of 6-12 chromosomes per nucleus.

EMBO J, 1985 Nov, 4(11), 2971 - 6
Identification of a sequence element in the promoter of the Drosophila melanogaster hsp23 gene that is required for its heat activation; Mestril R et al.; The expression of Drosophila melanogaster hsp23-Escherichia coli beta-galactosidase hybrid genes containing different segments of the 5' non-transcribed sequence of the hsp23 gene has been examined at the RNA and protein levels in Xenopus oocytes . Transcription of the hybrid genes is initiated correctly . Mutant genes with hsp23 gene promoter segments of at least 140 bp in length are strongly heat-activated while genes with shorter promoter segments are expressed constitutively and at low levels . This maps an element required for the heat-controlled expression of the D . melanogaster hsp23 gene to a region, approximately 140 bp upstream from the start of the transcription site, which contains a sequence (CGAGAAGTT-TCGTGT) that is closely related to the one responsible for the heat regulation of the hsp70 gene . These findings demonstrate the importance of this regulatory sequence for a second hsp gene and support the notion that hsp genes are heat-regulated by a common mechanism . The functional element in the hsp23 gene promoter is located greater than 80 bp further upstream from the TATA box than the relevant element in the hsp70 gene promoter . Even though other related sequences are present further upstream and downstream from the functional element, they play at most an auxiliary role in the regulation of hsp23 gene expression.

Proc Natl Acad Sci U S A, 1985 Nov, 82(21), 7178 - 82
Cloning of yeast TOP1, the gene encoding DNA topoisomerase I, and construction of mutants defective in both DNA topoisomerase I and DNA topoisomerase II; Goto T et al.; Rabbit antibodies specific to yeast DNA topoisomerase I were used in immunological screening of a Saccharomyces cerevisiae genomic DNA library in Escherichia coli . One of the clones identified by its expression of antigenic determinants of the yeast enzyme is shown to contain the coding sequence of the enzyme: no active DNA topoisomerase I is detectable in cell extracts when insertion or deletion mutations are introduced into a 2-kilobase-pair (kb) region of the sequence in a haploid yeast genome . Blot hybridizations show that there is a single copy of the cloned sequence per haploid and that the sequence is transcribed to give a 2.7-kb poly(A)+ message . Mutants in which 1.7 kb of the sequence is deleted are viable . Temperature-shift experiments using synchronously grown cells of a delta top1 top2 temperature-sensitive (ts) double mutant and its isogenic top2 ts strain show that, whereas mitotic blocks can prevent killing of the top2 ts mutant at a nonpermissive temperature, the same treatments are ineffective in preventing cell death of the delta top1 top2 ts double mutant . These experiments suggest that in yeast DNA topoisomerase I serves a role auxiliary to DNA topoisomerase II.

J Virol, 1985 Nov, 56(2), 607 - 12
Posttranslational processing of p21 ras proteins involves palmitylation of the C-terminal tetrapeptide containing cysteine-186; Chen ZQ et al.; The p21 proteins of ras oncogenes are synthesized as precursors in the cytosol . After processing, which involves acylation, the products are associated with the plasma membrane in eucaryotic cells . The p21 overproduced in Escherichia coli, however, is not processed by acylation . A synthetic tetrapeptide of the p21 C terminus is used to identify the acylation site in eucaryotic p21 as cysteine-186 . The same peptide of bacterial p21 is not acylated . Although p21 of Harvey murine sarcoma virus-transformed NRK cells can be metabolically labeled with either {3H}palmitate or {3H}myristate, the lipid moiety of the hydrophobic peptide is identified as palmitic acid . We suggest that the enzymatic mechanism for p21 palmitylation may be different from N-terminal myristylation of many other membrane proteins.

J Bacteriol, 1985 Nov, 164(2), 957 - 9
IS186: an Escherichia coli insertion element isolated from a cDNA library; Kothary RK et al.; Escherichia coli IS186 was isolated from cDNA libraries made from rainbow trout RNA and maintained in E . coli RR1 . The element was 1,347 base pairs in length, had a perfect inverted repeat of 25 base pairs, and had an open reading frame of 375 amino acids . The hypothetical protein sequence of IS186 had limited homology to the E . coli IS4 hypothetical protein I sequence . There were three copies of IS186 in E . coli RR1.

J Bacteriol, 1985 Nov, 164(2), 922 - 4
Initiation of chromosome replication in Escherichia coli after induction of dnaA gene expression from a lac promoter; Bremer H et al.; Escherichia coli HB282 carries a dnaA46(Ts) allele on the chromosome, a wild-type dnaA allele under the control of the lacUV5 promoter on the multicopy plasmid pBC32, and an overproducing lac repressor allele on an F' factor . When the plasmid dnaA gene is repressed, the strain is thermosensitive . After a temporary deficiency in active dnaA protein at nonpermissive temperature, the addition of isopropyl-beta-D-thiogalactopyranoside to the culture was found to produce a burst of initiations within 5 to 10 min at 30% of the origins in 90% of the cells . Initiations then continued at a rate slightly faster than the mass-doubling time such that after 2 h the origin-to-mass ratio of the control culture was restored.

J Bacteriol, 1985 Nov, 164(2), 872 - 7
Negative regulation of adenylate cyclase gene (cya) expression by cyclic AMP-cyclic AMP receptor protein in Escherichia coli: studies with cya-lac protein and operon fusion plasmids; Kawamukai M et al.; We constructed cya-lac protein and operon fusion plasmids in vitro . The effect of cyclic AMP (cAMP) on cya expression was examined by measuring the synthesis of beta-galactosidase in Escherichia coli cells containing fused plasmids . In the cya-lacZ fused protein system, cya expression was strongly repressed by exogenous cAMP . Functional cAMP receptor protein (CRP) was necessary for this effect . On the other hand, in a tet-lacZ fused protein as a control system, tet expression was not affected by cAMP . The inhibition of cya expression by cAMP was also observed in the cya-lac fused operon system, although it was necessary to increase the amount of cAMP or CRP in the cells to detect the effect . The results indicate that cAMP-CRP is a negative regulator of cya expression at the level of transcription.

J Bacteriol, 1985 Nov, 164(2), 797 - 801
Construction of a series of ompF-ompC chimeric genes by in vivo homologous recombination in Escherichia coli and characterization of the translational products; Nogami T et al.; OmpF and OmpC are major outer membrane proteins . Although they are homologous proteins, they function differently in several respects . As an approach to elucidate the submolecular structures that determine the difference, a method was developed to construct a series of ompF-ompC chimeric genes by in vivo homologous recombination between these two genes, which are adjacent on a plasmid . The genomic structures of these chimeric genes were determined by restriction endonuclease analysis and nucleotide sequence determination . In almost all cases, recombination took place between the corresponding homologous regions of the ompF and ompC genes . Many of the chimeric genes produced proteins that migrated to various positions between the OmpF and OmpC proteins on polyacrylamide gel . On the basis of the results, a domain contributing to the mobility difference the OmpF and OmpC proteins was identified . Some chimeric genes did not accumulate outer membrane proteins, despite the fact that the fusion of the ompF and ompC genes was in frame . Bacterial cells possessing the chimeric proteins were also tested as to their sensitivity to phages which require either OmpF or OmpC as a receptor component . The chimeric proteins were either of the OmpF or OmpC type with respect to receptor activity . Based on the observations, the roles of submolecular domains in the structure, function, and biogenesis of the OmpF and OmpC proteins are discussed.

J Bacteriol, 1985 Nov, 164(2), 689 - 95
Isolation of insertion, deletion, and nonsense mutations of the uracil-DNA glycosylase (ung) gene of Escherichia coli K-12; Duncan BK; Two uracil-DNA glycosylase (ung) mutation selection procedures based upon the ability of uracil glycosylase to degrade the chromosomes of organisms containing uracil-DNA were devised to obtain a collection of well-defined ung alleles . In an enrichment procedure, lysogens were selected from Escherichia coli cultures infected with lambda pKanr phage containing uracil in their DNA . (These uracil-DNA phage were prepared by growth on host cells deficient in both dUTPase and uracil-DNA glycosylase.) The lysogenic Kanr population was enriched for uracil glycosylase-deficient mutants by a factor of 10(4) . In a phage suicide selection procedure, lambda pung+ phage were unable to form plaques on dut ung cells containing uracil-DNA in their chromosomes, and all of the progeny were lambda pung- . Deletion, insertion (ung::Mu and ung::Tn10), nonsense, and missense mutants were isolated by using these procedures . Extracts of three insertion mutants contained no detectable enzyme activity . All of the other mutant isolates had less than 1% of the normal uracil glycosylase specific activity . The previously studied ung-1 allele, which was derived by N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis, produced about 0.02% of the normal amount of uracil glycosylase activity . No significant phenotypic differences between ung-1 and ung::Tn10 alleles were observed . Variations of the lysogen selection procedure may be helpful for isolating other DNA glycosylase mutations in E . coli and other organisms.

J Bacteriol, 1985 Nov, 164(2), 653 - 8
Identification of the Escherichia coli recN gene product as a major SOS protein; Finch PW et al.; The recA+ lexA+-dependent induction of four Escherichia coli SOS proteins was readily observed by two-dimensional gel analysis . In addition to the 38-kilodalton (kDa) RecA protein, which was induced in the greatest amounts and was readily identified, three other proteins of 115, 62, and 12 kDa were seen . The 115-kDa protein is the product of the uvrA gene, which is required for nucleotide excision repair and has previously been shown to be induced in the SOS response . The 62-kDa protein, which was induced to high intracellular levels, is the product of recN, a gene required for recBC-independent recombination . The recA and recN genes were partially derepressed in a recBC sbcB genetic background, a phenomenon which might account for the recombination proficiency of such strains . The 12-kDa protein has yet to be identified.

J Bacteriol, 1985 Nov, 164(2), 578 - 84
Primary characterization of the protein products of the Escherichia coli ompB locus: structure and regulation of synthesis of the OmpR and EnvZ proteins; Comeau DE et al.; The ompB operon of Escherichia coli contains the structural genes for two proteins, OmpR and EnvZ, which control the osmoregulated biosynthesis of the porin proteins OmpF and OmpC . By inserting XbaI octamer linkers into the cloned ompB locus, four distinct frameshift mutants were isolated and subsequently characterized for their OmpR and EnvZ protein products and their outer membrane porin phenotype . In a minicell expression system, the wild-type products of the ompR and envZ genes were found to be approximately 28 and 50 kilodaltons in size, respectively, whereas the mutant proteins were either truncated or extended due to the frame shift . The identity of the envZ gene product was confirmed by immunoprecipitation . M13 dideoxy sequencing of the DNA around the wild-type ompR-envZ junction revealed an error in the sequence published for this operon; the complete corrected sequence is presented . A sequence, ATGA, was found that forms the termination codon for the OmpR reading frame and a possible initiation codon for the EnvZ protein; these sequences are consistent with the sizes of the proteins observed after sodium dodecyl sulfate-polyacrylamide gel electrophoresis . The translational activity of this ATG codon was confirmed by fusing the lacZ gene in frame with the putative EnvZ coding sequence . The implications of these results are discussed with respect to the regulation of synthesis of the ompB gene products.

Genetika, 1985 Nov, 21(11), 1799 - 805
{Large deletions in the region of the replication termination of Escherichia coli K-12 chromosome}; Kulakauskas ST et al.; The Escherichia coli strain PLK1427 (Henson, Kopp, Kuempel, 1984) was used in this work . It carries a deletion of 60 thousand pairs of nucleotides in the chromosomal region 30-31 min and a partially deleted prophage lambda rev cI875Sam7 integrated into the 30 min region, instead of the rac prophage . Among the mutants of PLK1427 strain selected for resistance to 42 degrees C, deletions extending about 4 min and affecting the loci nirR (29.3 min), zdc235::Tn10 (32.3 min) and zdd230::Tn9 (33.3 min) were found . Although the deletion mutants obtained affect the region of replication termination (terC) of the chromosome, they have no alterations in the growth rate . It was demonstrated that some deletions may be transferred and are capable of recombination, giving the wild type in transductional experiments with the mutant phage T4.

J Bacteriol, 1985 Nov, 164(2), 972 - 5
phs Locus of Escherichia coli, a mutation causing pleiotropic lesions in metabolism, is an rpoA allele; Rowland GC et al.; The phs mutation, which causes a pleiotropic growth defect, has been mapped and shown to be an allele of rpoA, the gene for the alpha subunit of RNA polymerase . The mutation is shown to cause a transcription defect in the arabinose operon, araBAD.

J Neurogenet, 1985 Nov, 2(5), 345 - 63
High-efficiency cloning of DNA sequences complementary to mouse neuroblastoma polyadenylated RNA; Sparkman DR et al.; A cDNA library was efficiently synthesized from mouse neuroblastoma poly(A)+RNA . Several modifications of the oligo(dC)(dG) tailing procedure were used . After first strand synthesis, a dATP tail was added to the 3'-end of the cDNA . The second strand was primed for synthesis with oligo(dT) . Blunt ends were produced on the cDNA by treatment with S1 nuclease . Size-enriched fractions of high molecular weight DNAs were obtained by passing the cDNA over a Sepharose CL-4B column . The optimal tailing time for each cDNA fraction was individually tested . Tailing reactions used terminal deoxynucleotidyl transferase and annealing reactions used a (G)-tailed Pst I cut pBR322 . E . coli K12 RR1 cells were transformed and 2.5-5 X 10(6) transformants per microgram cDNA insert were obtained for each size fraction . The transformants had an average insert size of 1200 base pairs and were 98% ampicillin sensitive . Our modifications in the method for cDNA library synthesis had 3 advantages . (1) Homopolymer-primed cDNA treated with S1 nuclease allowed the blunt ends to be tailed synchronously . This allowed a higher transformation efficiency without loss of 5'-sequences . (2) Time tailing determined the most efficient tail length and optimized the transformation efficiency in each size fraction . (3) A Sephadex G-50 mini-column was used to desalt and dry nitrogen was used to concentrate the ds cDNA instead of the usual ethanol precipitation . This resulted in almost 100% recovery of synthesized products at each step of this procedure.

J Bacteriol, 1985 Nov, 164(2), 904 - 10
Phenotypic properties of a unique rpoA mutation (phs) of Escherichia coli; Giffard PM et al.; The phs mutation of Escherichia coli has been suggested to affect the Na+/H+ antiport (D . Zilberstein, E . Padan, and S . Schuldiner, FEBS Lett . 168:327-330, 1980) . We have recently shown that the mutation affects the rpoA gene and thus affects transcription . The extent of the pleiotropy of the phs mutation was investigated . In addition to the previously reported growth defect on L-glutamate and melibiose, the mutation also affects at least two other metabolic systems . The transport and metabolism of arabinose is impaired and the transport of sulfate is reduced . The extent to which the effects of the phs mutation on metabolism are due to a defect in the Na+/H+ antiport was investigated, and no causal role for this transport system in the metabolic defects was found.

J Bacteriol, 1985 Nov, 164(2), 816 - 22
Role of glnB and glnD gene products in regulation of the glnALG operon of Escherichia coli; Bueno R et al.; We have isolated insertion and deletion mutants in glnB, the structural gene of PII, a member of the adenylylation system for glutamine synthetase of Escherichia coli, to study the role of PII in the regulation of the synthesis of glutamine synthetase and of histidase in response to nitrogen deprivation or excess . We have studied the effects of this mutation alone and combined with null mutations resulting from the insertion of transposons or from a deletion in the other genes affecting this regulation, glnD, glnF (ntrA), glnG (ntrC), and glnL (ntrB) . Our results confirm that only the products of glnF and glnG are essential for this regulation . In cells of the wild type, the response is mediated by the products of glnD and glnB via the product of glnL . In the condition of nitrogen excess, PII, the product of glnB, appears to convert the product of glnL to a form that prevents the activation of transcription of the structural genes for glutamine synthetase and for histidase by the products of glnF and glnG . During nitrogen deprivation, uridylyltransferase, the product of glnD, is activated by the intracellular excess of 2-ketoglutarate over glutamine and converts PII to PII-UMP and changes the form of the glnL product to one that stimulates the activation of transcription of glutamine synthetase and histidase by the products of glnF and glnG.

Infect Immun, 1985 Nov, 50(2), 517 - 22
Three-dimensional structure of fimbriae determines specificity of immune response; Karch H et al.; We recently described how a fraction of isolated fimbriae from a multifimbriated strain of Escherichia coli O7:K1:H6 (WF96) could be subdivided by sequential disaggregation in disrupting agents into individual subunits with different molecular weights . In this study, antibodies were raised in rabbits against these isolated fimbrial subunits and against purified intact WF96 fimbriae . These sera were tested by Western blot analysis or by enzyme-linked immunosorbent assays for reactivity against the following antigens: intact WF96 fimbriae, dissociated WF96 fimbriae, dissociated and reaggregated WF96 fimbriae, the WF96 21K fimbrial subunit, reaggregated WF96 21K subunits, the WF96 16K subunits, reaggregated WF96 16K subunits, intact fimbriae from four other E . coli strains, and deaggregated fimbriae from these strains . We found that antibody against intact WF96 fimbriae only reacted strongly with intact WF96 fimbriae, depolymerized and reaggregated WF96 fimbriae, or reaggregated fimbrial subunits; no reactions were evident with intact fimbriae from four other E . coli strains . Conversely, antisera prepared against the WF96 16K subunit and against the WF96 21K subunit did not react with intact WF96 fimbriae or with depolymerized and reaggregated WF96 fimbriae, but did react with homologous isolated subunits . One cross-reaction between fimbrial subunits was apparent: anti-WF96 16K subunit bound to a 21K subunit of deaggregated fimbriae, from another E . coli strain . Taken together, the findings indicate that the three-dimensional structure of the fimbrial preparation used to immunize animals determines the specificity of the immune response.

Mol Biol (Mosk), 1985 Nov-Dec, 19(6), 1633 - 42
{Secondary structure of total protein and 23S RNA in 50S ribosomal subunits and in the isolated state}; Gongadze GM et al.; Optical and sedimentational studies of isolated 23S RNA, total proteins and some RNP-complexes of the 50S subunits were carried out . It is shown that the secondary structure content of 23S RNA in the ribosome is lower than in the isolated state . Ribosomal proteins stabilize the 23S RNA structure and make it more compact . At the same time they cause some unwinding effect on the secondary structure of the 23S RNA and possibly fix some segments of the 23S RNA in the conformation necessary for its function . In turn, the 23S RNA increased somewhat the level of the total ordered secondary structure in the ribosomal proteins . There was no considerable change of the ratio between the alpha- and beta-structures in the proteins.

Mol Biol (Mosk), 1985 Nov-Dec, 19(6), 1445 - 56
{tRNA genes of pro- and eukaryotes . II . Gene expression and processing of gene products}; Venkstern TV; The up-to-date state of investigations on cytoplasmic tRNA genes expression and processing of gene products are reviewed . Special emphasize is given to data on RNAase P and splicing.

Cell, 1985 Nov, 43(1), 379 - 87
Tn10 protects itself at two levels from fortuitous activation by external promoters; Davis MA et al.; Tn10 rarely transposes, primarily because its IS10-encoded transposase protein is synthesized infrequently . Since the 5' end of the transposase gene is immediately adjacent to flanking host sequences, insertion of Tn10 into an actively transcribed operon could conceivably result in dramatically increased transposition . We show here that Tn10 is protected from such fortuitous activation; high levels of transcription from an upstream promoter actually decrease its rate of transposition . Protection operates at two levels . First, externally-initiated transcripts yield only a small amount of additional transposase protein, primarily because of inhibition at a posttranscriptional level . We suggest that the transposase gene start codon is sequestered in an mRNA secondary structure not present in transcripts initiated at the normal promoter . Second, transcription per se across an IS10 terminus inhibits its activity, thus negating any small transposase increase . These observations provide additional evidence that Tn10 has evolved specific mechanisms for keeping its transposition activity low.

Proc Natl Acad Sci U S A, 1985 Nov, 82(22), 7540 - 4
Expression in Escherichia coli of a cloned DNA sequence encoding the pre-S2 region of hepatitis B virus; Offensperger W et al.; A DNA sequence encoding the entire pre-S2 region (amino acids 120-174; serotype ayw) of human hepatitis B virus envelope protein has been inserted into the lacZ gene of the plasmid pSKS105 yielding a recombinant, pWS3 . Lac+ colonies of the Escherichia coli M182 delta (lacIOPZYA), isolated after transformation with pWS3, produced a pre-S2 peptide-beta-galactosidase fusion protein . This fusion protein, which comprised as much as 3% of the total bacterial protein, was purified to greater than 90% homogeneity by affinity chromatography on p-aminophenyl-beta-D-thiogalactoside-Sepharose . It is immunoprecipitable with rabbit antibodies to a synthetic peptide corresponding to amino acids 120-145 of the pre-S2 region of serotype adw {pre-S(120-145)} or with antibodies to hepatitis B virus . pre-S(120-145) completely blocked the binding of either antibody to the pre-S2 peptide-beta-galactosidase fusion protein . These results indicate that there are antigenic determinants on the fusion protein that are closely related to, if not identical to, determinants on synthetic pre-S(120-145) and on pre-S2 sequences of native hepatitis B virus particles . Thus, bacteria transformed with pWS3 can provide an abundant source of pre-S2-beta-galactosidase fusion protein, which may prove useful either as a diagnostic reagent possessing marker enzyme activity suitable for ELISA tests or as an immunogen with potential to contribute to active prophylaxis of hepatitis B.

J Biochem Biophys Methods, 1985 Nov, 11(6), 347 - 55
Quantitative determination of Coomassie R bound to proteins in polyacrylamide gel . A facilitated method for multi-spot analysis of electrograms; Tal M et al.; Dimethylsulfoxide (DMSO) was found to be an efficient solvent for extraction of Coomassie Blue R 250 (Coomassie R) from stained proteins on polyacrylamide gels . Kinetic measurements show that the extraction of the dye from a 2-D gel reached equilibrium in 48 h . Staining of E . coli ribosomal proteins by Coomassie R dissolved in trichloroacetic acid exhibited two types of dye-protein complexes, the majority of them yield a blue-purple colour, while the rest are stained with a light-blue tone and fluorescent appearance as well . The absorbance spectra of the complexes in the gel matrix differ significantly from each other . However, the DMSO-extracted Coomassie show identical absorbance profiles with lambda max at 602 nm, thus the amount of the bound dye can easily be measured spectrophotometrically.

J Bacteriol, 1985 Nov, 164(2), 950 - 3
Coupling between DNA replication and cell division mediated by the FtsA protein in Escherichia coli: a pathway independent of the SOS response, the "TER" pathway; Tormo A et al.; Inhibition of DNA synthesis prevented the recovery of cell division in filaments of D-3R {ftsA3(Ts) recA56} returned to the permissive temperature . The FtsA protein may be a signal involved in the "TER" pathway, a series of events that coordinate cell division with DNA replication, that is independent of the SOS pathway.

J Bacteriol, 1985 Nov, 164(2), 556 - 62
Characterization of the in vivo RNA product of the pOUT promoter of IS10R; Lee Y et al.; We characterized a single RNA species (RNAout1) which was the major in vivo RNA made from pOUT of IS10R . RNAout1 was 70 nucleotides long; its 5' end corresponded exactly to the in vitro start of pOUT transcription . The concentration of RNAout1 was estimated at 5 to 10 molecules per cell containing the single-copy plasmid NR1 . RNA sequences from pOUT of IS10L were detected at a much lower (less than one molecule per cell) steady-state concentration and may be preferentially degraded in vivo . We suggest that the low level of the IS10L transcript led to the inability of IS10L sequences to translationally inhibit Tn10 transposition.

Proc Soc Exp Biol Med, 1985 Nov, 180(2), 240 - 5
Differential response of muscle and gastric histidine decarboxylase to compound 48/80 and dietary calcium; Greene SM et al.; Previous work from this laboratory had indicated that in vivo, histidine decarboxylase (HDC) activity was stimulated by compound 48/80 in rat leg muscle, and that high dietary calcium had a stimulating effect on gastric HDC activity . In the present investigations the 48/80 effect was also observed in vitro in leg muscle extracts from rats, chicks, and guinea pigs . Compound 48/80 had no effect in vitro on histamine metabolism of gastric tissue homogenates in any of the animal species studied . A dietary effect of high calcium intake was noted in rat gastric tissue but not in rat leg muscle . In vitro addition of 48/80 and/or calcium had no stimulatory effect on bacterial HDC or on muscle carnosinase activity . These findings, in conjunction with a comparison of stomach and leg muscle mast cell populations, confirm an HDC stimulatory role for 48/80 in muscle, in addition to its histamine-releasing function from mast cells.

Virology, 1985 Oct 30, 146(2), 292 - 301
Molecular cloning and physical mapping of the genome of fish lymphocystis disease virus; Darai G et al.; A defined and complete gene library of the fish lymphocystis disease virus (FLDV) genome was established . FLDV DNA was cleaved with EcoRI, BamHI, EcoRI/BamHI and EcoRI/HindIII and the resulting fragments were inserted into the corresponding sites of the pACYC184 or pAT153 plasmid vectors using T4 DNA ligase . Since FLDV DNA is highly methylated at CpG sequences (Darai et al., 1983; Wagner et al., 1985), an Escherichia coli GC-3 strain was required to amplify the recombinant plasmids harboring the FLDV DNA fragments . Bacterial colonies harboring recombinant plasmids were selected . All cloned fragments were individually identified by digestion of the recombinant plasmid DNA with different restriction enzymes and screened by hybridization of recombinant plasmid DNA to viral DNA . This analysis revealed that sequences representing 100% of the viral genome were cloned . Using these recombinant plasmids, the physical maps of the genome were constructed for BamHI, EcoRI, BestEII, and PstI restriction endonucleases . Although the FLDV genome is linear, due to circular permutation the restriction maps are circular.

Biochim Biophys Acta, 1985 Oct 29, 810(1), 62 - 72
Assignment of ESR signals of Escherichia coli terminal oxidase complexes; Hata A et al.; The ESR signals of all the major components of the aerobic respiratory chain of Escherichia coli were measured and assigned at liquid helium temperature . Cytochrome b-556 gives a weak high-spin signal at g = 6.0 . The terminal oxidase cytochrome b-562 . o complex gives signals at g = 6.0, 3.0 and 2.26, and the terminal oxidase cytochrome b-558 . d complex gives signals at g = 6.0, 2.5 and 2.3 . A signal derived from cupric ions in the purified cytochrome b-562 . o complex was observed near g = 2.0 . It was shown by the effects of KCN or NaN3 on cytochromes under the air-oxidized conditions that cytochrome o has a high-spin heme and cytochrome d has a low-spin heme . The E'm values for cytochromes b-558 and d, respectively, determined by potentiometric titration of the ESR signals were 140 and 240 mV in the membrane preparation, and 30 and 240 mV in the purified preparation . The oxidized cytochrome d gave intense low-spin signals at g = 2.5 and 2.3, while cytochrome d under the air-oxidized conditions gave corresponding signals of only very low intensity . These results suggested that most of the cytochrome d under the air-oxidized conditions contains a diamagnetic iron atom with a bound dioxygen.

FEBS Lett, 1985 Oct 28, 191(2), 278 - 82
Identification of proline carrier in Escherichia coli K-12; Hanada K et al.; Proline carrier, a product of the putP gene of Escherichia coli, was identified as a 35 kDa cytoplasmic membrane protein by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) . Its identification was based on the following evidence: First, the density of the band corresponding to a 35 kDa protein correlated with the proline-binding activity of cytoplasmic membranes from putP-deficient and putP-amplified strains . Second, by the differential labeling method, the 35 kDa protein was specifically labeled with radioactive N-ethyl-maleimide . The 35 kDa protein was found to aggregate on heat treatment and to show abnormal mobility on SDS-PAGE.

J Biol Chem, 1985 Oct 25, 260(24), 12968 - 9
Crystallization and preliminary x-ray investigation of purine-nucleoside phosphorylase from Escherichia coli; Cook WJ et al.; Crystals of purine-nucleoside phosphorylase from Escherichia coli have been grown from solutions of ammonium sulfate . The crystals are hexagonal with space group P6(1)22 or P6(5)22; the axes are alpha = 106.5 A and c = 241.3 A . The crystals are moderately stable to x-rays and diffract beyond 3.0-A resolution . It appears that the molecule, which is a hexamer, utilizes the 2-fold symmetry of the space group, resulting in three subunits/asymmetric unit.

Nucleic Acids Res, 1985 Oct 25, 13(20), 7395 - 412
Reactions of the UVRABC excision nuclease with DNA damaged by diamminedichloroplatinum(II); Beck DJ et al.; Mutants of Escherichia coli, which are blocked in excision repair (uvrA6, uvrB5, or uvrC34) are exceptionally sensitive to the antitumor drug cis-Pt(II)(NH3)2Cl2 (cis-DDP) but not the trans isomer . Plasmid DNA, damaged by either the cis or trans compound and treated with the UVRABC excision nuclease was cut as shown by conversion of supercoiled DNA to relaxed forms . All three protein products of the uvrA, uvrB, and uvrC genes were required for incision . End-labeled fragments damaged with cis-DDP and reacted with the UVRABC nuclease were cut at the 8th phosphodiester bond 5' and at the 4th phosphodiester bond 3' to adjacent GG's . DNA treated with trans-DDP was not cut appreciably at adjacent GG's by the repair enzyme as subsequent analysis of reaction products after enzyme digestion gave a pattern similar to those obtained with control untreated fragments . The results indicate that the UVRABC nuclease may promote cell survival by the removal of adjacent GG's which are crosslinked by cis-Pt(II)(NH3)2Cl2.

J Biol Chem, 1985 Oct 25, 260(24), 13281 - 5
M13 procoat inserts into liposomes in the absence of other membrane proteins; Geller BL et al.; Procoat, the precursor form of the major coat protein of coliphage M13, assembles into the Escherichia coli inner membrane and is cleaved to mature coat protein by leader peptidase . This assembly process has previously been reconstituted using lipids and purified leader peptidase in a cell-free protein synthesis reaction (Watts, C., Silver, P., and Wickner, W . (1981) Cell 25, 347-353; Ohno-Iwashita, Y., and Wickner, W . (1983) J . Biol . Chem . 258, 1895-1900) . We now report that procoat can also cross a liposomal membrane composed of only purified phospholipids; leader peptidase is not needed to catalyze insertion . When procoat is synthesized in vitro in the presence of liposomes with encapsulated chymotrypsin, the procoat inserts spontaneously through the membrane and is degraded . The protease was shown by several criteria to be in the lumen of the liposomes . These results demonstrate that the precursor form of an E . coli integral membrane protein can cross a membrane without the aid of leader peptidase or any other membrane proteins.

Nucleic Acids Res, 1985 Oct 25, 13(20), 7457 - 72
A novel expression selection approach allows precise mapping of the hepatitis B virus enhancer; Tognoni A et al.; We have used a novel approach called expression selection to precisely define the hepatitis B virus (HBV) enhancer . Expression selection is based on a shuttle vector containing an enhancerless SV40 T antigen gene, the SV40 origin of replication and a plasmid replicon . This vector is linearized, ligated with the sonicated DNA to be analyzed and transfected into eukaryotic cells, where only plasmids which have incorporated an enhancer can express T antigen and therefore replicate . Vectors amplified by replication are selectively rescued in E . coli and their inserts analyzed . When we performed this protocol with HBV DNA we rescued two overlapping fragments of 166 and 214 bp which in HBV DNA map about 500 bp upstream of the core antigen mRNA initiation site and 1150 bp downstream of the surface antigen mRNA initiation site . These results were confirmed by conventional deletion mapping . When compared to the SV40 enhancer in nonhepatic cell lines, the HBV enhancer is only 5 to 10% as active; nevertheless, it also acts in an orientation-independent manner and in a position downstream of a gene . The HBV enhancer is situated in the coding region of the potential reverse transcriptase, and thus is the first enhancer identified to map in a protein-coding region.

J Biol Chem, 1985 Oct 25, 260(24), 12987 - 92
The polymerase subunit of DNA polymerase III of Escherichia coli . II . Purification of the alpha subunit, devoid of nuclease activities; Maki H et al.; The alpha subunit (140 kDa) of DNA polymerase III (pol III) holoenzyme has been purified to near-homogeneity from a plasmid-carrying Escherichia coli strain which overproduced the alpha subunit about 20-fold . Pol III core (containing only the alpha, epsilon, and theta subunits), produced at twice the normal level, was also purified in good yield . The isolated alpha subunit has DNA polymerase activity, which is completely inhibited by 10 mM N-ethylmaleimide or 150 mM KCl as observed in the pol III core or holoenzyme . The alpha subunit has an apparent turnover number of 7.7 nucleotides polymerized per s, compared to 20 for pol III core, and is more thermolabile . The alpha subunit lacks the 3'----5' exonuclease (proofreading) activity of pol III core; neither alpha subunit nor core (nor holoenzyme) possesses any of the previously reported 5'----3' exonuclease activity . Thus, the alpha polypeptide is the polymerase subunit and epsilon (27 kDa) is the proofreading subunit (Scheuermann, R . H., and Echols, H . (1984) Proc . Natl . Acad . Sci . U . S . A . 81, 7747-7751) . Together with the theta polypeptide (10 kDa), of unknown function, they form a pol III core with greater stability and catalytic efficiency.

J Biol Chem, 1985 Oct 25, 260(24), 12954 - 6
Regulation of the manganese-containing superoxide dismutase is independent of the inducible DNA repair system in Escherichia coli; Hancock LC et al.; Studies on the induction of the manganese-containing superoxide dismutase in several strains of Escherichia coli with different mutations in recA and lexA revealed that the inductions of the Mn-isozyme and of the SOS system by oxygen free radicals are not coregulated . We also studied the synthesis of the manganese-superoxide dismutase in the temperature-dependent, protease-constitutive strain recA441(tif-1) that also contained a lac fusion in an SOS gene . A shift to the temperature at which recA441 has constitutive protease activity did not induce Mn-superoxide dismutase but did induce beta-galactosidase . The data clearly demonstrate that induction of the Mn-superoxide dismutase is independent of the SOS system.

J Biol Chem, 1985 Oct 25, 260(24), 12982 - 6
The polymerase subunit of DNA polymerase III of Escherichia coli . I . Amplification of the dnaE gene product and polymerase activity of the alpha subunit; Maki H et al.; The Escherichia coli dnaE gene, which encodes the alpha subunit of DNA polymerase III (pol III) holoenzyme, has been cloned in a plasmid containing the PL promoter of phage lambda and thermally induced to overproduce the alpha subunit . In cells carrying this plasmid (pKH167), the alpha subunit was amplified, after heat induction, to a level of about 0.2% of the total cellular protein . Polymerase activity was assayed in three ways: (i) gap-filling by pol III holoenzyme and subassemblies of it, (ii) the extensive replication of a primed, single-stranded DNA circle only by pol III holoenzyme, and (iii) complementation of a crude, inactive pol III holoenzyme (temperature-sensitive dnaE mutant fraction) in replication of a primed, single-stranded DNA circle . Amplification of the alpha subunit raised the polymerase level 10-fold in assay (i), indicative of the dependence of pol III gap-filling activity on this polypeptide; pol III holoenzyme activity remained unaffected (assay (ii)), but the complementation activity was raised 5-fold (assay (iii)) . Thus, the elevated alpha subunit (free or in a subassembly form) can substitute in vitro for a defective alpha subunit in pol III holoenzyme, but cannot increase the in vivo level of about eight pol III holoenzyme molecules per cell . This low level of pol III holoenzyme is fixed in wild type cells (bearing no plasmid) despite the presence of a 5-fold excess of the alpha subunit, as inferred from the various assays . These results suggest that the low level of pol III holoenzyme is determined by a factor or factors other than the level of the alpha subunit.

Nucleic Acids Res, 1985 Oct 25, 13(20), 7289 - 97
Attenuation and processing of RNA from the rpsO-pnp transcription unit of Escherichia coli; Takata R et al.; Ribosomal protein S15 and polynucleotide phosphorylase of E . coli are encoded by two adjacent genes, rpsO and pnp, respectively . Analysis of in vivo transcripts from these two genes shows that they are within the same operon (S15 operon) . By correlating the 5' and 3' ends of their in vivo transcripts with the DNA sequence, we have identified several features of the operon structure . These features include a promotor upstream from rpsO, an attenuator downstream from rpsO and an RNA processing site between these two genes.

Biochemistry, 1985 Oct 22, 24(22), 6163 - 9
Fluorine NMR studies on stereochemical aspects of reactions catalyzed by transcarboxylase, pyruvate kinase, and enzyme I; Hoving H et al.; The stereochemistry of the transcarboxylase-catalyzed carboxylation of 3-fluoropyruvate has been studied by using fluorine NMR of unpurified reaction mixtures . When the product 3-fluorooxaloacetate was trapped by using malate dehydrogenase, only the 2R,3R diastereomer of 3-fluoromalate was formed . The fluoromethyl group of fluoropyruvate does not take up deuterium label from the solvent during the reaction . These results confirm and extend those obtained previously by Walsh and co-workers {Goldstein, J . A., Cheung, Y . F., Marletta, M . A., & Walsh, C . (1978) Biochemistry 17, 5567-5575} showing that transcarboxylase is specific for one of the two prochiral hydrogens in fluoropyruvate . Transcarboxylase, coupled to malate dehydrogenase, has been used to analyze samples of chiral fluoropyruvate obtained by dephosphorylation of (Z)-fluorophosphoenolpyruvate in D2O in the presence of either pyruvate kinase or enzyme I from the Escherichia coli sugar transport systems . Analysis of the fluoromalate produced showed that fluoroenolpyruvate is deuterated from opposite faces by these two enzymes: enzyme I protonates (deuterates) fluoroenolpyruvate exclusively from the 2-re face and pyruvate kinase does so mainly from the 2-si face . Fluoropyruvate is carboxylated by transcarboxylase with absolute retention of configuration.

Biochemistry, 1985 Oct 22, 24(22), 6245 - 52
Primary structure of the succinyl-CoA synthetase of Escherichia coli; Buck D et al.; The primary structure of the succinyl-CoA synthetase of Escherichia coli has been deduced from the nucleotide sequence of a 2451-base-pair segment of DNA containing the corresponding sucC (beta subunit) and sucD (alpha subunit) genes . The genes are located at one end of a gene cluster that encodes several citric acid cycle enzymes: gltA-sdhCDAB-sucABCD; gltA, citrate synthase; sdh, succinate dehydrogenase; sucA and sucB, the dehydrogenase (E1) and succinyltransferase (E2) components of the 2-oxoglutarate dehydrogenase complex . The sucC and sucD genes are separated from the sucA and sucB genes by a 273-base-pair segment containing four palindromic units, but they appear to be expressed from a sucABCD read-through transcript that extends from the suc promoter to a potential rho-independent terminator at the distal end of sucD . The stop codon of the sucC gene overlaps the sucD initiation codon by a single nucleotide, indicating close translational coupling of the sucC and sucD genes . The sucC gene comprises 1161 base pairs (388 codons, excluding the stop codon), and it encodes a polypeptide of Mr 41 390 corresponding to the beta subunit of succinyl-CoA synthetase . The sucD gene comprises 864 base pairs (288 codons, excluding the start and stop codons), and it encodes a product of Mr 29 644, corresponding to the alpha subunit of succinyl-CoA synthetase . The alpha subunit contains a 12-residue amino acid sequence that is identical with the histidine peptide previously isolated from the phosphoenzyme . This sequence forms part of one of the two potential nucleotide binding sites detected in the alpha subunit.

Proc R Soc Lond B Biol Sci, 1985 Oct 22, 226(1242), 43 - 58
The nuclear location signal; Smith AE et al.; A short sequence of predominantly basic amino acids Pro-Pro-Lys-Lys-Lys-Arg-Lys-Val from SV40 Large T is responsible for the normal nuclear location of the protein . Alteration of Lys-128 to each of six different residues other than Arg renders Large T cytoplasmic, whereas single amino acid changes in the surrounding region impair but do not prevent nuclear accumulation . When transposed to the amino terminus of cytoplasmic Large T species, or Escherichia coli beta-galactosidase or of chicken muscle pyruvate kinase, the sequence around Lys-128 of Large T is able to direct the recipient protein to the nucleus . This demonstrates that these amino acids can be sufficient for nuclear location and can act as a nuclear location signal . A computer search of over 2500 proteins reveals that some other nuclear proteins (for example, BK virus Large T, SV40 VP2 and adenovirus 72kDa DNA binding protein) contain very similar basic tracts, but so too do some presumed non-nuclear proteins (for example, poliovirus VP3) . We suggest that the related sequence acts as the nuclear location signal in the other nuclear proteins but that the sequence does not function in all cases, perhaps because it is not accessible . A similar, but shorter or less basic sequence, was detected in a number of other nuclear proteins, for example, polyoma virus Large T, SV40 VP1 and several histones . However, such sequences were also found in many other proteins . Perhaps the shorter basic sequences can also act as nuclear location signals, but to be functional they need to be exposed (for example, at the amino terminus of the protein as in SV40 VP1) or to be present in multiple copies.

Proc R Soc Lond B Biol Sci, 1985 Oct 22, 226(1242), 25 - 42
Cellular targets for SV40 large T-antigen; Lane DP et al.; SV40 virus infection is able to induce tumours in newborn hamsters and to transform a wide range of eukaryotic cells in in vitro culture . This is achieved by integration of the viral DNA into the host cell DNA and expression of the virus-encoded Large T-antigen . The expression of Large T, a 708 amino acid phosphoprotein, is required both to induce and maintain the transformed state . The Large T protein initiates viral DNA synthesis and regulates viral transcription, apparently by binding in a specific manner to viral DNA sequences at and near the viral origin of replication . SV40 Large T also affects cellular DNA synthesis and transcription and this may account for its oncogenic activity . A novel immunochemical procedure has permitted the isolation of cellular DNA sequences occupied by SV40 Large T in the chromatin of SV40 transformed cells . Some of the cellular sequences contain high affinity binding sites for SV40 Large T, and hybridize to messenger RNAs expressed in SV40 transformed but not in normal cells . A second type of cellular target for Large T is the cell coded p53 protein that it binds to and stabilizes . A range of monoclonal antibodies to p53 has been isolated and characterized . They demonstrate that p53 is in the cytoplasm of normal cells but is located in the nucleus of transformed cells . One of the antibodies recognizes an epitope on p53 that is stabilized or induced by binding to Large T . Further studies on the T-p53 protein complex have been facilitated by constructing bacterial plasmids that direct the synthesis of substantial quantities of Large T-beta-galactosidase and p53-beta-galactosidase fusion proteins in bacteria . The results are discussed in the context of our current knowledge of oncogene action.

FEBS Lett, 1985 Oct 21, 191(1), 72 - 4
Functionally important site in the vicinity of the amino-terminus of the Escherichia coli RNA polymerase beta subunit; Nikiforov VG et al.; We have analyzed the interaction of monoclonal antibodies against Escherichia coli RNA polymerase with products of its limited proteolysis . Two major proteolytic fragments of molecular masses 107 and 43 kDa originate as a result of a single cleavage in the vicinity of the 980th amino acid residue . Anti-beta subunit monoclonal antibody PYN-2 inhibiting RNA polymerase activity at the stage of RNA elongation reacts with an epitope located between the amino-terminus and the 50th amino acid residue of the beta subunit . DNA sequencing has shown that the RNA polymerase mutation rpoB22 converts the Gln(1111) codon of the beta subunit gene into the amber codon . An epitope for the monoclonal antibody PYN-6 was located between the major site of proteolytic cleavage and Gln(1111) of the beta subunit.

J Mol Biol, 1985 Oct 20, 185(4), 743 - 54
Genetic reconstruction and functional analysis of the repeating lipoyl domains in the pyruvate dehydrogenase multienzyme complex of Escherichia coli; Guest JR et al.; The dihydrolipoamide acetyltransferase component (E2p) of the pyruvate dehydrogenase complex of Escherichia coli contains three highly homologous sequences of about 100 residues that are tandemly repeated to form the N-terminal half of the polypeptide chain . All three sequences include a lysine residue that is a site for lipoylation and they appear to form independently folded functional domains . These lipoyl domains are in turn linked to a much larger (about 300 residues) subunit-binding domain of the E2p chain that aggregates to form the octahedral inner core of the complex and also contains the acetyltransferase active site . In order to investigate whether individual lipoyl domains play different parts in the enzymic mechanism, selective deletions were made in vitro in the dihydrolipoamide acetyltransferase gene (aceF) so as to excise one or two of the repeating sequences . This was facilitated by the high degree of homology in these sequences, which allowed the creation of hybrid lipoyl domains that closely resemble the originals . Pyruvate dehydrogenase complexes incorporating these genetically reconstructed E2p components were purified and their structures were confirmed . It was found that the overall catalytic activity, the system of active site coupling, and the ability to complement pyruvate dehydrogenase complex mutants, were not significantly affected by the loss of one or even two lipoyl domains per E2p chain . No special role can be attached thus far to individual lipoyl domains . On the other hand, certain genetic deletions affecting the acetyltransferase domain caused inactivation of the complex, highlighting particularly sensitive areas of that part of the E2p chain.

J Mol Biol, 1985 Oct 20, 185(4), 689 - 99
Nuclear magnetic resonance study of the proton exchange rate in the operator-promoter DNA sequence of the trp operon of Escherichia coli; Lefevre JF et al.; The dynamic behavior of a palindromic oligonucleotide (C-G-T-A-C-T-A-G-T-T-A-A-C-T-A-G-T-A-C-G) representative of the operator sequence and containing the Pribnow box of the trp operon of Escherichia coli was investigated . The resonances of the imino protons and of the H2 protons of the adenosine residues were all assigned . The opening rate constants of the base-pairs were calculated by monitoring the exchange rate of the observable imino protons (nine out of ten), using selective temperature (T1) measurements, which avoid the complication of cross-relaxation and spin diffusion . These measurements have to be performed in conditions where the exchange process is much faster than the opening and closing of the base-pairs, so that the observed exchange rate is equal to the opening rate . It is shown that the catalysis of the exchange process by phosphate dianions is not very efficient (kB approximately equal to 7 X 10(4) M-1 S-1) . Hence, in phosphate buffer, the necessary opening-rate limiting condition is met only at high pH values (approximately equal to 9.5) where efficient catalysis by OH- takes place, or at very high buffer concentration . While G X C base-pairs show very little exchange, acting in the sequence as molecular "staples", the A X T base-pairs that are protected from the fraying have very different opening and closing rates, depending on the sequence . Although it seems possible that the opening process could occur at the base-pair level, it is localized at most to two base-pairs in that particular sequence . The activation energies for the opening process of all non-fraying base-pairs are very similar (19 +/- 1 kcal mol-1; 1 cal = 4.184 J), and the differences in the opening rates are essentially due to differences in the activation entropies . With regard to the role of this sequence in the promoter, it is observed that the end of the Pribnow box exchanges relatively easily, and that the activation parameters for the "breathing" process and for the isomerization step of the promoter--RNA polymerase are not very different.

J Mol Biol, 1985 Oct 20, 185(4), 755 - 67
Copy number of the broad host-range plasmid R1162 is determined by the amounts of essential plasmid-encoded proteins; Kim K et al.; DNA of the broad host-range plasmid R1162 contains a 1700 base-pair segment essential for plasmid maintenance . This region, RepI, consists of two cotranscribed genes encoding polypeptides with molecular weights of 29,000 and 31,000 . Fusion of RepI to the strong tac promoter results in greatly increased amounts of at least one of these polypeptides . In trans, this construction has two other properties: it can raise the copy number of R1162, and it can protect this plasmid from loss due to incompatibility . Both effects require intact RepI genes . These properties of the RepI region, along with those of an origin-linked region described earlier, are discussed with respect to current models for control of plasmid copy number.

J Mol Biol, 1985 Oct 20, 185(4), 713 - 20
Processing enzyme ribonuclease E specifically cleaves RNA I . An inhibitor of primer formation in plasmid DNA synthesis; Tomcsanyi T et al.; When the RNA processing enzyme RNAase E is inactivated in an Escherichia coli strain carrying derivatives of the colicin E1 plasmid, a small RNA, about 100 nucleotides long, accumulates . Structural analysis of this RNA showed that it is RNA I, the RNA that inhibits plasmid DNA synthesis . RNA I is a specific substrate for RNAase E and the cleavage takes place between the fifth and sixth nucleotides from the 5' end of the molecule . This is only the second natural RNA substrate that has been found, so far, for the RNA processing enzyme ribonuclease E, the other being a precursor for 5 S ribosomal RNA . It is remarkable that nine nucleotides around the cleavage sites are identical in both substrates: (Formula: see text) . Therefore, we suggest that at least part of the interaction between RNAase E and its substrate is controlled by these nine nucleotides.

Biochim Biophys Acta, 1985 Oct 18, 831(3), 288 - 96
Purification and properties of ornithine decarboxylase from Tetrahymena pyriformis; Sklaviadis TK et al.; In Tetrahymena pyriformis, ornithine decarboxylase (L-ornithine carboxy-lyase, EC 4.1.1.17) activities are present in the cytosolic and nuclear fractions and reach maximal values in the middle and late log phases of growth, respectively . The two activities have been purified to homogeneity by ammonium sulfate fractionation (20-45%), anion-exchange chromatography (DEAE-Bio-Gel A), gel-filtration (Sephadex G-150 and Sephadex G-100 superfine) and hydrophobic chromatography (Phenyl-Sepharose) . Both the crude and the purified enzyme preparations are inactivated irreversibly by alpha-difluoromethylornithine, a suicide inhibitor of mammalian ornithine decarboxylase . The enzyme preparations from the nucleus and cytosol each showed a single band on polyacrylamide gel electrophoresis under native and denaturing conditions and on acrylamide gel electrofocusing . Both activities show the same pH optima (8.6) isoelectric point (5.3), molecular weight (64 000) and Kmorn (4.7 microM) . The Km for L-lysine is 0.5 mM . The two activities also cross-react with acidic antizyme extracted from E . coli mutant MA 255 . Based on the physicochemical properties, one can safely conclude that cytosolic and nuclear activities reside on the same protein molecule.

Biochim Biophys Acta, 1985 Oct 18, 831(3), 330 - 4
The active-site and amino-terminal amino acid sequence of bovine intestinal alkaline phosphatase; Culp JS et al.; The active site of bovine intestinal alkaline phosphatase (orthophosphoric-monoester phosphohydrolase (alkaline optimum), EC 3.1.3.1) was labeled with {32P}Pi, a radioactive CNBr peptide was isolated and the amino acid sequence was determined . The sequence of the active-site peptide has limited homology (26%) with the active-site sequence of Escherichia coli alkaline phosphatase except for the ten residues immediately flanking the active-site serine (70%) . A possible amino acid sequence deduced from the amino acid composition of an active-site tryptic peptide from human placental alkaline phosphatase is very similar to the bovine intestinal active-site sequence . The amino-terminal sequence of bovine intestinal alkaline phosphatase is homologous (69%) with the human placental enzyme but not with the E . coli phosphatase.

Biochim Biophys Acta, 1985 Oct 18, 831(3), 275 - 80
Temperature-dependent structural rearrangement of apotryptophanase in potassium phosphate; Lachmann G et al.; Apotryptophanase (L-tryptophan indole-lyase, EC 4.1.99.1) from Escherichia coli B/1t7A shows, in the presence of potassium phosphate, a temperature-dependent structural rearrangement which is not observed in the presence of sodium phosphate or imidazole plus KC1 . This rearrangement can be described by a two-state equilibrium between two forms of the apoenzyme . The midpoint temperature of the rearrangement (TM) and the van't Hoff enthalpy (delta H) at different potassium phosphate concentrations and pH values, respectively, were determined by measuring the temperature-dependence of the ultraviolet absorbance of apotryptophanase . Increasing the potassium phosphate concentration at pH 7.8 causes a simultaneous increase in total absorbance and the delta H value, whereas the TM increases between pH 7.0 and 7.8 but starts to decrease at pH values above 7.8 . In 0.1 M potassium phosphate at the pH optimum of the enzyme (7.8) TM and delta H were found to be 293.1 K and 167 kJ X mol-1, respectively . Moreover, the tyrosine residues of apotryptophanase dissociate in potassium phosphate and in imidazole plus KCl with pK values of 8.6 and 9.8, respectively, indicating that potassium phosphate favors the formation of tyrosinate . The rearrangement might be interpreted as the formation of specific hydrogen bonds between tyrosine and potassium phosphate which are ruptured at higher temperature . Such hydrogen bonds cannot be formed at all or only to a small extent in the presence of imidazole plus KCl or sodium phosphate . Those hydrogen bonds stabilize the structure of apotryptophanase . In contrast, holotryptophanase requires only K+ for enzymatic activity.

Biochim Biophys Acta, 1985 Oct 18, 831(3), 340 - 6
Circular dichroism studies on synthetic signal peptides; Reddy GL et al.; Circular dichroism studies on synthetic peptides corresponding to the signal sequences of chicken lysozyme and Escherichia coli proteins, lambda-receptor and lipoprotein, have been carried out in trifluoroethanol . The peptides, (CH3)3-C-O-CO-Thr-Leu-Lys-Lys-Leu-Pro-Leu-Ala-Val-Ala-Val-Ala-Ala-Gly- Val-Met-Thr-Ala- Ala-Met-Ala-OCH3, (CH3)3-C-O-CO-Met-Lys-Ser-Leu-Leu-Ile-Leu-Val-Leu-Cys(benzyl)- Phe-Leu-Pro- Leu-Ala-Ala-Leu-Gly-OH and (CH3)3-C-O-CO-Leu-Val-Leu-Gly-Ala-Val-Ile-Leu-Gly- Thr-Thr-Leu-Leu- Ala-Gly-OCH3, corresponding to the signal sequences of lambda-receptor, lysozyme and the hydrophobic region of lipoprotein, respectively, show two negative bands at approx . 205 and 220 nm, characteristic of an alpha-helical conformation . Secondary structural features are discernible even in the shorter, 12-residue carboxy-terminal fragments of these signal peptides . A comparison of the conformation of the amino-terminal, central and carboxy-terminal fragments of lipoprotein signal sequence indicates that the central octapeptide fragment is more structurally ordered compared to the amino- and carboxy-terminal fragments.

Nature, 1985 Oct 17-23, 317(6038), 643 - 5
Porin channel triplets merge into single outlets in Escherichia coli outer membranes; Engel A et al.; Previous observations on the structural and functional properties of porin, the matrix protein of Escherichia coli, have indicated that the channel-forming trimers span the outer membranes of the bacterial cell, forming a molecular sieve . By using electron microscopy and image reconstruction, we demonstrate here that three channels on the outer surface of the cell merge into a single channel at the periplasmic face . Conductance measurements using conditions under which single activated triplets could be observed led us to conclude that the three individual consecutive closing steps reflect three channels within a single trimeric unit . Statistical analysis of conductance levels revealed that the first relaxation step is distinctly smaller than the two subsequent channel closings . This functional observation can be explained if the channels of porin trimers coalesce.

Carbohydr Res, 1985 Oct 15, 142(2), 269 - 76
Structure of the K95 antigen from Escherichia coli O75:K95:H5, a capsular polysaccharide containing furanosidic KDO-residues; Dengler T et al.; The structure of the K95 antigenic capsular polysaccharide (K95 antigen) of Escherichia coli O75:K95:H5 was elucidated by determination of the composition, 1H- and 13C-n.m.r . spectroscopy, periodate oxidation, and methylation analysis . The K95 polysaccharide, which contains furanosidic 3-deoxy-D-manno-2-octulosonic acid (KDOf) residues, consists of----3)-beta-D-Rib-(1----8)-KDOf-(2----repeating units, has a molecular weight of approximately 25,000 (approximately 65 repeating units), and is randomly O-acetylated (1 acetyl group per repeating unit at unknown positions).

Biochem Biophys Res Commun, 1985 Oct 15, 132(1), 232 - 9
Molecular cloning of DNA complementary to mRNA of rat liver serine dehydratase; Noda C et al.; A cDNA clone containing sequences complementary to the mRNA cording for rat hepatic serine dehydratase was isolated to study the multihormonal regulation of this enzyme . Serine dehydratase mRNA was partially purified (50-fold enrichment, 8.2% of the total mRNA activity) from the liver of rats fed high protein diet by polysome immunoadsorption followed by oligo(dT)-cellulose column chromatography . This preparation was used as template for synthesis of cDNA . Double-stranded cDNA sequences were inserted into the plasmid pBR322 and cloned in Escherichia coli DH1 . Of 860 transformants screened, 6 clones containing DNA complementary to serine dehydratase mRNA were identified by differential colony hybridization and hybrid-selected translation . The length of serine dehydratase mRNA was estimated to be 1,500 bases by Northern blot analysis . One cloned cDNA comprised about 1,000 base pairs, or 65% of the length of the mRNA . The amount of the mRNA was greatly increased in the liver of rats given high protein diet.

Biochem Biophys Res Commun, 1985 Oct 15, 132(1), 217 - 22
Determination of the metabolic origin of the sulfur atom in thiamin of Escherichia coli by mass spectrometry; DeMoll E et al.; In this study cells were grown in 34S-sulfate or L-{sulfane-34S}thiocystine, and the effects of unlabeled methionine and cystine on incorporation of sulfur into methionine, cystine and thiamin were determined . Unlabeled methionine effectively suppresses the incorporation of 34S into methionine but not into cysteine or thiamin . In contrast, cystine blocks incorporation of 34S only to approximately the relative ratio of 32S to 34S indicating, that cysteine is closely related to the origin of the sulfur in thiamin, and therefore the sulfane sulfur of thiocystine is also an effective source of the thiamin sulfur.

Biochem Biophys Res Commun, 1985 Oct 15, 132(1), 126 - 33
Expression of intact Ki-ras p21 protein in Escherichia coli; Tamaoki T et al.; We have constructed recombinant plasmids capable of expressing in Escherichia coli the intact ras p21 protein encoded by Kirsten murine sarcoma virus . The Ki-ras gene was inserted into an expression vector carrying the E . coli tryptophan promoter and E . coli lipoprotein transcriptional terminator . The resulting plasmids direct the synthesis of large quantities of p21 protein, which represented 20% of the total cellular protein . The Ki-ras p21 protein is immunoprecipitated with monoclonal antibody to p21, and exhibits guanine nucleotide binding activity and autophosphorylation activity . The purified Ki-ras p21 expressed in E . coli has shown to have intact N-terminal and C-terminal amino acid sequences predicted by the nucleotide sequences and migrate as -23K in SDS/polyacrylamide gels.

J Biol Chem, 1985 Oct 15, 260(23), 12720 - 4
Pathways for the incorporation of exogenous fatty acids into phosphatidylethanolamine in Escherichia coli; Rock CO et al.; Two distinct pathways for the incorporation of exogenous fatty acids into phospholipids were identified in Escherichia coli . The predominant route originates with the activation of fatty acids by acyl-CoA synthetase followed by the distribution of the acyl moieties into all phospholipid classes via the sn-glycerol-3-phosphate acyltransferase reaction . This pathway was blocked in mutants (fadD) lacking acyl-CoA synthetase activity . In fadD strains, exogenous fatty acids were introduced exclusively into the 1-position of phosphatidylethanolamine . This secondary route is related to 1-position fatty acid turnover in phosphatidylethanolamine and proceeds via the acyl-acyl carrier protein synthetase/2-acylglycerophosphoethanolamine acyltransferase system . The turnover pathway exhibited a preference for saturated fatty acids, whereas the acyl-CoA synthetase-dependent pathway was less discriminating . Both pathways were inhibited in mutants (fadL) lacking the fatty acid permease, demonstrating that the fadL gene product translocates exogenous fatty acids to an intracellular pool accessible to both synthetases . These data demonstrate that acyl-CoA synthetase is not required for fatty acid transport in E . coli and that the metabolism of exogenous fatty acids is segregated from the metabolism of acyl-acyl carrier proteins derived from fatty acid biosynthesis.

Biochem Biophys Res Commun, 1985 Oct 15, 132(1), 162 - 70
cys154 Is important for lac permease activity in Escherichia coli; Menick DR et al.; The lac Y gene of Escherichia coli which encodes the lac permease has been modified by oligonucleotide-directed, site-specific mutagenesis such that cys154 is replaced with either gly or ser . Permease with gly in place of cys154 exhibits essentially no transport activity, while substitution of cys154 with ser also causes marked, though less complete loss of activity . The findings suggest that cys154 plays an important role in lactose:H+ symport.

Eur J Biochem, 1985 Oct 15, 152(2), 411 - 8
Identification of surface residues in the trp repressor of Escherichia coli; Lane AN et al.; A subset of the spin systems assigned in the 1H NMR spectrum of the trp repressor in the first paper in this series (our penultimate preceding paper in this journal) can be identified as surface or buried residues on the basis of four independent types of measurement: selective spin-lattice relaxation times; the dependence of line widths on temperature and the concentration of manganous ion; fluorescence quenching; and titration behaviour . Criteria are developed for distinguishing surface and buried residues . The significance for the function of DNA binding proteins is discussed.

Eur J Biochem, 1985 Oct 15, 152(2), 405 - 9
Preliminary identification of the secondary structure of the trp repressor from Escherichia coli; Lane AN et al.; The probable secondary structure content of the trp repressor from Escherichia coli has been inferred from NMR and circular dichroic measurements; the results are compared with those of prediction algorithms . 70% of the amide protons have exchange rate constants orders of magnitude smaller than the intrinsic rate constants, identifying them as participating in hydrogen bonds . The exchange rate constants fall into two distinct classes, one having half-lives of 20 min and the other more than 24 h . The latter class, consisting of 50% of all amide protons, indicates a stable core . The exchange data are consistent with circular dichroism and predictions that suggest that about 55% of the peptides from alpha helix, and 20% form beta sheets and turns . The NMR spectrum further indicates that there is little beta sheet, suggesting that the secondary structure class is alpha.

Eur J Biochem, 1985 Oct 15, 152(2), 395 - 404
NMR studies of the trp repressor from Escherichia coli . Characterisation and assignments of residue types; Lane AN et al.; High-resolution proton nuclear magnetic resonance spectra of the trp repressor of Escherichia coli under various conditions are reported and analysed . The spectrum of the denatured state agrees with that predicted from the amino acid composition, with the exception of the two histidine residues, which have different chemical shifts although they titrate normally . The spectrum of the native protein shows the presence of extensive secondary and tertiary structure . Using information from chemical shifts, numbers of protons, titration behaviour, homonuclear chemical-shift-correlated spectroscopy and nuclear Overhauser enhancement correlated spectroscopy, most of the aromatic protons have been assigned to residue type . Further, about 30% of the aliphatic protons have been assigned to residue type by two-dimensional spectroscopy . Nuclear Overhauser enhancements establish that high-field methyl groups belonging to a valine residue lie directly over an aromatic ring.

Eur J Biochem, 1985 Oct 15, 152(2), 387 - 93
The EcoDXX1 restriction and modification system of Escherichia coli ET7 . Purification, subunit structure and properties of the restriction endonuclease; Piekarowicz A et al.; The Escherichia coli plasmid pDXX1 codes for a new restriction-modification system . The specific restriction endonuclease coded by this system has been purified by a procedure that includes phosphocellulose and heparin-agarose chromatography . Sedimentation on glycerol gradients showed one peak of activity with a value of about 12 S . The highly purified enzyme require ATP and Mg2+ for activity as well as S-adenosylmethionine, although some S-adenosylmethionine molecules are probably bound to the enzyme . The enzyme does not cleave lambda DNA at well-defined sites and has a strong non-modified DNA-dependent ATPase activity . The enzyme has also methylase activity acting against non-modified DNA.

J Biol Chem, 1985 Oct 15, 260(23), 12803 - 9
Polyoma virus major capsid protein, VP1 . Purification after high level expression in Escherichia coli; Leavitt AD et al.; We have expression-cloned in Escherichia coli the major polyoma virus capsid protein, VP1 . Under the inducible control of the hybrid tac promoter, VP1 constituted between 2 and 3% of the total host cell protein . The expressed VP1 was purified to near homogeneity with initial yields to 10% . Optimal expression was temperature-dependent, and significant intracellular degradation could be demonstrated . The final product was obtained as one predominant isoelectric focusing species, without the pattern of post-translational modification seen in virus-infected eukaryotic cells . The purified VP1 from E . coli will be useful as a substrate for the purification of VP1 modification enzymes and in the study of inter-VP1 oligomerization.

J Biol Chem, 1985 Oct 15, 260(23), 12635 - 40
ATP synthesis and hydrolysis by a hybrid system reconstituted from the beta-subunit of Escherichia coli F1-ATPase and beta-less chromatophores of Rhodospirillum rubrum; Gromet-Elhanan Z et al.; Photophosphorylation and ATPase activities were restored to beta-less Rhodospirillum rubrum chromatophores by their reconstitution with purified beta-subunits of either R . rubrum F1-ATPase (Rr beta) or Escherichia coli F1-ATPase (Ec beta) . In the homologous reconstituted system both activities were restored to the same extent, whereas in the hybrid system ATP synthesis was restored to about 10% when the hydrolysis was restored to 200% . This difference in rates of synthesis and hydrolysis was not due to any general uncoupling effect of Ec beta leading to an increased membrane permeability to protons, because with both hybrid and homologous systems an identical light-induced quenching of quinacrine fluorescence was observed . They differed, however, in ATP-driven quenching of quinacrine fluorescence, which was much lower in the hybrid system . These results suggest that the hybrid has a decreased capacity for proton-translocation through the membrane-bound Fo channel during ATP hydrolysis, and probably also during ATP synthesis . The very high ATPase activity of the hybrid system indicates that it might enable the released protons to leak to the outside medium rather than to move inside through the Fo channel . The activities restored by Rr beta and Ec beta exhibit a similar sensitivity to dicyclohexylcarbodiimide, but different sensitivities to oligomycin and to an anti-E . coli F1 (EcF1) antibody . Oligomycin inhibited only the homologous R . rubrum system whereas anti-EcF1 was a much more effective inhibitor of the hybrid system . It is therefore concluded that Rr beta plays a role, that the Ec beta cannot fulfill, in conferring oligomycin sensitivity to the RrFo X F1-ATP synthase-ATPase complex.

J Biol Chem, 1985 Oct 15, 260(23), 12600 - 6
Purification of a liver alkaline protease which degrades oxidatively modified glutamine synthetase . Characterization as a high molecular weight cysteine proteinase; Rivett AJ; A nonlysosomal alkaline protease which degrades the oxidatively modified form of Escherichia coli glutamine synthetase has been purified to apparent homogeneity from rat and mouse liver acetone powders . Its molecular weight was determined to be 300,000 by Sephacryl S-300 gel filtration but results of further studies using high pressure liquid chromatography gel filtration suggest a value of 650,000 . Examination of the subunit structure by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed multiple bands of molecular weights between 22,000 and 34,000 . The alkaline protease was inhibited by thiol reagents . Phenylmethylsulfonyl fluoride, aprotinin, leupeptin, antipain, and chymostatin partially inhibited the protease . The inhibition by phenylmethylsulfonyl fluoride was prevented by dithiothreitol, and alpha 1-antitrypsin and soybean trypsin inhibitor did not inhibit . No inhibition was observed with metalloprotease inhibitors . The alkaline protease is active over a broad range of pH with optimum activity for the degradation of oxidized glutamine synthetase around pH 9.0 . Its activity is not stimulated by MgATP . A study of the products of insulin B chain degradation demonstrated major cleavage sites at Gln13-Ala14, Leu15-Tyr16, Cys(SO3H)19-Gly20, Gln4-His5, and Leu17-Val18 . Based on its endopeptidase activity and its inhibitor specificity, the alkaline protease should be classified as a cysteine proteinase . It appears to be distinct from previously described proteinases and is likely involved in nonlysosomal mechanisms of intracellular protein turnover.

J Biol Chem, 1985 Oct 15, 260(23), 12884 - 9
Complete replication of templates by Escherichia coli DNA polymerase III holoenzyme; O'Donnell ME et al.; DNA polymerase III holoenzyme (holoenzyme) processively and rapidly replicates a primed single-stranded DNA circle to produce a duplex with an interruption in the synthetic strand . The precise nature of this discontinuity in the replicative form (RF II) and the influence of the 5' termini of the DNA and RNA primers were analyzed in this study . Virtually all (90%) of the RF II products primed by DNA were nicked structures sealable by Escherichia coli DNA ligase; in 10% of the products, replication proceeded one nucleotide beyond the 5' DNA terminus displacing (but not removing) the 5' terminal nucleotide . With RNA primers, replication generally went beyond the available single-stranded template . The 5' RNA terminus was displaced by 1-5 nucleotides in 85% of the products; a minority of products was nicked (9%) or had short gaps (6%) . Termination of synthesis on a linear DNA template was usually (85%) one base shy of completion . Thus, replication by holoenzyme utilizes all, or nearly all, of the available template and shows no significant 5'----3' exonuclease action as observed in primer removal by the "nick-translation" activity of DNA polymerase I.

J Biol Chem, 1985 Oct 15, 260(23), 12875 - 83
Dynamics of DNA polymerase III holoenzyme of Escherichia coli in replication of a multiprimed template; O'Donnell ME et al.; Movements of DNA polymerase III holoenzyme (holoenzyme) in replicating a template multiprimed with synthetic pentadecadeoxynucleotides (15-mers) annealed at known positions on a single-stranded circular or linear DNA have been analyzed . After extension of one 15-mer on a multiprimed template, holoenzyme moves downstream in the direction of chain elongation to the next primer . Holoenzyme readily traverses a duplex, even 400 base pairs long, to exploit its 3'-hydroxyl end as the next available primer . This downstream polarity likely results from an inability to diffuse upstream along single-stranded DNA . These holoenzyme movements, unlike formation of the initial complex with a primer, do not require ATP . Time elapsed between completion of a chain and initiation on the next downstream primer is rapid (1 s or less); dissociation of holoenzyme to form a complex with another primed template is slow (1-2 min) . Thus, holoenzyme diffuses rapidly only on duplex DNA, probably in both directions, and forms an initiation complex with the first primer encountered . Based on these findings, schemes can be considered for holoenzyme action at the replication fork of a duplex chromosome.

FEBS Lett, 1985 Oct 14, 190(2), 319 - 23
Identification of the plasmid-encoded immunity protein for colicin E1 in the inner membrane of Escherichia coli; Goldman K et al.; A set of plasmids containing portions of the Col E1 plasmid were transformed into recA- cells . These cells, after UV irradiation, only incorporate labelled amino acids into plasmid-encoded proteins . UV-irradiated cells label a 14.5 kDa band if they are phenotypically immune to colicin E1, and do not contain this band if they are sensitive to colicin E1 . We conclude that the 14.5 kDa protein is the colicin E1 immunity protein . When the inner and outer membranes of these cells are fractionated, the labelled band appears in the inner membrane . The immunity protein must be an intrinsic inner membrane protein, confirming the predictions made by hydrophobicity calculations from primary sequence data.

FEBS Lett, 1985 Oct 14, 190(2), 227 - 31
The oxygen reaction of the cytochrome d-terminated respiratory chain of Escherichia coli at sub-zero temperatures . Kinetic resolution by EPR spectroscopy of two high-spin cytochromes; Kumar C et al.; The oxygen reaction of the fully reduced respiratory chain in membranes from oxygen-limited Escherichia coli was studied at sub-zero temperatures using EPR spectroscopy . Laser photolysis of CO-liganded cytochrome oxidase d precedes oxidation of at least 2 kinetically separable high-spin cytochromes . At -120 to -100 degrees C, a rhombic signal appears, attributable to cytochrome d, followed at above -100 degrees C, by appearance of a second, axial signal near g = 6, here assigned to cytochrome(s) b, and changes in the redox state of iron-sulphur clusters . The data kinetically resolve the 2 high-spin signals attributed to the oxidase complex and suggest schemes for electron flow to oxygen.

FEBS Lett, 1985 Oct 14, 190(2), 189 - 98
The prosthetic groups of succinate dehydrogenase: 30 years from discovery to identification; Singer TP et al.; Recent studies using magnetic circular dichroism at cryogenic temperatures, electron paramagnetic resonance (EPR) and linear electric field effect-EPR (LEFE) of succinate dehydrogenase in membranes and in soluble, homogeneous preparations demonstrated the presence of 3 different Fe-S clusters in the mammalian enzyme, as well as in a similar bacterial enzyme, fumarate reductase from Escherichia coli . There is one each of the 2Fe, 3Fe, and 4Fe clusters . Thus, succinate dehydrogenase is the first enzyme which has been shown to contain all 3 of these Fe-S clusters . The enzyme also contains 1 mol 8 alpha-{N(3)-histidyl}-FAD . It has taken the combined expertise of many laboratories and 15 years of effort to identify the flavin component, and nearly 3 decades to identify the Fe-S clusters . The data from physical methods appear to be internally consistent, in harmony with the results of chemical analysis, and provide a rational explanation for earlier results by the cluster extrusion method . There remains, however, a number of interesting and substantive questions for future investigations . This review traces the tortuous path, the many pitfalls and false leads, which have led us from the discovery of nonheme iron and 'bound' flavin in the enzyme to elucidation of their structures.

FEBS Lett, 1985 Oct 14, 190(2), 275 - 8
The properties of the complex between ribosomal protein L2 and tRNA; Remme J et al.; Escherichia coli ribosomal protein L2 interacts with fMet-tRNAfMet and NacPhe-tRNAPhe in solution, protecting their 3'-ends from enzymatic degradation . At the same time L2 enhances the rate of spontaneous hydrolysis of the ester bonds between terminal riboses and amino acyl moieties of these two peptidyl-tRNA analogues . L2 has, however, only a slight effect on the rate of spontaneous deacylation of aminoacyl-tRNAs . We suggest that the role of L2 is in the fixation of the aminoacyl stem of tRNA to the ribosome at its P-site, and speculate that this protein is directly involved in the peptidyl transferase (PT) reaction.

Nucleic Acids Res, 1985 Oct 11, 13(19), 7067 - 77
Stereospecific removal of methyl phosphotriesters from DNA by an Escherichia coli ada+ extract; Weinfeld M et al.; The ada+ gene product, a DNA methyltransferase present in extracts from an Escherichia coli strain constitutive for the adaptive response, removes only half of the methyl phosphotriesters from alkylated DNA . Since DNA phosphotriesters occur in two isomeric configurations (denoted Rp and Sp), we examined whether this reflects a stereospecific mode of repair by the methyltransferase . Analysis by reverse-phase HPLC, phosphorus NMR and circular dichroism established that only triesters in the Sp configuration are acted upon by the E . coli extract.

Nucleic Acids Res, 1985 Oct 11, 13(19), 6867 - 80
Cloning of eukaryotic protein synthesis initiation factor genes: isolation and characterization of cDNA clones encoding factor eIF-4A; Nielsen PJ et al.; Monoclonal antibodies directed against rabbit reticulocyte protein synthesis initiation factor 4A (eIF-4A) were used to isolate mouse cDNA clones expressing eIF-4A protein sequences in E . coli . The identity of cDNA clones encoding eIF-4A sequences was confirmed by hybrid-selected translation and peptide mapping of the translation product . Analysis of the mRNA coding for eIF-4A from mouse liver and HeLa cells by Northern hybridization revealed two discrete mRNA species of approximately 2000 and 1600 nucleotides in length . The existence of two mRNAs in mouse and HeLa cells encoding eIF-4A was confirmed by cDNA sequencing.

Nucleic Acids Res, 1985 Oct 11, 13(19), 7139 - 51
Cloning and sequencing of the adenylate kinase gene (adk) of Escherichia coli; Brune M et al.; Adenylate kinase, the product of the adk locus in Escherichia coli K12, catalyzes the conversion of AMP and ATP to two molecules of ADP . The gene has been cloned by complementation of an adk temperature sensitive mutation . The DNA sequence of the complete coding region and of 5'- and 3'-untranslated regions were determined . The resulting protein sequence was found to contain several regions of high homology with cytosolic adenylate kinase of pig muscle (AK1), whose three-dimensional structure has been determined . The most significant of the amino acid exchanges is the replacement of histidine 36 with glutamine . This residue is believed to play a role in catalysis through metal ion binding . The codon usage pattern and the determination of adenylate kinase molecules per cell shows that the enzyme is one of the more abundant soluble proteins of the bacterial cells.

Nucleic Acids Res, 1985 Oct 11, 13(19), 7025 - 39
DNA sequence and characterization of the Escherichia coli serB gene; Neuwald AF et al.; We have determined the sequence of a DNA fragment containing the Escherichia coli serB gene . An open reading frame of 966 nucleotides was identified that encodes a polypeptide of 322 amino acids with a molecular weight of 35,002 daltons . The transcription start site was determined by Mung Bean nuclease mapping . The -10 and -35 regions of the serB promoter lack homology to the consensus sequences . In addition, the -35 region of the serB promoter overlaps the -35 region of a second divergent promoter . Frameshift mutations were constructed at three different sites within the serB gene . When plasmids carrying these mutations were used as templates in a minicell system, mutations closer to the proposed transcription and translation start sites resulted in smaller polypeptides than those further away, confirming the proposed direction of transcription and translation . The observed sizes of the truncated and native polypeptides were in agreement with those predicted from the DNA sequence . A very stable stem and loop structure (delta G= -32 kcal/mole) that does not fit the criteria of known transcription terminators was found one nucleotide downstream from the putative UAA translation stop codon.

Nucleic Acids Res, 1985 Oct 11, 13(19), 6999 - 7014
Inhibition of HeLa cell DNA topoisomerase I by ATP and phosphate; Low RL et al.; The relaxation activity of DNA topoisomerase I from HeLa cell nuclei is strongly inhibited by a variety of purine nucleotides in the presence but not absence of 1 mM potassium phosphate . For ATP, 3-4 mM causes nearly complete inhibition . The 2'-and 3'-AMP isomer are active as well in the presence of 1 mM phosphate, but the 5'-AMP isomer and adenosine are inert . At 3 mM ATP, the titration curve for phosphate is sigmoidal with inhibition beginning abruptly at about 0.5 mM . The negatively-supercoiled DNA isolated from an "inhibited" reaction is relaxed as well as the standard DNA template in the absence of ATP and phosphate suggesting that inhibition does not result from an alteration of the template which protects against its relaxation . Relaxation of positively-supercoiled DNA is also inhibited . Catalysis by E . coli DNA topoisomerase I and HeLa DNA topoisomerase II is not inhibited at concentrations of ATP and phosphate sufficient to cause 80-90% inhibition of HeLa type 1 enzyme.

Nucleic Acids Res, 1985 Oct 11, 13(19), 6919 - 36
Investigation of the tertiary folding of Escherichia coli 16S RNA by in situ intra-RNA cross-linking within 30S ribosomal subunits; Atmadja J et al.; Intra-RNA cross-links were introduced into E . coli 30S ribosomal subunits by mild ultraviolet irradiation . The subunits were partially digested with cobra venom nuclease, followed in some cases by a second partial digestion with ribonuclease H in the presence of the hexanucleotide d-(CTTCCC) . The cross-linked RNA complexes were separated by two-dimensional gel electrophoresis and the sites of cross-linking analysed by our published procedures . Tertiary structural cross-links in the 16S RNA were identified between positions 31 and 48, between oligonucleotides 1090-1094 and 1161-1164, and between oligonucleotides 1125-1127 and 1280-1281 . The first of these imposes a rigid constraint on the relative orientations of helices 3 and 4 of the 16S secondary structure . A further tertiary cross-link (which could not be precisely localised) was found between regions 1-72 and 1020-1095, and secondary structural cross-links were identified between positions 497 and 545-548, and positions 1238-1240 and 1298.

Nucleic Acids Res, 1985 Oct 11, 13(19), 6847 - 66
The distribution and properties of RNA primed initiation sites of DNA synthesis at the replication origin of Escherichia coli chromosome; Kohara Y et al.; RNA-linked DNA molecules were obtained from E . coli dnaCts cells synchronously initiating a new round of chromosome replication . The deoxynucleotides at the transition from primer RNA to DNA were 32P-labeled, and their positions were located on the nucleotide sequence of 1.4 kb genomic region (position -906 to +493) including the oriC and its leftside flanking region . In the r-strand (the counterclockwise strand), many strong transition sites were mapped in the left half portion of the oriC and a few weak sites in the left outside region . In the 1-strand (the clockwise strand), no transition sites were found inside the oriC but many weak sites were found in the left outside region . The results support the initiation mechanism in which the first leading strand synthesis starts with the r-strand counterclockwise from the oriC that is followed by the 1-strand synthesis on the displaced template strand on the left of oriC . Primer RNA molecules attached to the strong r-strand transition sites were only a few residues in length . Properties of the transition sites were discussed.

Biochim Biophys Acta, 1985 Oct 10, 819(2), 231 - 40
Electron spin resonance studies of lipid fluidity changes in membranes of an uncoupler-resistant mutant of Escherichia coli; Herring FG et al.; The fluidity of the lipids in membrane preparations from a mutant of Escherichia coli resistant to the uncoupler CCCP, grown at different temperatures with and without CCCP, was examined by electron spin resonance using the spin probe 5-doxyl stearic acid . The fluidity of the membrane lipids at the growth temperature, as estimated using electron spin resonance, was less in cells grown at lower temperatures . Precise homeoviscous adaptation was not observed . Growth in the presence of CCCP resulted in a decrease in membrane lipid fluidity, particularly in the inner (cytoplasmic) membrane . There was no change in the proportion of phosphatidylethanolamine, phosphatidylglycerol and cardiolipin in the cell envelope . However, there was an increase in the proportion of unsaturated fatty acids in membranes from cells grown with uncoupler . This was reflected in the increased fluidity of the lipids extracted from these membranes . This result is contrary to that expected from measurements of the fluidity of the lipid in these membranes . The decreased fluidity of the lipid in these membranes may be a consequence of the observed increase in the ratio of protein to phospholipid.

Biochemistry, 1985 Oct 8, 24(21), 5723 - 8
Cyclobutane pyrimidine dimers and (6-4) photoproducts block polymerization by DNA polymerase I; Chan GL et al.; Bipyrimidine cyclobutane dimers and 6-4'-(pyrimidin-2'-one)-pyrimidine photoproducts are the major adducts formed in DNA following exposure to ultraviolet light . The relationship between the type and frequency of UV-induced DNA damage and the effects of such damage on DNA replication were investigated . UV-irradiated M13 phage DNA was employed in polymerization reactions with the Kenow fragment of Escherichia coli DNA polymerase I . The locations and frequencies of polymerase termination events occurring within a defined sequence of M13 DNA were compared with measurements of the locations and frequencies of UV-induced DNA damage of the same DNA sequence by using UV-specific enzymatic and chemical methods . The results indicate that both cyclobutane dimers and (6-4) photoproducts quantitatively block polymerization by DNA polymerase I.

Biochemistry, 1985 Oct 8, 24(21), 6020 - 4
Cytidine deaminase from Escherichia coli B . Purification and enzymatic and molecular properties; Vita A et al.; Cytidine deaminase (cytidine aminohydrolase, EC 3.5.4.5) from Escherichia coli has been purified to homogeneity through a rapid and efficient two-step procedure consisting of anion-exchange chromatography followed by preparative electrophoresis . The final preparation is homogeneous, as judged by a single band obtained by disc gel electrophoresis performed in the absence and presence of denaturing agents . The native protein molecular weight determined by gel filtration is 56 000 . Sodium dodecyl sulfate disc gel electrophoresis experiments conducted upon previous incubation of the enzyme with dimethyl suberimidate suggest an oligomeric structure of two identical subunits of 33 000 molecular weight . The absorption spectrum of the protein reveals a maximum at 277 nm and a minimum at 255 nm . The isoelectric point is at pH 4.35 . Amino acid analysis indicates an excess of acidic amino acid residues as well as six half-cystine residues . No interchain disulfide groups have been evidenced . According to Cleland's nomenclature, kinetic analysis shows a rapid-equilibrium random Uni-Bi mechanism . Cytidine deaminase is competitively inhibited by various nucleosides . Km values for cytidine, deoxycytidine, and 5-methylcytidine are 1.8 X 10(-4), 0.9 X 10(-4), and 12.5 X 10(-4) M, respectively.

Biochemistry, 1985 Oct 8, 24(21), 5899 - 906
High-resolution differential scanning calorimetric analysis of the subunits of Escherichia coli aspartate transcarbamoylase; Edge V et al.; The thermal denaturation of the catalytic (c3) and regulatory (r2) subunits of Escherichia coli aspartate transcarbamoylase (c6r6) in the absence and presence of various ligands has been studied by means of highly sensitive differential scanning calorimetry . The denaturation of both types of subunit is irreversible as judged by the facts that the proteins coagulate when heated and that no endotherm is observed when previously scanned protein is rescanned . Despite this apparent irreversibility, there is empirical justification for analyzing the calorimetric data in terms of equilibrium thermodynamics as embodied in the van't Hoff equation . The observed curves of excess apparent specific heat vs . temperature are asymmetric and can be expressed within experimental uncertainty as the sums of sequential two-state steps, a minimum of two steps being required for r2 and three for c3 . As previously reported {Vickers, K . P., Donovan, J . W., & Schachman, H . K . (1978) J . Biol . Chem . 253, 8493-8498}, the addition of the effectors ATP and CTP raises the denaturation temperature of r2 and lowers that of c3 while the addition of the bisubstrate analogue N-(phosphonoacetyl)-L-aspartate raises the denaturation temperature of c3 and lowers that of r2 . These effects vary with ligand concentration in the manner expected from the van't Hoff equation, indicating that they are simply manifestations of Le Chatelier's principle rather than being due to "stabilization" or "destabilization" of the proteins . The denaturational enthalpy is increased in those cases of ligand binding in which the denaturation temperature is increased, because of the contribution from the enthalpy of dissociation of the ligand.

Biochemistry, 1985 Oct 8, 24(21), 5810 - 7
On the fidelity of DNA replication: manganese mutagenesis in vitro; Beckman RA et al.; Manganese is mutagenic in vivo and in vitro in studies with a variety of enzymes and templates . Using Escherichia coli DNA polymerase I with poly{d(A-T)} and phi X174 DNA templates, we analyzed the mechanism of manganese mutagenesis by determining the dependence of error rate on free Mn2+ concentration and comparing this to measured dissociation constants of Mn2+ from enzyme, template, and deoxynucleoside triphosphate substrates . This comparison suggests several conclusions: (1) At very low Mn2+ concentrations, the enzyme is activated at high fidelity . Thus, it is unlikely that activation with manganese per se significantly alters the conformation of the enzyme so as to affect nucleotide selection . (2) At low free Mn2+ concentrations (less than 100 microM), manganese causes errors in incorporation via its interaction with the DNA template . The concentration dependence of mutagenesis is determined by the strength of binding Mn2+ to the particular DNA template used . The data do not allow one to rule out the possibility that Mn2+-deoxynucleoside triphosphate interactions contribute to mutagenesis in selected situations . This range of free Mn2+ concentrations is the one of greatest relevance for in vivo mutagenesis . (3) At higher concentrations (between 500 microM and 1.5 mM), further mutagenesis by Mn2+ occurs . This mutagenesis probably is due either to binding of manganese to single-stranded regions within the DNA or to weak accessory sites on the enzyme.

Biochemistry, 1985 Oct 8, 24(21), 5776 - 80
Slow transacylation of peptidyladenosine allows analysis of the 2'/3'-isomer specificity of peptidyltransferase; Taiji M et al.; 2'-O-(N-acetyl-L-phenylalanyl-L-phenylalanyl)adenosine and 3'-O-(N-acetyl-L-phenylalanyl-L-phenylalanyl)adenosine (Ac-Phe-Phe-Ado) were chemically synthesized, and these two isomers were clearly separated from each other by high-performance liquid chromatography (HPLC) on an ODS column . By this HPLC method, the abundance ratio of the 2'-isomer and 3'-isomer in equilibrium in aqueous solution at pH 7.0 and 0 degrees C was found to be 0.30:0.70, and the equilibration rate was determined as 0.59 +/- 0.04 min-1 . Thus, the rate of transacylation between the 2'-isomer and 3'-isomer of peptidyl-tRNA was found to be much slower than that for the two isomers of aminoacyl-tRNA . The HPLC method was used for isomer analysis of the product of the Escherichia coli ribosomal peptidyltransferase reaction . By the use of an isomerizable analogue, 2'(3')-O-L-phenylalanyladenosine (Phe-Ado), as the acceptor of the N-acetyl-L-{3H}phenylalanine (Ac-{3H}Phe) group in the Ac-{3H}Phe-tRNAPhe.poly(U).70S ribosome system, the reaction product was found exclusively to be the 3'-isomer of Ac-{3H}Phe-Phe-Ado . Thus, the slow transacylation of peptidyladenosine allows the analysis of the 2'/3'-isomer specificity of peptidyltransferase.

Biochemistry, 1985 Oct 8, 24(21), 5729 - 34
Incorporation of biotin-labeled deoxyuridine triphosphate into DNA during excision repair and electron microscopic visualization of repair patches; Hunting DJ et al.; Biotin-labeled deoxyuridine triphosphate (BiodUTP) has the potential to be a useful affinity probe for studies on DNA repair, if it can be incorporated into DNA repair patches and does not inhibit subsequent steps in the excision repair pathway . We have synthesized BiodUTP by an improved procedure and have used permeable normal human fibroblasts to determine the effect of substituting BiodUTP for thymidine triphosphate on several steps in the excision repair pathway: incision, polymerization, ligation, and nucleosome rearrangement . The results demonstrate that BiodUTP is efficiently incorporated into repair patches and has little or no effect on the repair process . The presence of BiodUMP in ligated repair patches has been used to visualize the repair patches by electron microscopy following incubation with ferritin-labeled avidin . This approach has been used to estimate the maximum size of repair patches induced by ultraviolet radiation.

Nature, 1985 Oct 31-Nov 6, 317(6040), 782 - 6
The three-dimensional structure of trp repressor; Schevitz RW et al.; The crystal structure of the Escherichia coli trp repressor has been solved to atomic resolution . The dimeric protein has a remarkable subunit interface in which five of each subunit's six helices are interlinked . The binding of L-tryptophan activates the aporepressor indirectly by fixing the orientation of the second helix of the helix-turn-helix motif and by moulding the details of the repressor's structure near the DNA binding surface.

J Biol Chem, 1985 Oct 5, 260(22), 12308 - 12
The direction of RecA protein assembly onto single strand DNA is the same as the direction of strand assimilation during strand exchange; Register JC 3rd et al.; The RecA protein of Escherichia coli optimally promotes DNA strand exchange reactions in the presence of the single strand DNA-binding protein of E . coli (SSB protein) . Under these conditions, assembly of RecA protein onto single-stranded DNA (ssDNA) occurs in three steps . First, the ssDNA is rapidly covered by SSB protein . The binding of RecA protein is then initiated by nucleation of a short tract of RecA protein onto the ssDNA . Finally, cooperative polymerization of additional RecA protein accompanied by displacement of SSB protein results in a ssDNA-RecA protein filament (Griffith, J . D., Harris, L . D., and Register, J . C . (1984) Cold Spring Harbor Symp . Quant . Biol . 49, 553-559) . We report here that RecA protein assembly onto circular ssDNA yields RecA protein-covered circles in which greater than 85% are completely covered by RecA protein with no remaining SSB protein-covered segments (as detected by electron microscopy) . However, when linear ssDNA is used, 90% of the filaments contain a short segment at one end complexed with SSB protein . This suggests that RecA protein assembly is unidirectional . Visualization of the assembly of RecA protein onto either long ssDNA tails (containing either 5' or 3' termini) or ssDNA gaps generated in double strand DNA allowed us to determine that the RecA protein polymerizes in the 5' to 3' direction on ssDNA and preferentially nucleates at ssDNA-double strand DNA junctions containing 5' termini.

J Biol Chem, 1985 Oct 5, 260(22), 12060 - 4
Differential expression of the multiple forms of rat prekininogen mRNAs after acute inflammation; Kageyama R et al.; Responses of the rat liver prekininogen mRNAs after induction of acute inflammation were examined by blot-hybridization and S1 nuclease protection analyses with the aid of cDNA probes specific for rat kininogens . Marked changes in the relative levels of the low molecular weight (LMW) prekininogen mRNAs were observed after administration of Escherichia coli lipopolysaccharide, and the mRNA levels increased with a half-maximal dose of approximately 100 ng of lipopolysaccharide/100 g body weight . At maximum level of induction, the LMW prekininogen mRNAs comprised about 1% of total liver mRNA, thus representing a major component of the liver mRNA in the acutely inflamed rat . Differences in the inflammatory responses of various forms of the prekininogen mRNAs were then investigated by S1 nuclease protection analysis with the use of three different cDNA probes, each specific for either K-prekininogen or two types of T-prekininogens . Both of the T-prekininogen mRNAs increased progressively during the first 24 h after induction of inflammation, and at maximum level of induction, these two mRNAs increased about 10- and 13-fold over their normal level . In contrast, neither of the high molecular weight and LMW K-prekininogen mRNAs exhibited such an increase after induction of inflammation . Thus, the expressions of the rat T- and K-prekininogen mRNAs are differentially regulated in response to the induction of acute inflammation.

J Mol Biol, 1985 Oct 5, 185(3), 535 - 43
Properties of lac P2 in vivo and in vitro . An overlapping RNA polymerase binding site within the lactose promoter; Peterson ML et al.; The Escherichia coli lac promoter has been shown to contain an RNA polymerase binding site (P2) that overlaps with, and is shifted 22 base-pairs upstream from the normal lac promoter (P1) . In this paper, we provide RNA polymerase protection data obtained in vitro that show that, in the absence of CAP-cAMP, in vitro P2 is the preferred polymerase binding site on the P+ template . In the presence of CAP-cAMP, polymerase binding to P2 is reduced and more polymerase is bound at P1 . Two lac P1 "-35 region" mutations, L157 and 4, which increase the homology between this region and the consensus "-10 region" sequence, are both shown to have an increased affinity for polymerase binding at P2 . CAP-cAMP is also able to decrease the amount of polymerase bound to P2 and to increase the amount bound to P1 on these mutant promoter fragments . P2 does not initiate transcription efficiently in vivo . Nuclease S1 mapping experiments detect only a low level of transcription from one of the P2 "up" mutations, but no beta-galactosidase synthesis is directed by this mutant . Mutations such as L157 and 4, which alter the P2-10 region, also alter lac P sensitivity to CAP-cAMP in vivo, suggesting that the P2 sequence plays a role in CAP-cAMP regulation of lac P . Possible roles for P2 in vivo are discussed.

J Biol Chem, 1985 Oct 5, 260(22), 12313 - 9
The FLP protein of the 2-micron plasmid of yeast . Purification of the protein from Escherichia coli cells expressing the cloned FLP gene; Babineau D et al.; Most laboratory strains of the yeast Saccharomyces cerevisiae contain many copies of an autonomously replicating plasmid called 2-micron circle DNA . This plasmid codes for a site-specific recombinase, the FLP protein which promotes recombination across two 599-base pair inverted repeats of the plasmid DNA . We have cloned the FLP gene under the control of a strong Escherichia coli promoter and have hyperproduced the protein in that organism . Cell-free extracts from this source promote highly efficient site-specific recombination in vitro and we have used this activity to purify the FLP protein substantially . The enzyme acts efficiently on circular and linear substrates and requires only monovalent or divalent cations for activity.

J Biol Chem, 1985 Oct 5, 260(22), 12092 - 8
Molecular cloning and sequencing of the gene for CDP-diglyceride hydrolase of Escherichia coli; Icho T et al.; Previous work from this laboratory had demonstrated that CDP-diglyceride hydrolase of Escherichia coli is encoded by the cdh gene that maps near minute 88 (Bulawa, C . E., and Raetz, C . R . H . (1984) J . Biol . Chem . 259, 11257-11264) . We now report the construction of hybrid plasmids and the sequencing of a 1,243-base pair insert carrying cdh . The further construction of BAL31 deletions of this insert, in conjunction with maxicell experiments and in vitro enzyme assay, has led to the identification of a 756-base pair coding sequence for the cdh polypeptide . The molecular weight of the primary translation product deduced from the DNA sequence of the cdh gene is 28,450, in agreement with maxicell experiments . Parallel purification of the enzyme from extracts of wild-type and overproducing strains confirms the presence of a 27-kDa polypeptide in the overproducer, as judged by polyacrylamide gel electrophoresis of the most purified fractions . Inspection of the DNA sequence reveals a very hydrophobic N-terminal domain that may be either a signal peptide or a special region, anchoring the hydrolase to the membrane . In contrast to the CDP-diglyceride synthetase, the overall amino acid composition of the CDP-diglyceride hydrolase is not extraordinarily hydrophobic . Although both CDP-diglyceride synthetase and CDP-diglyceride hydrolase can transfer the CMP moiety of CDP-diglyceride to a suitable acceptor, the primary structures and mechanisms of action of these two enzymes are very different.

J Biol Chem, 1985 Oct 5, 260(22), 12084 - 91
Purification and properties of the membrane-bound CDP-diglyceride synthetase from Escherichia coli; Sparrow CP et al.; The enzyme CDP-diglyceride synthetase (CTP: phosphatidate cytidylyltransferase; EC 2.7.7.41) has been purified to 90% homogeneity from Escherichia coli cells that overproduce the enzyme 50-fold through the use of recombinant DNA technology . The purification required the use of different detergents at each step, illustrating the refractory hydrophobic nature of this protein . Apparent physical effects of EDTA on the enzyme were also utilized in the purification . The enzyme has an apparent minimum subunit mass of 27,000 daltons, as estimated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate . The amino acid composition of the protein was determined, and it correlates well with the theoretical protein product of the cds gene, the sequence of which is reported in the accompanying paper (Icho, T., Sparrow, C . P., and Raetz, C . R . H . (1985) J . Biol . Chem . 260, 12078-12083) . The pure enzyme displays surface dilution kinetics when assayed in the presence of Triton X-100 . As previously suggested on the basis of studies using partially purified preparations, the enzyme mechanism is sequential, and computer-calculated kinetic constants are reported herein . The substrate specificity of the enzyme is also investigated . This is the first time this enzyme has been purified to homogeneity from any source, despite the fact that it is essential for phospholipid biosynthesis in all organisms.

J Biol Chem, 1985 Oct 5, 260(22), 12078 - 83
Molecular cloning and sequencing of the gene for CDP-diglyceride synthetase of Escherichia coli; Icho T et al.; The cds gene of Escherichia coli codes for the enzyme CDP-diglyceride synthetase . We now report the construction of plasmids which carry cds . Using these plasmids, we have sequenced 1274 base pairs of DNA, including a 750-base pair open reading frame which is the coding region of the cds gene . This DNA sequence allows the deduction of the primary peptide sequence for CDP-diglyceride synthetase . The protein is very hydrophobic, and, assuming no processing or modification, has a molecular weight of 27,570 . Furthermore, there is a second open reading frame immediately after cds, implying that cds may be part of an operon . We have also constructed a runaway replication cds-plasmid that directs approximately 50-fold overproduction of CDP-diglyceride synthetase . This overproduction has been utilized in the purification of the enzyme to homogeneity, as described in the accompanying paper (Sparrow, C.P., and Raetz, C.R.H., J . Biol . Chem . 260, 12084-12091) . Finally, the molecular cloning work reported herein allows the exact placement of the cds gene on the E . coli genetic map.

J Biol Chem, 1985 Oct 5, 260(22), 11994 - 2000
A soluble ATP-dependent system for protein degradation from murine erythroleukemia cells . Evidence for a protease which requires ATP hydrolysis but not ubiquitin; Waxman L et al.; A soluble ATP-dependent system for protein degradation has been demonstrated in reticulocyte lysates, but not in extracts of nucleated cells . We report that extracts of undifferentiated murine erythroleukemia (MEL) cells contain a labile ATP-stimulated proteolytic system . The addition of ATP to MEL cell extracts at alkaline pH enhances degradation of endogenous cell proteins and various radiolabeled exogenous polypeptides from 2-15-fold . Nonhydrolyzable ATP analogs had no effect . In reticulocytes, one role of ATP in proteolysis is for ubiquitin conjugation to protein substrates . MEL cells also contain ubiquitin and extracts can conjugate 125I-ubiquitin to cell proteins; however, this process in MEL cells seems unrelated to protein breakdown . After removal of ubiquitin from these extracts by DEAE- or gel chromatography, the stimulation of proteolysis by ATP was maintained and readdition of purified ubiquitin had no further effect . In addition, these extracts degraded in an ATP-dependent fashion casein whose amino groups were blocked and could not be conjugated to ubiquitin . After gel filtration or DEAE-chromatography of the MEL cell extracts (unlike those from reticulocytes), we isolated a high molecular weight (600,000) ATP-dependent proteolytic activity, which exhibits many of the properties of energy-dependent proteolysis seen in crude cell extracts . For example, both the protease and crude extracts are inhibited by hemin and N-ethylmaleimide and both hydrolyze casein, globin, and lysozyme rapidly and denatured albumin relatively slowly . The protease, like the crude extracts, is also stimulated by UTP, CTP, and GTP, although not as effectively as ATP . Also, nonhydrolyzable ATP analogs and pyrophosphate do not stimulate the protease . Thus, some mammalian cells contain a cytosolic proteolytic pathway that appears independent of ubiquitin and involves and ATP-dependent protease, probably similar to that found in Escherichia coli or mitochondria.

J Biol Chem, 1985 Oct 5, 260(22), 12266 - 72
Mutational analysis of primosome assembly sites . Evidence for alternative DNA structures; Greenbaum JH et al.; Primosome assembly sites are complex DNA structures that share common functions (they elicit the DNA-dependent ATPase of replication factor Y from Escherichia coli and serve as origins of complementary strand DNA synthesis), but display little sequence homology . In order to ascertain a common basis for factor Y-DNA recognition, a primosome assembly site and its mutated derivatives have been functionally and structurally analyzed . Under conditions in which they lose the capacity to function as ATPase effectors these DNA templates have been (i) assayed for their ability to bind factor Y, and (ii) probed, with pancreatic DNase, for structural alterations . In this ATPase-inactivating environment (suboptimal concentrations of MgCl2 and NaCl, and high levels of the E . coli single-stranded DNA binding protein), factor Y does not bind to its cognate DNA and the DNase cleavage pattern characteristic of this site is perceptibly changed: compared to the DNase digest obtained under activating conditions, cleavage is notably decreased in the 5' half of the site and enhanced at the 3' end . The results of this study strongly indicate that the structure of the primosome assembly site under analysis consists of two hairpins which interact with each other . When the sites of pancreatic DNase attack are plotted on the proposed double hairpin structure, the 5' cleavage sites all map to one duplex while the 3' sites map to the other . The observation that, under factor Y ATPase-activating conditions, the 3' hairpin is largely refractory to the action of pancreatic DNase indicates that tertiary interactions between the two duplexes render a portion of the DNA structure inaccessible to the nuclease.

J Biol Chem, 1985 Oct 5, 260(22), 12029 - 34
The role of ATP hydrolysis in the breakdown of proteins and peptides by protease La from Escherichia coli; Goldberg AL et al.; The energy requirement for protein breakdown in Escherichia coli appears to be due to protease La, the lon gene product, which hydrolyzes proteins and ATP in a coupled process . This novel enzyme was investigated with small peptides, identified as substrates in the preceding manuscript . Although the degradation of proteins to acid-soluble material requires hydrolysis of a nucleoside triphosphate, cleavage of small fluorogenic substrates, such as glutaryl-Ala-Ala-Phe-methoxynaphthylamine, was found to require only binding of nucleotides to the enzyme . Nonhydrolyzable analogs of ATP, slowly hydrolyzed nucleotides, and even inorganic triphosphate and pyrophosphate stimulate the breakdown of these peptides but not of large proteins such as casein or serum albumin . In addition, vanadate, an inhibitor of the enzyme's ATPase activity, prevents protein degradation, but vanadate does not inhibit and can even stimulate peptide hydrolysis . Degradation of natural oligopeptides or of small polypeptides (less than 10,000 Da) also does not require hydrolysis of the nucleotide . Furthermore, although protein substrates promote ATP cleavage, the fluorogenic peptides inhibit this process . Also, no evidence was obtained for phosphorylation of the protease or of the substrate during ATP hydrolysis . These findings suggest that protein breakdown involves a cyclical series of reactions: 1) ATP binds to the protease and activates it allosterically, thus allowing peptide bond cleavage; 2) the hydrolysis of ATP must occur subsequently and should prevent further peptide bond cleavage until additional nucleoside triphosphates are bound; 3) with proteins as substrates, this reaction cycle probably occurs repeatedly until small peptides are generated.

J Mol Biol, 1985 Oct 5, 185(3), 525 - 33
Lactose promoter mutation Pr115 activates an overlapping promoter within the lactose control region; Peterson ML et al.; The Escherichia coli lac promoter mutation Pr115, an A X T to T X A transversion at +1 (the transcription initiation site of the lac wild-type and lac UV5 promoters), creates a new "-10 region"-like sequence starting at +1 . We show that this mutation activates a new RNA polymerase binding site (P115) that overlaps with, and is shifted 12 base-pairs downstream from, the wild-type RNA polymerase binding site (P1) . Nuclease S1 mapping studies and RNA polymerase protection experiments in vitro indicate that, in the absence of CAP-cAMP, this new site is used preferentially over the P1 site . In vivo, beta-galactosidase assays of the Pr115 mutation in combination with mutations of the P1 "-35 region" demonstrate that the P1 -35 region sequences are not involved in the interaction between RNA polymerase and P115 in the absence of CAP-cAMP; therefore P115 is an independent binding site . The presence of CAP-cAMP in vivo stimulates polymerase binding and initiation at P1, which serves to block polymerase from binding at P115.

Science, 1985 Oct 4, 230(4721), 78 - 82
A model for the tertiary structure of p21, the product of the ras oncogene; McCormick F et al.; A model was developed for the structure of p21, the protein with a molecular weight of 21,000 that is produced by the ras genes . This model predicts that p21 consists of a central core of beta-sheet structure, connected by loops and alpha helices . Four of these loops comprise the guanine nucleotide binding site . The phosphoryl binding region is made up of amino acid sequences from 10 to 16 and from 57 to 63 of p21 . The latter sequence may contain a site for magnesium binding . Amino acids defining guanine specificity are Asn-116 and Asp-119, and sequences around amino acid 145 may contribute to guanine binding . The model makes it possible to visualize how oncogenic mutations of p21 affect interaction with guanine nucleotides.

Science, 1985 Oct 4, 230(4721), 32 - 6
Structure of the GDP domain of EF-Tu and location of the amino acids homologous to ras oncogene proteins; Jurnak F; A 2.7 angstrom resolution x-ray diffraction analysis of a trypsin-modified form of the Escherichia coli elongation factor Tu reveals that the GDP-binding domain has a structure similar to that of other nucleotide-binding proteins . The GDP ligand is located at the COOH-terminal end of the beta sheet and is linked to the protein via a Mg2+ ion salt bridge . The location of the guanine ring is unusual; the purine ring is located on the outer edge of the domain, not deep within a hydrophobic pocket . The amino acids from Pro10 to Arg44 and from Gly59 to Glu190 have been assigned to the electron density with computer graphic techniques, and the resulting model is consistent with all known biochemical data . An analysis of the structure reveals that four regions of the amino acid sequence that are homologous with the family of ras oncogene proteins, termed p21, are located in the vicinity of the GDP-binding site, and most of the invariant amino acids shared by the proteins interact directly with the GDP ligand.

Jpn J Antibiot, 1985 Oct, 38(10), 2821 - 6
{Transfer of latamoxef into human burn blister fluid and its pharmacokinetic analysis}; Aoyama H et al.; Latamoxef (LMOX) (50 mg/kg) was administrated intravenously to burned patients over 1 hour period . Burn blister fluid and serum were taken during 8 hours after injection, and concentrations of LMOX in burn blister fluid and serum were determined by bioassay using E . coli as a test organism . The serum concentrations of LMOX were 170.8 +/- 30.6 micrograms/ml at 30 minutes, 227.0 +/- 19.8 micrograms/ml at 1 hour, 90.3 +/- 21.4 micrograms/ml at 2 hours, 52.9 +/- 14.6 micrograms/ml at 3 hours, 38.7 +/- 13.3 micrograms/ml at 4 hours, 25.1 +/- 8.1 micrograms/ml at 5 hours, 20.5 +/- 8.1 micrograms/ml at 6 hours, 13.0 +/- 5.5 micrograms/ml (mean +/- S.D., n = 5) at 8 hours after the injection . The LMOX concentrations in burn blister fluid were 36.9 +/- 32.8 micrograms/ml at 30 minutes, 77.5 +/- 42.2 micrograms/ml at 1 hour, 85.4 +/- 19.6 micrograms/ml at 2 hours, 76.4 +/- 18.5 micrograms/ml at 3 hours, 63.5 +/- 17.8 micrograms/ml at 4 hours, 54.9 +/- 17.1 micrograms/ml at 5 hours, 34.8 +/- 10.3 micrograms/ml at 6 hours, 25.2 +/- 4.8 micrograms/ml (mean +/- S.D., n = 7) at 8 hours after the injection . The data obtained were analysed pharmacokinetically . The serum levels were analysed by two-compartment model, and the LMOX levels in burn blister fluid were analysed by the model, in which blister was considered as a small part of the peripheral compartment . In results, Tmax and Cmax of LMOX levels in burn blister fluid were calculated as 1.81 hours and 90.6 micrograms/ml, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Biokhimiia, 1985 Oct, 50(10), 1639 - 45
{Association of eukaryotic aminoacyl-tRNA-synthases with polyribosomes}; Fedorov AN et al.; Upon fractionation of a mitochondria-free extract of rabbit reticulocytes into a ribosome-free extract and mono- and polyribosomes the bulk of the aminoacyl-tRNA synthetase activity was found in the fraction of mono- and polyribosomes . All the fifteen aminoacyl-tRNA synthetases were revealed, although in somewhat different quantities, in both fractions of the mitochondria-free reticulocyte extract . Aminoacyl-tRNA synthetases of the ribosome-free extract are found in two forms: RNA-binding one, and, the one having no affinity for high molecular weight RNAs . Aminoacyl-tRNA synthetases dissociated from the complexes with polyribosomes exist only in the RNA-binding form . All aminoacyl-tRNA synthetases can be removed from such complexes by an addition of 16S rRNA of E . coli, poly(U) or tRNA of rabbit reticulocytes . This testifies to labile association of aminoacyl-tRNA synthetases with the RNA-component of polyribosomes as well as to a rather nonspecific character of their interaction . After EDTA-induced dissociation of polyribosomes, the aminoacyl-tRNA synthetase activity was detected in the complex with both ribosomal subunits.

Microbiol Sci, 1985 Oct, 2(10), 299 - 302
Plasmid partitioning in Escherichia coli; Mann NH; The mechanisms by which plasmids are stably maintained in bacterial populations are not yet completely understood . However, it is apparent that interactions with the cell envelope, site-specific recombination and modulation of the host cell's division process may be involved in plasmid maintenance.

Mol Immunol, 1985 Oct, 22(10), 1145 - 50
Antibodies to the first constant-region domain of the murine mu-chain induced by a synthetic peptide; Ghose AC et al.; A peptide corresponding to the N-terminal 13 amino acid residues of the murine C mu 1 domain was synthesized by the solid-phase method and was coupled to carrier proteins through an additional cysteine residue . Rabbit antisera to these peptide-carrier conjugates were found to react with intact mouse IgM as well as its Fab mu fragment . These antisera also reacted with the isolated mu-chain and the V mu fragment of the heavy chain . This fragment consists of the VH-domain and the N-terminal residues of the C mu 1 domain preceding the interchain half-cystine . No significant reactivity of these antisera was found with the IgM of human and equine species or with murine IgG isotypes . Apart from their utility in the purification of the V mu fragment, these and similar antisera can be used to probe structure and function relationships of immunoglobulin domains . Furthermore, such antisera may be used in the study of expression vectors with heavy-chain genes to detect the expression of truncated forms of heavy chain in E . coli and other hosts.

Food Addit Contam, 1985 Oct-Dec, 2(4), 253 - 7
Detection and quantitation of aflatoxin B1 in orange juice by SOS-Chromotest; Riesenfeld G et al.; A new method for the detection and quantitation of aflatoxin B1 in liquids is described . The method is based on the SOS Chromotest, in which damage caused by aflatoxin B1 to the DNA of suitably engineered E . coli induces beta-galactosidase . Aflatoxin B1 developing in orange juice inoculated with spores of Aspergillus parasiticus is detectable equally well by TLC as by the SOS-Chromotest.

Proc Natl Acad Sci U S A, 1985 Oct, 82(19), 6561 - 5
Identification of a nuclear localization signal of a yeast ribosomal protein; Moreland RB et al.; To identify a signal involved in transporting a ribosomal protein to the nucleus, we constructed hybrid genes encoding amino-terminal segments of yeast ribosomal protein L3 joined to the amino-terminal end of the entire Escherichia coli beta-galactosidase molecule . The subcellular locations of the corresponding hybrid proteins in yeast were determined by in situ immunofluorescence . The first 21 amino acids of L3 were sufficient to localize beta-galactosidase to the nucleus . This region shows limited homology to portions of other nuclear proteins identified as essential for their transport . Larger fusion proteins were also localized to the nucleus . However, a hybrid protein containing all but the 14 carboxyl-terminal amino acids from L3 initially failed to localize; this defect was corrected by inserting a glycine- and proline-containing bridge between the L3 and beta-galactosidase moieties . The renovated protein was able to associate with ribosomes, suggesting that, in addition to entering the nucleus, this hybrid polypeptide was assembled into 60S ribosomal subunits that were subsequently exported to the cytoplasm.

Proc Natl Acad Sci U S A, 1985 Oct, 82(19), 6455 - 9
Conserved features of eukaryotic hsp70 genes revealed by comparison with the nucleotide sequence of human hsp70; Hunt C et al.; We have determined the nucleotide sequence of the human hsp70 gene and 5' flanking region . The hsp70 gene is transcribed as an uninterrupted primary transcript of 2440 nucleotides composed of a 5' noncoding leader sequence of 212 nucleotides, a 3' noncoding region of 242 nucleotides, and a continuous open reading frame of 1986 nucleotides that encodes a protein with predicted molecular mass of 69,800 daltons . Upstream of the 5' terminus are the canonical TATAAA box, the sequence ATTGG that corresponds in the inverted orientation to the CCAAT motif, and the dyad sequence CTGGAAT/ATTCCCG that shares homology in 12 of 14 positions with the consensus transcription regulatory sequence common to Drosophila heat shock genes . Comparison of the predicted amino acid sequences of human hsp70 with the published sequences of Drosophila hsp70 and Escherichia coli dnaK reveals that human hsp70 is 73% identical to Drosophila hsp70 and 47% identical to E . coli dnaK . Surprisingly, the nucleotide sequences of the human and Drosophila genes are 72% identical and human and E . coli genes are 50% identical, which is more highly conserved than necessary given the degeneracy of the genetic code . The lack of accumulated silent nucleotide substitutions leads us to propose that there may be additional information in the nucleotide sequence of the hsp70 gene or the corresponding mRNA that precludes the maximum divergence allowed in the silent codon positions.

J Bacteriol, 1985 Oct, 164(1), 321 - 30
Two modes of control of pilA, the gene encoding type 1 pilin in Escherichia coli; Orndorff PE et al.; Type 1 piliation in Escherichia coli is subject to metastable regulation at the transcriptional level (B . I . Eisenstein, Science 214:337-339, 1981) . However, the genes controlling in this fashion are not known . We present evidence that the pilA gene, encoding the structural subunit of type 1 pili, is subject to metastable transcriptional regulation . A pilA'-lacZ fusion, constructed in vitro on a recombinant plasmid, was used in conjunction with a recBC sbcB mutant of E . coli K-12 to introduce the fusion into the chromosomal region encoding Pil . This fusion was found to be subject to metastable transcriptional control . The rate of switching from the Lac+ to the Lac- phenotype was 4 X 10(-4) per cell per generation and 6.2 X 10(-4) in the opposite direction . A ca . 10-fold difference in beta-galactosidase activity was observed between phenotypically "ON" (Lac+) and "OFF" (Lac-) populations . P1 transduction experiments showed that the element determining the ON or OFF phenotype was tightly linked to pilA . In addition to the metastable regulation of pilA, a second type of transcriptional regulation was effected by the product of a gene, hyp, adjacent to pilA . By using a recombinant plasmid containing just a pilA'-lacZ fusion and the putative pilA promoter, we found that a lesion in hyp conferred a beta-galactosidase activity about fivefold higher than that of a strain possessing the parental hyp gene . Mutants constructed to have a pilA'-lacZ fusion and a hyp::Tn5-132 mutation in the chromosome exhibited a frequency of switching from Lac+ to Lac- and vice versa indistinguishable from that of the parental strain . However, in the ON mode, hyp::Tn5-132 mutants showed a twofold-higher beta-galactosidase activity . Thus, hyp does not appear to affect metastable variation but does affect the level of transcription of the pilA gene in the ON (transcribed) mode.

Chest, 1985 Oct, 88(4 Suppl), 268S - 270S
Vasoconstriction and remodeling in pulmonary hypertension; Meyrick B et al.; In one group of sheep, Escherichia coli endotoxin was given intravenously three times per week for ten weeks, and in another group the cyclooxygenase inhibitor indomethacin was given subcutaneously two times per day for three weeks . Both groups developed the structural and functional changes of modest but sustained pulmonary hypertension and showed granulocyte sequestration in the peripheral lung . Indomethacin enhanced pulmonary vasoreactivity, but endotoxin depressed reactivity transiently . Prolonged inflammation of the lung may be associated with alterations in vasoreactivity and the development of chronic pulmonary hypertension.

J Biomol Struct Dyn, 1985 Oct, 3(2), 281 - 97
Use of "loss-of-contact" substitutions to identify residues involved in an amino acid-base pair contact: effect of substitution of Gln18 of lac repressor by Gly, Ser, and Leu; Ebright RH; A procedure to identify which base pair of lac operator (lacO) a suspected contacting amino acid of Lac repressor (LacR) interacts with is presented . The procedure is to eliminate the ability of the amino acid under study to contact DNA, and then to determine at which base pair--if any--specificity is eliminated . To implement this procedure, four sets of Escherichia coli K-12 strains have been constructed . These strains permit: (i) the substitution of a selected amino acid of LacR by, respectively, Gly, Ser, Leu, or Gln, and (ii) the analysis of the specificity of the resulting substituted LacR with respect to base pairs 5, 6, 7, 8, 9, and 10 of lacO . This procedure has been applied to Gln18 of LacR . The preliminary data indicate that LacR (Gln18----Gly) is unable to distinguish between the O+ base pair G:C and the Oc base pair T:A at position 7 of lacO (KDOc/KDO+ = 0.93) . In contrast, LacR(Gln18----Gly) discriminates O+ from Oc by a factor of 13 to 23 at each other position . The same qualitative pattern of results was obtained with LacR(Gln18----Ser) and LacR (Gln18----Leu) . Therefore, I propose that Gln18 contacts base pair 7 of lacO . This proposal is consistent with the contact predicted in Ebright, R . in Protein Structure, Folding, and Design . D . Oxender ed., Alan R . Liss, New York (1985), in press.

J Biomol Struct Dyn, 1985 Oct, 3(2), 387 - 408
Temperature jump relaxation studies on the interactions between transfer RNAs with complementary anticodons . The effect of modified bases adjacent to the anticodon triplet; Houssier C et al.; We have used the temperature-jump relaxation technique to determine the kinetic and thermodynamic parameters for the association between the following tRNAs pairs having complementary anticodons: tRNA(Ser) with tRNA(Gly), tRNA(Cys) with tRNA(Ala) and tRNA(Trp) with tRNA(Pro) . The anticodon sequence of E . coli tRNA(Ser), GGA, is complementary to the U*CC anticodon of E . coli tRNA(Gly(2} (where U* is a still unknown modified uridine base) and A37 is not modified in none of these two tRNAs . E . coli tRNA(Ala) has a VGC anticodon (V is 5-oxyacetic acid uridine) while tRNA(Cys) has the complementary GCA anticodon with a modified adenine on the 3' side, namely 2-methylthio N6-isopentenyl adenine (mS2i6A37) in E . Coli tRNA(Cys) and N6-isopentenyl adenine (i6A37) in yeast tRNA(Cys) . The brewer yeast tRNA(Trp) (anticodon CmCA) differs from the wild type E . coli tRNA(Trp) (anticodon CCA) in several positions of the nucleotide sequence . Nevertheless, in the anticodon loop, only two interesting differences are present: A37 is not modified while C34 at the first anticodon position is modified into a ribose 2'-O methyl derivative (Cm) . The corresponding complementary tRNA is E.coli tRNA(Pro) with the VGG anticodon . Our results indicate a dominant effect of the nature and sequence of the anticodon bases and their nearest neighbor in the anticodon loop (particularly at position 37 on the 3' side); no detectable influence of modifications in the other tRNA stems has been detected . We found a strong stabilizing effect of the methylthio group on i6A37 as compared to isopentenyl modification of the same residue . We have not been able so far to assess the effect of isopentenyl modification alone in comparison to unmodified A37 . The results obtained with the complex yeast tRNA(Trp)-E.coli tRNA(Pro) also suggest that a modification of C34 to Cm34 does not significantly increase the stability of tRNA(Trp) association with its complementary anticodon in tRNA(Pro) . The observations are discussed in the light of inter- and intra-strand stacking interactions among the anticodon triplets and with the purine base adjacent to them, and of possible biological implications.

Mol Cell Biol, 1985 Oct, 5(10), 2860 - 5
Production of human c-myc protein in insect cells infected with a baculovirus expression vector; Miyamoto C et al.; A cDNA fragment coding for human c-myc was inserted into the genome of the baculovirus Autographa californica nuclear polyhedrosis virus adjacent to the strong polyhedrin promoter . Insect cells infected with the recombinant virus produced significant amounts of c-myc protein, which constituted the major phosphoprotein component in these cells . By immunoprecipitation and immunoblot analysis, two proteins of 61 and 64 kilodaltons were detected with c-myc-specific antisera . The insect-derived proteins were compared with recombinant human c-myc-encoded proteins synthesized in Escherichia coli and Saccharomyces cerevisiae cells . The c-myc gene product was found predominantly in the nucleus by subcellular fractionation of infected insect cells.

Avian Dis, 1985 Oct-Dec, 29(4), 1108 - 17
In vitro and in vivo characterization of avian Escherichia coli . III . Immunization; Rosenberger JK et al.; Broiler breeder hens were vaccinated once at 20 weeks or twice at 20 and 25 weeks of age with a formalin-inactivated oil-emulsion Escherichia coli bacterin composed of serogroups O2, O78, and O35 . Serological responses as assessed by microagglutination documented an increase in serotype-specific antibody in vaccinated birds . Challenge of progeny from vaccinates and nonvaccinates with homologous E . coli demonstrated that maternally derived antibody could protect against mortality and/or lesions for as long as 2 weeks post-hatching.

J Microsc, 1985 Oct, 140 ( Pt 1), 55 - 63
Low temperature embedding with Lowicryl resins: two new formulations and some applications; Carlemalm E et al.; Lowicryl K4M and HM20 are methacrylate/acrylate based low temperature embedding resins for biological material which can be used in conjunction with either the progressive lowering of temperature (PLT) technique or with freeze-substitution . K4M and HM20 are applicable over a very extended temperature range, approximately 220 K to 340 K . With two new resins, K11M and HM23, one can reach even lower temperatures, c . 200 K . Freeze-substitution combined with low temperature embedding allows for very mild or no chemical fixation which seems to increase the sensitivity of immunocytochemical localization of antigens on sections.

Zh Mikrobiol Epidemiol Immunobiol, 1985 Oct, (10), 69 - 76
{Changes in the oxygen-dependent metabolic activity of polymorphonuclear leukocytes from the peripheral blood and macrophages from the peritoneal exudate in the experimental infection of mice with the causative agent of plague}; Isin ZhM; The subcutaneous infection of C57BL/6J mice and noninbred white mice with 40 LD100 of Y . pestis virulent strain has been found to produce synchronous changes in the oxygen-dependent metabolism (ODM) of peripheral blood neutrophils in the spontaneous or zymosan-, E . coli- and Y . pestis-stimulated variants of the NBT test . These changes can be divided into three phases: (I) the phase of a sharp drop in ODM activity; (II) the phase of the increase of this activity, occurring simultaneously with the penetration of Y . pestis cells into the blood stream; and (III) the phase of the terminal decrease of ODM activity as the cytotoxic lesion of phagocytic cells occurs . Peritoneal exudate macrophages show a more gradual decrease in ODM activity . The infection of the animals with 40,000 LD100 of Y . pestis has been found to produce an increase in the ODM activity of neutrophils, rapidly followed by its decrease to the zero level . Macrophages show phasic changes in their ODM activity, identical to changes in the ODM values of neutrophils in mice infected with 40 LD100 of Y . pestis.

Zh Mikrobiol Epidemiol Immunobiol, 1985 Oct, (10), 66 - 9
{Mechanisms of the mediated neutrophil reactivity to Escherichia coli peptidoglycan}; Azov NA et al.; The opsonic properties of normal serum with respect to E . coli peptidoglycan was studied under the actual conditions of the oxygen-dependent metabolism of neutrophils . In the course of the differentiated study of the influence of antibodies, the classical and the alternative cascades of complement the serum was heated, treated with ethylenediaminetetraacetate and ethylene glycol tetraacetate, exhausted in the cold . In serial experiments the opsonic activity of purified fibronectin was studied . The indirect reactions were shown to be the leading mechanisms of the neutrophil-stimulated activity of E . coli peptidoglycan . IgG was found to be in the center of the opsonic cooperation and thus to determine the quantitative manifestation of the total phenomenon . Complement proved to be of lesser importance; depending on the conditions of the experiment, the activation of complement occurred by the alternative way (after the removal of antibodies) or the classical way (whole serum) . The actual contribution of IgG-independent and complement-independent opsonins was insignificant . Fibronectin in physiological concentrations showed no opsonic activity.

Environ Health Perspect, 1985 Oct, 62, 329 - 35
Is 2,3,7,8-TCDD (dioxin) a carcinogen for humans?
Ayres SM, Webb KB, Evans RG, Mikes J.
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) has suddenly become the focal point of controversy over the relationship of chemical waste to human health . Specific concern exists regarding its potential association with human malignancy . Subcellular, cellular, and whole-animal experiments suggest that TCDD exerts much of its activity by inducing enzymes that protect the intact organism from the assault of environmental contamination . TCDD is a potent inducer of aryl hydrocarbon hydroxylase, although wide variations between species do exist . Conventional tests for mutagenicity have produced conflicting results . Animal experiments have shown the development of tumors following chronic low level ingestion of TCDD . The human evidence regarding the potential carcinogencity of TCDD comes from occupational, military and environmental exposures . Several studies have come out of Sweden suggesting an association between sarcoma and exposure to herbicides . Although there is little solid evidence that 2,3,7,8-TCDD produces substantial chronic disability or premature death in man, a significant body of experimental evidence for its carcinogenicity makes it likely that a small number of human malignancies may be due to its action . Since 2,3,7,8-TCDD is an unwanted contaminant it could be eliminated with little measurable consequence.

Environ Health Perspect, 1985 Oct, 62, 171 - 6
Extrachromosomal probes for mutagenesis by carcinogens: studies on the mutagenic activity of O6-methylguanine built into a unique site in a viral genome; Essigmann JM et al.; This work examines the mutagenic activity of O6-methylguanine (O6MeGua), a DNA adduct formed by certain carcinogenic alkylating agents . A tetranucleotide, 5'-HOTpm6GpCpA-3', was synthesized and ligated into a four-base gap in the unique Pst I site of the duplex genome of the E . coli virus, M13mp8 . The double-stranded ligation product was converted to single-stranded form and used to transform E . coli to produce progeny phage . The mutation frequency of O6MeGua was defined as the percentage of progeny phage with mutations in their Pst I site, and this value was determined to be 0.4% . To determine the impact of DNA repair on mutagenesis, cellular levels of O6MeGua-DNA methyltransferase (an O6MeGua-repair protein) were depleted by treatment of host cells for virus replication with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) prior to viral DNA uptake . In these host cells, the mutation frequency due to O6MeGua increased markedly with increasing MNNG dose (the highest mutation frequency observed was 20%) . DNA sequence analysis of mutant genomes revealed that in both MNNG treated and untreated cells, O6MeGua induced exclusively G to A transitions.

Environ Health Perspect, 1985 Oct, 62, 163 - 9
Quantitative comparison of genetic effects of ethylating agents on the basis of DNA adduct formation . Use of O6-ethylguanine as molecular dosimeter for extrapolation from cells in culture to the mouse; van Zeeland AA et al.; DNA-adduct formation and induction of gene mutations were determined simultaneously after treatment with the four ethylating agents, ethyl methanesulfonate (EMS), ethylnitrosourea (ENU), diethyl sulfate (DES), and N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG) . Both, in E . coli K-12 (NAL-resistance) and in V79 Chinese hamster cells in culture (HPRT-deficiency), the frequencies of mutation induction by all chemicals were the same when plotted against the amount of O6-ethylguanine formed in DNA, suggesting that this DNA adduct can be used as a common dosimeter for the comparisons of the frequencies of gene mutations induced by ethylating agents in various mutagenicity assay systems . Using ENU, such a comparison was performed between mutation induction in V79 cells in vitro and in the specific-locus assay in the mouse . The data indicate that at equal levels of O6-ethylguanine in the DNA of V79 cells and in testicular DNA from male mice treated with ENU, the frequencies of induced mutants in both assay systems were quite similar . These results support the concept that the determination of premutagenic DNA adducts in vivo can be used to monitor exposure to chemical mutagens and that genetic risk estimations may ultimately be performed on the basis of such measurements and of comparative mutagenesis in vitro and in vivo.

Environ Health Perspect, 1985 Oct, 62, 115 - 7
Cellular responses to DNA damage; Walker GC et al.; For many years, the study of the regulation of the SOS network was complicated by both the complexities of the responses and the interrelationships of the key regulatory elements . However, recently the application of powerful genetic and molecular biological techniques has allowed us to gain a detailed picture of the regulation of this complex network . The network is now known to consist of more than 17 genes, each of which is repressed by the LexA protein . Induction of the genes in the SOS network occurs when the RecA protein becomes activated in response to a signal generated by DNA damage . Two of the genes in this network, umuD and umuC, are absolutely required for mutagenesis by UV and various carcinogens . The umuD and umuC genes have molecular weights of 16,000 and 45,000 daltons, respectively, and are organized in an operon repressed by LexA . The mutagenesis-enhancing plasmid pKM101 carries two genes mucA and mucB, which are analogs of the umuD and umuC genes, respectively.

Anal Biochem, 1985 Oct, 150(1), 243 - 8
Bulk preparation and crystallization of the Escherichia coli elongation factor Tu-Ts complex; Yoder M et al.; A simple procedure for the preparation of 10-500 mg of the Escherichia coli elongation Tu-Ts complex is described . The protocol is based on the separate purification and quantitation of EF-Tu-GDP and EF-Ts, followed by mixing of equimolar amounts of each protein and removal of the displaced GDP by dialysis . Single crystals grown from the final product have been analyzed by X-ray diffraction techniques . The procedure is also applicable to the bulk preparation and crystallization of the trypsin-modified Tu-Ts complex . Quantitation of the elongation factors by three methods is presented.

Anal Biochem, 1985 Oct, 150(1), 121 - 4
beta-Hydroxydecanoylthioester dehydrase: a rapid, convenient, and accurate product distribution assay; Schwab JM et al.; High-performance liquid chromatography on silica gel has been used to separate the products from incubation of substrates with beta-hydroxydecanoylthioester dehydrase (Escherichia coli) . Peaks are detected by their absorbances at 230 nm . Following correction for differences in extinction coefficients, comparison of the peak areas reveals the relative amounts of beta-hydroxydecanoate, E-2-decenoate, and Z-3-decenoate thioesters of N-acetylcysteamine.

Mol Cell Biochem, 1985 Oct, 68(2), 121 - 30
Redox interconversion of Escherichia coli glutathione reductase . A study with permeabilized and intact cells; Mata AM et al.; The redox interconversion of Escherichia coli glutathione reductase has been studied both in situ, with permeabilized cells treated with different reductants, and in vivo, with intact cells incubated with compounds known to alter their intracellular redox state . The enzyme from toluene-permeabilized cells was inactivated in situ by NADPH, NADH, dithionite, dithiothreitol, or GSH . The enzyme remained, however, fully active upon incubation with the oxidized forms of such compounds . The inactivation was time-, temperature-, and concentration-dependent; a 50% inactivation was promoted by just 2 microM NADPH, while 700 microM NADH was required for a similar effect . The enzyme from permeabilized cells was completely protected against redox inactivation by GSSG, and to a lesser extent by dithiothreitol, GSH, and NAD(P)+ . The inactive enzyme was efficiently reactivated in situ by physiological GSSG concentrations . A significant reactivation was promoted also by GSH, although at concentrations two orders of magnitude below its physiological concentrations . The glutathione reductase from intact E . coli cells was inactivated in vivo by incubation with DL-malate, DL-isocitrate, or higher L-lactate concentrations . The enzyme was protected against redox inactivation and fully reactivated by diamide in a concentration-dependent fashion . Diamide reactivation was not dependent on the synthesis of new protein, thus suggesting that the effect was really a true reactivation and not due to de novo synthesis of active enzyme . The glutathione reductase activity increased significantly after incubation of intact cells with tert-butyl or cumene hydroperoxides, suggesting that the enzyme was partially inactive within such cells . In conclusion, the above results show that both in situ and in vivo the glutathione reductase of Escherichia coli is subjected to a redox interconversion mechanism probably controlled by the intracellular NADPH and GSSG concentrations.

J Clin Microbiol, 1985 Oct, 22(4), 637 - 40
Determination of immunoglobulin M antibodies for hepatitis B core antigen with a capture enzyme immunoassay and biotin-labeled core antigen produced in Escherichia coli; Vilja P et al.; A new capture enzyme immunoassay for the determination of immunoglobulin M (IgM) antibodies against hepatitis B core antigen (HBcAg) is described . Core antigen produced in Escherichia coli was labeled with biotin and subsequently detected by an avidin-biotin-peroxidase complex . The biotin-labeled core antigen was effective at concentrations as low as 20 ng/ml . Of 561 serum samples from different groups of patients that were tested, 465 samples were negative for other hepatitis B virus markers and also for anti-HBcAg IgM . Sera from the early stages of hepatitis B infection had high levels of anti-HBcAg IgM, and a clear correlation with the acuteness of the disease was observed in 45 follow-up sera from 23 patients with acute or recent hepatitis B . Sera from 21 patients with past hepatitis B were all negative for anti-HBcAg IgM . Twenty serum samples from chronic carriers of hepatitis B surface antigen showed slightly elevated antibody levels for anti-HBcAg IgM . Ten sera which were positive for anti-HBcAg IgG antibodies and had high levels of rheumatoid factor were negative for anti-HBcAg IgM.

J Clin Microbiol, 1985 Oct, 22(4), 626 - 8
Evaluation of antisera used for detecting enterotoxigenic Escherichia coli in Sao Paulo; Guth BE et al.; The usefulness of antisera in detecting enterotoxigenic Escherichia coli (ETEC) strains in Sao Paulo was evaluated . Polyvalent antisera detected 49% of ETEC isolates and were more effective in identifying E . coli that produced heat-labile and heat-stable enterotoxins and in strains that produced only heat-stable enterotoxin . ETEC strains not detected by the antisera belonged to different serogroups not isolated in Sao Paulo before; 34% of these strains had undetermined O antigens, and most of them produced only heat-labile toxin . A variation of serogroups over time was especially observed among strains that produced heat-stable toxin . The importance of H-antigen determinations in the effectiveness of ETEC diagnosis by serological methods became evident, as non-ETEC strains were also detected by polyvalent antisera, but their serotypes were different from those of ETEC strains . Although antisera can be used to identify O:H types of ETEC strains with accuracy, serotyping cannot be recommended for routine diagnosis . However, such a procedure may be useful for studying outbreaks of ETEC diarrhea if the involved serotypes are already known.

J Clin Microbiol, 1985 Oct, 22(4), 576 - 81
Enterotoxigenic Escherichia coli in a population of infants with diarrhea in Chile; Aguero ME et al.; The incidence of enterotoxigenic Escherichia coli (ETEC) was investigated in 95 E . coli strains isolated from 48 infants with diarrhea in Santiago, Chile . By using standard biological assays and DNA-DNA hybridization procedures, ETEC was found in 31.2% of the cases: 14 strains produced heat-stable enterotoxin (ST) only, three strains produced heat-labile enterotoxin (LT) and ST, and two strains produced LT only . DNA probes detected all enterotoxin producers except one ST-producing strain . The ST strains hybridized with one or both of the human ST probes (ST Ib and ST A2) . Two of the LT-ST strains hybridized with the ST Ia and ST Ib probes, and the third strain did not hybridize with any of the ST probes . Only the ST group expressed multiple resistance (85.7%) and colonization factor antigen I (CFA I) (92.8%); CFA II was found in two of three LT-ST strains . The O153:H45 serotype was found in 10 of 14 ST strains, and O6:K15:H16 was found in one LT strain and in two LT-ST strains . These findings suggest that ETEC, especially strains that produce ST, may be an important cause of diarrhea among Chilean infants.

J Biochem (Tokyo), 1985 Oct, 98(4), 921 - 6
A simple preparation method for apoaspartate aminotransferase from Escherichia coli B, and its application for the assay of pyridoxal and pyridoxamine 5'-phosphate; Yagi T et al.; A simple and rapid preparation method for apoaspartate aminotransferase from Escherichia coli B was developed . A crude extract of the bacterial cells was treated batchwise with DEAE-cellulose . The enzyme fraction obtained was then applied to a pyridoxamine-Sepharose column . Apoaspartate aminotransferase was eluted with 50 mM potassium phosphate buffer (pH 7.0), and found to be electrophoretically homogeneous . The apoenzyme preparation thus obtained showed very low holoenzyme activity (only 0.4% of the activity seen in the fully saturated condition with pyridoxal 5'-phosphate) and was successfully used for assaying pyridoxal and pyridoxamine 5'-phosphate.

J Biochem (Tokyo), 1985 Oct, 98(4), 1117 - 25
Purification and characterization of lysophospholipase L2 of Escherichia coli K-12; Karasawa K et al.; Lysophospholipase L2, which is bound to the inner membrane of Escherichia coli K-12, was produced in a large amount in cells bearing its cloned structural gene . Starting from these cells, the lysophospholipase L2 was purified approximately 700-fold to near homogeneity by solubilization with KCl, ammonium sulfate fractionation, chromatofocusing in the presence of a zwitterionic detergent, CHAPS (3-{(3-cholamidopropyl)dimethylammonio}-1-propanesulfonate), and heparin-Sepharose affinity column chromatography . The final preparation showed a single protein band with a molecular weight of 38,500 daltons in SDS-polyacrylamide gel electrophoresis . The amino acid sequence of the NH2-terminal portion of the purified enzyme was determined . It was in complete agreement with that deduced from the nucleotide sequence of the structural gene, pldB {Kobayashi, T., Kudo, I., Karasawa, K., Mizushima, H., Inoue, K., & Nojima, S . (1985) J . Biochem . 98, 1017-1025.} The purified enzyme hydrolyzes 2-acyl glycerophosphoethanolamine (GPE) and 2-acyl glycerophosphocholine (GPC) more effectively than 1-acyl GPE and 1-acyl GPC, but does not attack diacylphospholipids . The enzyme also catalyzes the transfer of an acyl group from lysophospholipid to phosphatidylglycerol for formation of acyl phosphatidylglycerol . The acyl group was more effectively transferred from 2-acyl lysophospholipid than from the 1-acyl derivative . This enzyme was heat-labile and was inactivated at 55 degrees C within 5 min . The present paper shows clearly that lysophospholipase L2 is a different enzyme protein from lysophospholipase L1 which was formerly purified from the supernatant of the wild strain of E . coli K-12 homogenates {Doi, O . & Nojima, S . (1975) J . Biol . Chem . 250, 5208-5214}.

J Biochem (Tokyo), 1985 Oct, 98(4), 1017 - 25
Nucleotide sequence of the pldB gene and characteristics of deduced amino acid sequence of lysophospholipase L2 in Escherichia coli; Kobayashi T et al.; The nucleotide sequence of the pldB gene of Escherichia coli K-12, which codes for lysophospholipase L2 located in the inner membrane, was determined . The deduced amino acid sequence of lysophospholipase L2 contains 340 amino acid residues, resulting in a protein with a molecular weight of 38,934 . It is characterized by a high content of arginine residues (36 out of 340 residues) . The amino acid sequence near the NH2-terminus of the protein is composed of a large number of polar or charged amino acid residues, suggesting that this region cannot be a signal peptide . The hydropathy profile of the deduced amino acid sequence of lysophospholipase L2 was studied . Most of the region was rather hydrophilic, and there was no stretch of hydrophobic amino acid region, such as might be predicted to traverse the lipid bilayer . These results are consistent with the experimental observation that lysophospholipase L2 is extracted by salt solution from the membrane fraction, and it may be classified as a peripheral membrane protein . Computer analysis showed that there is no homology in amino acid sequences between lysophospholipase L2 and other extracellular phospholipases, as well as detergent-resistant phospholipase A, which is another membrane-bound phospholipase in E . coli and whose DNA sequence was determined (Homma, H., Kobayashi, T., Chiba, N., Karasawa, K., Mizushima, H., Kudo, I., Inoue, K., Ideka, H., Sekiguchi, M., & Nojima, S . (1984) J . Biochem . 96, 1655-1664) . This is the first report of the primary structure of a lysophospholipase.

DNA, 1985 Oct, 4(5), 351 - 5
Nucleotide sequence of the partition function of Escherichia coli plasmid ColE1; Leung DW et al.; The DNA nucleotide sequence of a 382-bp Hpa II fragment containing cer (ColE1 resolution) function responsible for ColE1 plasmid stability in dividing Escherichia coli was determined . The partition (par) region of pSC101 and the cer region have similar biological functions, as they both maintain plasmid stability through plasmid monomerization . Both regions contain 40- to 70-bp hairpin-loop structures that resemble bidirectional transcription terminators and share sequence homology with each other . Deletion mapping of the cer fragment shows that sequences extending beyond both sides of the terminator-like structure are also involved in the plasmid partition process.

Microbiologica, 1985 Oct, 8(4), 391 - 4
Enhancement of mutagenesis in gamma ray exposed Escherichia coli by plasmids of different incompatibility groups; Eftimiadi C et al.; We have analyzed the correlation between plasmid incompatibility group and enhanced mutagenesis in gamma ray exposed Escherichia coli WP2 . The data reported indicate that plasmids of different incompatibility groups are involved in DNA repair mechanisms and suggest that the damage of the DNA produced by gamma rays or other physical agents and by chemical mutagens stimulates a common mechanism of bacterial DNA repair.

J Gen Microbiol, 1985 Oct, 131 ( Pt 10), 2847 - 50
Antigenic alteration of contaminating lipopolysaccharide during extraction of Escherichia coli outer-membrane proteins from polyacrylamide gels; Chart H et al.; An antiserum raised to the ferric enterobactin receptor protein of Escherichia coli, isolated from SDS-polyacrylamide gels, contained high-titre antibodies to the lipopolysaccharide (LPS) of E . coli O111 . This antiserum was used to show that proteins dissected from polyacrylamide gels can be contaminated with comigrating LPS at levels below those detectable by very sensitive silver staining methods . Using this antiserum it was also shown that the procedures used to extract proteins from polyacrylamide gels can alter the molecular structure and, consequently, the antigenic properties of the contaminating LPS.

J Gen Microbiol, 1985 Oct, 131 ( Pt 10), 2771 - 82
Isolation, characterization and complementation analysis of nirB mutants of Escherichia coli deficient only in NADH-dependent nitrite reductase activity; MacDonald H et al.; Mutants have been isolated which lack NADH-dependent nitrite reductase activity but retain NADPH-dependent sulphite reductase and formate hydrogenlyase activities . These NirB- strains synthesize cytochrome c552 and grow normally on anaerobic glycerol-fumarate plates . The defects map in a gene, nirB, which is extremely close to cysG, the gene order being crp, nirB, cysG, aroB . Complementation studies established that nirB+ and cysG+ can be expressed independently . The data strongly suggest that nirB is the structural gene for the 88 kDal NADH-dependent nitrite oxidoreductase apoprotein (EC 1.6.6.4) . The nirB gene is apparently defective in the previously described nirD mutant, LCB82 . The nirH mutant, LCB197, was unable to use formate as electron donor for nitrite reduction, but NADH-dependent nitrite reductase was extremely active in this strain and a normal content of cytochrome c552 was detected . Strains carrying a nirE, nirF or nirG mutation gave normal rates of nitrite reduction by glucose, formate or NADH.

J Gen Microbiol, 1985 Oct, 131 ( Pt 10), 2753 - 7
Isolation and mapping of Escherichia coli K12 mutants defective in phenylacetate degradation; Cooper RA et al.; Mutants of Escherichia coli K12 unable to grow on phenylacetate have been isolated and mapped . The mutations were located in the relatively 'silent' region of the E . coli K12 chromosome at min 30.4 on the genetic map, with the gene order rac pac-1 pac-2 trg.

J Lipid Res, 1985 Oct, 26(10), 1277 - 82
Chemical synthesis of all-trans retinoyl beta-glucuronide; Barua AB et al.; All-trans retinoyl fluoride reacts with the 3,6-lactone of glucuronic acid in slightly alkaline solution to give the lactone of retinoyl beta-glucuronide, along with other retinoyl glucurono-lactones, in about 60% yield . Hydrolysis of the lactone with very dilute alkali gives the free acid, retinoyl beta-glucuronide, in about 80% yield . Pure all-trans retinoyl beta-glucuronide (overall yield: 20-25%) was obtained free from other isomeric and anomeric forms by reverse-phase high pressure liquid chromatography . Retinoyl beta-glucuronide was characterized by UV-visible, infrared, and 1H-NMR spectra, by elementary analysis, by mass spectra, and by its susceptibility to hydrolysis by bacterial beta-glucuronidase.

J Hyg (Lond), 1985 Oct, 95(2), 353 - 61
Toxin production and haemagglutination in strains of Escherichia coli from diarrhoea in Brescia, Italy; Bisicchia R et al.; Two hundred and ninety-nine different strains of Escherichia coli, isolated from 172 patients with diarrhoea and 113 healthy subjects, were examined for enterotoxin, cytotoxin and haemolysin (Hly) production and for mannose-resistant haemagglutination (MRHA) and invasive properties . Three strains proved enterotoxigenic, none enteroinvasive; cytotoxin and Hly production was shown in 25 strains from patients and in 3 from controls . Ten strains produced the cytotoxic necrotizing factor (CNF), 6 released other factors which kill cell cultures . Hly production was shown in 21 strains, 9 of which were also positive for CNF . MRHA was detected in 26% of strains from diarrhoea compared with 14% of strains from healthy people . A strong association between toxin production and MRHA was demonstrated . Serotyping results showed that the strains exhibiting virulence traits mostly belonged to serogroups commonly involved in extra-intestinal infections . The possible role of strains of E . coli showing one or more virulence factors as opportunistic pathogens in diarrhoeal diseases is discussed.

Biochem J, 1985 Oct 1, 231(1), 209 - 12
Preliminary results for the primary structure of bacterioferritin of Escherichia coli; Tsugita A et al.; Bacterioferritins are type-b cytochromes which resemble ferritin . Amino acid analysis combined with chemical modification and partial sequence analysis characterize bacterioferritin of Escherichia coli in terms of its primary structure . It is a protein composed of one kind of polypeptide chain that commences with methionine and terminates with glutamic acid . The length of the polypeptide chain is, tentatively, 146 residues . Besides the N-terminal methionine residue there are three more methionine residues, which yield four CNBr peptides, which have been aligned . The identity of the following positions in the sequence has been ascertained: residues 1-25, 30-37, 83-88, 127-132 and 143-146 . No homology with ferritin was found.

Am J Vet Res, 1985 Oct, 46(10), 2157 - 62
Effect of intrauterine infusion of Escherichia coli endotoxin in postpartum pony mares; Blanchard TL et al.; Fifteen pony mares were assigned to 1 of 3 treatment groups after foaling: Group 1, 35 ml of sterile saline solution was infused into the uterine lumen within 24 hours after parturition (6 mares); group 2, 300 mg of Escherichia coli endotoxin was infused into the uterine lumen within 24 hours after parturition (6 mares); and group 3, 300 mg of E coli endotoxin was infused into the uterine lumen between 72 and 96 hours after parturition (3 mares) . Rectal temperatures were taken at -1, -0.5, 0, 0.5, 1, 1.5, 2, 3, 4, and 5 hours after treatment . Venous blood samples were also taken at these times for routine WBC counts . Data were analyzed as a repeated measurement design with linear and quadratic orthogonal contrasts performed where significant time and interaction with time occurred . Pretreatment averages of total WBC and neutrophil counts were compared with their nadir posttreatment averages by a t test when treatment-by-time interaction was significant for the parameter . Rectal temperature (37.9 +/- 0.1 C) remained stable and did not vary among treatment groups after intrauterine infusions . In contrast, total WBC and neutrophil counts did vary among treatment groups across time . However, for treatment groups 1 and 3, neither blood total WBC count nor neutrophil count after intrauterine infusions was different from pretreatment observations . In group 2, total WBC count decreased (P less than 0.10) from a pretreatment average of 11.5 +/- 0.4 X 10(3) cells/mm3 to a nadir concentration of 10.0 +/- 0.6 X 10(3) cells/mm3 by 60 minutes after infusion of endotoxin into the uterus.(ABSTRACT TRUNCATED AT 250 WORDS)

Am J Vet Res, 1985 Oct, 46(10), 2067 - 71
Effects of Escherichia coli heat-stable enterotoxin, atropine, clonidine, and morphine on chloride efflux from isolated enterocytes; Ahrens FA et al.; The effects of Escherichia coli heat-stable enterotoxin (ST) on chloride efflux rate were investigated in 3 fractions of enterocytes isolated in a villus-to-crypt gradient from porcine jejunum . There was no difference in chloride efflux rates between mature and immature cells from controls . Heat-stable enterotoxin significantly increased chloride efflux in all fractions . Morphine inhibited ST-augmented secretion in mature enterocytes . Atropine or clonidine had no effect . Calcium efflux rates and glucose or glutamic acid metabolism were not altered by ST . The results indicate that ST may stimulate chloride secretion in both villus and crypt cells and that opiates inhibit intestinal secretion by a direct action on villus epithelial cells.

Mol Pharmacol, 1985 Oct, 28(4), 364 - 70
Purification and characterization of some metabolic effects of S-neplanocylmethionine; Keller BT et al.; Our laboratory has previously demonstrated that treatment of mouse L929 cells with 1 microM neplanocin A results in the metabolic formation of S-neplanocylmethionine (Keller, B.T., and R.T . Borchardt, Biochem . Biophys . Res . Commun . 120:131-137 (1984} . The present study describes an efficient procedure for the purification of this analog from L cells based on its inherent chemical stability in alkaline conditions . Several metabolic effects of S-neplanocylmethionine are also reported . In L cells, S-neplanocylmethionine was determined to have an apparent half-life of 13 hr compared to 1 hr for S-adenosylmethionine during the initial 2 hr of a cycloleucine block . Analysis of polyamine levels in neplanocin A-treated cells showed a 3.8-fold decrease in putrescine and a 1.7-fold decrease in spermidine by 24 hr, reflecting a decrease in the cell growth rate in response to neplanocin A rather than a direct effect of S-neplanocylmethionine on the cellular S-adenosylmethionine decarboxylase . Consistent with these results are our findings that S-neplanocylmethionine does not significantly inhibit purified rat prostate or Escherichia coli S-adenosylmethionine decarboxylase and that {carboxy-14C}S-neplanocylmethionine exhibits no substrate activity with either enzyme . Purified S-neplanocylmethionine was observed to be a weak inhibitor of both S-adenosylmethionine-dependent protein carboxymethyltransferase and lipid methyltransferase in L cell extracts, having an IC50 value of 205 microM (S-adenosylmethionine = 10 microM) . Similar studies with {methyl-3H}S-neplanocylmethionine indicate that the analog has little substrate activity in these two L cell methylation reactions and thus appears to act as a poor competitive inhibitor.

J Bone Joint Surg Am, 1985 Oct, 67(8), 1195 - 201
Infection about the spine associated with low-velocity-missile injury to the abdomen; Romanick PC et al.; We reviewed the cases of twenty patients who had a fracture or disruption of the disc space of a lower thoracic or lumbar vertebra that was associated with a low-velocity-missile wound to the abdomen . All of the patients underwent an exploratory laparotomy at the time of admission and all received broad-spectrum antibiotics for a minimum of two days . None of the patients had an immediate laminectomy or an immediate debridement of the paraspinal area . In eight patients the gastrointestinal tract was not perforated, and none of them had evidence of infection . In four patients the stomach and small bowel were perforated by the bullet before it struck the vertebral column, and none of them had meningitis, paraspinal infection, or osteomyelitis . In contrast, meningitis, paraspinal infection, or osteomyelitis did develop in seven of eight patients in whom the bullet perforated the colon before it hit the vertebra . Perforation of the colon by a low-velocity missile before the missile fractured the thoracic or lumbar vertebra was associated with a high incidence of infection . The appropriate management may require early operative intervention . This is in contrast to the non-operative approach that has been advocated for low-velocity gunshot wounds to the spine . We agree that a non-operative approach is indicated for gunshot wounds of the abdomen that do not involve the colon.

Cryobiology, 1985 Oct, 22(5), 427 - 33
Genetic damage is not produced by normal cryopreservation procedures involving either glycerol or dimethyl sulfoxide: a cautionary note, however, on possible effects of dimethyl sulfoxide; Ashwood-Smith MJ; Cryopreservation of chinese hamster ovary cells in tissue culture with either glycerol or dimethyl sulfoxide did not result in chromosome damage as measured by the sister chromatid exchange technique . These results are consistent with earlier negative reports in which the freezing and thawing of mammalian cells did not increase the frequency of micronuclei . No increases in the spontaneous mutation rates of several bacterial strains at different genetic loci were observed during the course of a number of years of storage at -196 degrees C . It is concluded that standard cryopreservation procedures are without genetic hazards . However, the well-documented effects of dimethyl sulfoxide on cell fusion and gene differentiation suggest caution in its use as a cryopreservative for animal and human embryos.

Biochem Pharmacol, 1985 Oct 1, 34(19), 3537 - 42
Induction of DNA strand breaks by RSU-1069, a nitroimidazole-aziridine radiosensitizer . Role of binding of both unreduced and radiation-reduced forms to DNA, in vitro; Silver AR et al.; {2-14C}-RSU-1069 {1-(2-nitro-1-imidazolyl)-3-(1-aziridino)-2-propanol}, either as a parent (unreduced) or following radiation reduction, binds to calf thymus DNA in vitro . Radiation-reduced RSU-1069 binds to a greater extent and more rapidly than the parent compound . RSU-1137, a nonaziridino analogue of RSU-1069, binds following radiation reduction . Radiation-reduced misonidazole (1-(2-nitro-1-imidazolyl)-3-methoxy-2-propanol) exhibits binding ratios a thousand-fold less than those of reduced RSU-1069 . There is no evidence for binding of parent misonidazole . Both parent and reduced RSU-1069 cause single strand breaks (ssbs) in pSV2 gpt plasmid DNA with the reduced compound causing a greater number of breaks . Parent and reduced RSU-1137 and misonidazole do not cause ssbs . It is inferred that the aziridine moiety present in both parent and reduced RSU-1069 is required for ssb production . RSU-1069 reacts with inorganic phosphate probably via nucleophilic ring-opening of the aziridine fragment . Incubation of plasmid DNA with reduced RSU-1069 in the presence of either phosphate or deoxyribose-5-phosphate at concentrations greater than 0.35 mol dm-3 prevents strand breakage, whereas 1.2 mol dm-3 deoxyribose does not protect against strand breakage formation . From these findings it is proposed that the observed binding to DNA occurs via the aziridine and the reduced nitro group of RSU-1069 and that these two have different target sites . Binding to DNA via the reduced nitro group may serve to increase aziridine attack due to localization at or near its target.

Arch Biochem Biophys, 1985 Oct, 242(1), 90 - 103
Synthesis of messenger-like RNA in avian erythrocyte nuclei; Wiersma PA et al.; Cell ghosts have been prepared from mature chicken erythrocytes using 0.05% saponin . Such preparations are capable of incorporating label from {3H}UTP and provide a system, where the nucleus is permeable to nucleotides and macromolecules, for studying the low-level RNA synthesis characteristic of these cells . RNase A (50 micrograms/ml) eliminated all radioactivity binding to DE-81 filters, indicating that the product was RNA; and DNase (10 micrograms/ml) and actinomycin D (10 micrograms/ml) each inhibited UMP incorporation by 70%, suggesting that the synthesis was DNA-dependent . Polymerization was inhibited 90% by 0.1 microgram/ml alpha-amanitin, and maximum synthesis occurred in the presence of high salt (0.175 M KCl) and Mn2+ (0.5 mM) . Polyacrylamide gel electrophoresis indicated that the newly synthesized RNA was heterogeneous in size, having a distribution from 5 to 60 S with a significant fraction migrating as 8-12 S . Approximately 15% of the total RNA was bound by an oligo(dT)-cellulose column, suggesting that some RNA processing was occurring, although attempts to detect the incorporation of label from {alpha-32P}GTP into a 5'-cap structure were unsuccessful . In comparison to RNA synthesis in reticulocyte nuclei, both the rate and extent of transcription in erythrocyte nuclei were much reduced . Moreover, about 25-30% of the reticulocyte nascent RNA was released from the nuclei during a 60-min incubation, while no release was observed for the erythrocyte nuclei . Hybridization of radiolabeled RNA to excess chicken DNA indicated that the majority (80%) of the in vitro transcripts were complementary to unique sequence DNA (C0t1/2 = 4.5 X 10(3)) . When RNA synthesized by either erythrocyte or reticulocyte nuclei was hybridized to cDNA complementary to reticulocyte polysomal mRNA, about 8% of the reticulocyte nuclear RNA but less than 1% of the erythrocyte nuclear RNA were resistant to RNase A digestion . Taken together, these data suggest that nuclei prepared by saponin lysis of chicken erythrocytes synthesize messenger-like RNA via endogenous polymerase II activity . A fraction of this RNA is polyadenylated but contains few, if any, globin sequences or other transcripts found on reticulocyte polysomes.

Proc Natl Acad Sci U S A, 1985 Oct, 82(20), 6807 - 10
Cloning and characterization of a nonmuscle myosin heavy chain cDNA; DeLozanne A et al.; Despite many biochemical and structural similarities between muscle and nonmuscle myosins, their genes appear to have completely diverged, since muscle myosin molecular clones will not hybridize to RNA from nonmuscle sources . Here we report the isolation and characterization of a partial myosin heavy chain (MHC) cDNA clone from the slime mold Dictyostelium discoideum . We have isolated this clone from a lambda gt11 expression cDNA library by antibody screening . In contrast to the highly conserved sarcomeric muscle MHC multigene families in other organisms, there appears to be only one gene encoding MHC in the Dictyostelium genome . The cloned portion of this gene does not hybridize to the genomic DNAs of other eukaryotic organisms . Analysis of the predicted amino acid sequence of the partial Dictyostelium MHC clone shows that while there is no sequence homology to known striated muscle MHCs, the structure- and coiled-coil-forming capacities have been conserved.

Proc Natl Acad Sci U S A, 1985 Oct, 82(20), 6765 - 8
Site-specific mutagenesis of histidine residues in the lac permease of Escherichia coli; Padan E et al.; The lacY gene of Escherichia coli, which encodes the lac permease, has been modified by oligonucleotide-directed, site-specific mutagenesis such that each of the four histidine residues in the molecule is replaced with an arginine residue . Replacement of histidine-35 and histidine-39 with arginine has no apparent effect on permease activity . In contrast, replacement of either histidine-205 or histidine-322 by arginine causes a dramatic loss of transport activity, although the cells contain a normal complement of permease molecules, as determined by immunoadsorption assays . Interestingly, although substitution of histidine-205 or histidine-322 by arginine results in the loss of ability to catalyze active lactose transport, permease molecules with arginine at residue 322 appear to facilitate downhill lactose movements at high concentrations of the disaccharide . The results provide strong support for the contention that histidine residues in the lac permease play an important role in the coupling between lactose and proton translocation.

Proc Natl Acad Sci U S A, 1985 Oct, 82(19), 6414 - 8
DNA sequence of the lactose operon: the lacA gene and the transcriptional termination region; Hediger MA et al.; The lac operon of Escherichia coli spans approximately 5300 base pairs and includes the lacZ, lacY, and lacA genes in addition to the operator, promoter, and transcription termination regions . We report here the sequence of the lacA gene and the region distal to it, confirming the sequence of thiogalactoside transacetylase and completing the sequence of the lac operon . The lacA gene is characterized by use of rare codons, suggesting an origin from a plasmid, transposon, or virus gene . UUG is the translation initiation codon . A preliminary examination of 3' end of the lac messenger in the region distal to the lacA gene indicates several endpoints . A predominant one is located at the 3' end of a G + C-rich hairpin structure, which may be involved in termination of transcription or in post-transcriptional processing . An open reading frame of 702 base pairs is present on the complementary strand downstream from lacA.

Mutat Res, 1985 Oct-Nov, 158(1-2), 89 - 95
Condensed tannins induce micronuclei in cultured V79 Chinese hamster cells; Ferguson LR et al.; The tannins, delphinidin and procyanidin were isolated from flowers of white clover (Trifolium repens) and the leaves of Arnot Bristly Locust (Robina fertilis) respectively, and tested for mutagenic properties in a range of systems . There was no evidence for either compound causing significant levels of frameshift or base-pair mutagenesis in bacterial mutagenicity assays, although both were weakly positive in a bacterial DNA-repair test . Both compounds very slightly increased the frequency of petite mutagenesis in Saccharomyces cerevisiae strain D5 . In V79 Chinese hamster cells, both were efficient inducers of micronuclei . In each of these test systems, increasing the potential of the compound for metabolic activation by addition of 'S9' mix had little effect on toxicity or mutagenicity of either tannin . It would seem that potential chromosome-breaking activity of condensed tannins could represent a carcinogenic hazard for animals grazing on pastures of white clover in flower . It may also have wider implications for human carcinogenesis by some, if not all, condensed tannins.

J Bacteriol, 1985 Oct, 164(1), 484 - 6
Maintenance of plasmid pSC101 in Escherichia coli requires the host primase; Ely S et al.; The abilities of three Escherichia coli strains with thermosensitive dnaG alleles to maintain plasmids pSC101 or pBR322 or an RP4 derivative were studied at elevated growth temperatures . Under these conditions, pSC101 segregated from cells to a greater extent than did pBR322 . No segregation of the primase-encoding RP4 derivative was observed.

J Bacteriol, 1985 Oct, 164(1), 470 - 2
Isolation of a dihydrofolate reductase-deficient mutant of Escherichia coli; Singer S et al.; A strain of Escherichia coli was isolated in which dihydrofolate reductase was not detected by an enzyme assay or by competition for antibody . This strain requires methionine, glycine, a purine, and thymidine for growth in addition to the auxotrophic requirements of the parent strain . It was found to be useful as a recipient of plasmids harboring dihydrofolate reductase genes.

Infect Immun, 1985 Oct, 50(1), 328 - 32
Mucosal antitoxin response in volunteers to immunization with a synthetic peptide of Escherichia coli heat-stable enterotoxin; Klipstein FA et al.; Peroral immunization of volunteers on four weekly occasions with 750 micrograms of a conjugate containing 3,000 antigen units of a synthetically produced peptide of hyperantigenic Escherichia coli heat-stable (ST) toxin, conjugated with the heat-labile toxin B subunit as a carrier, raised serum immunoglobulin G antitoxin titers to ST by fourfold and intestinal immunoglobulin A antitoxin titers to ST by sevenfold over control values at five weeks postimmunization . The ability of jejunal aspirates from the immunized volunteers to neutralize ST in the suckling mouse assay correlated with the intestinal immunoglobulin A ST antitoxin response determined by enzyme-linked immunosorbent assay.

Infect Immun, 1985 Oct, 50(1), 317 - 9
Intracellular distribution of heat-labile enterotoxin in a clinical isolate of Escherichia coli; Clements JD et al.; The intracellular distribution of heat-labile enterotoxin in a human isolate of enterotoxigenic Escherichia coli varied significantly as a result of changing incubation time, media, and degree of aeration . Direct comparison with a K-12 plasmid recipient revealed a similar but less dramatic response to environmental factors.

Eur J Biochem, 1985 Oct 1, 152(1), 35 - 41
Hybrid pyruvate dehydrogenase complexes reconstituted from components of the complexes from Escherichia coli and Azotobacter vinelandii; de Kok A et al.; The pyruvate dehydrogenase complex of Escherichia coli was isolated in a simple three-step procedure . Its chain stoichiometry, determined by trinitrobenzoate modification was found to be 1.4 E1:1 E2:0.6 E3 . It was reproducible within 10% from preparation to preparation . The E . coli complex was resolved by chromatography on activated thiol Sepharose . Reconstitution of activity yielded a stoichiometry of 1.0 E1:1 E2:0.5 E3 . The optimum binding stoichiometry of E1E2 and E2E3 subcomplexes was determined by sedimentation experiments and found to be 2.0 E1:1 E2 and 2.5 E3:1 E2, respectively . Competition between E1 and E3 was observed in the binding experiments, but not in the kinetic experiments . Hybrid active complexes could be reconstituted from either an E1E2 subcomplex from Azotobacter vinelandii and the E3 component from E . coli or from E2E3 subcomplex from E . coli and the E1 component from A . vinelandii . Low activity and weak binding was observed when E1 from E . coli was recombined with an E2E3 subcomplex from A . vinelandii or when E3 from A . vinelandii was recombined with an E1E2 subcomplex from E . coli . The association behaviour and stoichiometry of the reconstituted complexes is determined by the nature of the E2 component . The formation of hybrid complexes indicates a considerable structural similarity between the complexes from both sources, despite the differences in size and stoichiometry.

Eur J Biochem, 1985 Oct 1, 152(1), 151 - 5
Intermediates in the synthesis of TolC protein include an incomplete peptide stalled at a rare Arg codon; Misra R et al.; TolC is a minor outer membrane protein of Escherichia coli K 12 and is initially synthesized as a precursor . A distinct intermediate polypeptide of Mr about 46 000 was consistently observed at the initial stages of biosynthesis . The further elongation of this peptide can be blocked by chloramphenicol . We have investigated the cause of the temporary accumulation of the 46 000-Mr intermediate and we postulate that the presence of a rare codon AGA (Arg) at codon 402 of the tolC mRNA halts translating ribosomes owing to a limiting amount of the tRNAArg (AGA) species in the cell . The translation of tolC mRNA can be increased by providing T4 tRNAArg (AGA), encoded on a plasmid.

Clin Orthop, 1985 Oct, (199), 201 - 6
Arthroscopic debridement of the knee for septic arthritis; Ivey M et al.; Sixteen knees with hematogenous septic arthritis in 12 adult patients were treated by arthroscopic decompression, debridement, and irrigation with motorized instruments plus suction drainage . Infectious disease consultants supervised the administration of intravenous antibiotics, and range-of-motion exercises were instituted when the drains were removed 48 hours after surgery . Patients were protected from bearing weight for six weeks . There were no treatment complications, and no patient required a repeated drainage procedure . Two patients died of diseases unrelated to knee infection . The 11 infected knees of the ten surviving patients were evaluated subjectively, functionally, objectively, and roentgenographically . With an average 34-month follow-up evaluation, all patients regained their preoperative functional status without loss of motion or roentgenographic evidence of cartilage loss . Arthroscopic debridement of the knee for septic arthritis is a safe, efficient method of joint decompression with minimal morbidity.

Br J Surg, 1985 Oct, 72(10), 792 - 5
Effects of bile, infection and pressure on pancreatic duct integrity; Armstrong CP et al.; Ionic flux, potential difference and mucosal ultrastructure have been studied in the rat bile-pancreatic duct and the effects of pressure, bile and infection on the duct evaluated . The duct remained stable after perfusion with control solution under low pressure and high pressure produced widening of intercellular spaces only . Perfusion with a bacterial solution of Escherichia coli did not effect significant changes . Sterile human bile disturbed the integrity of the duct by increasing ionic flux, altering potential difference and producing reversible ultrastructural changes of cell oedema . High pressure increased these changes . Infected human bile under high or low pressure was by far the most toxic substance tested . Perfusion with infected bile led to irreversible duct damage and complete loss of duct integrity . Pressure and infected bile may have a role in damaging duct integrity and could thus play an integral part in the genesis of acute gallstone pancreatitis.

Blood, 1985 Oct, 66(4), 859 - 65
Stimulation of bone marrow-derived and peritoneal macrophages by a T lymphocyte-derived hemopoietic growth factor, persisting cell-stimulating factor; Crapper RM et al.; Several lines of evidence indicated that P cell-stimulating factor (PSF), a T lymphocyte-derived lymphokine known to stimulate the growth of hemopoietic stem and progenitor cells, also acted on macrophages . PSF was absorbed from medium that had been mixed for two hours at 0 degrees C with either resident or thioglycollate-elicited peritoneal cells, suggesting the presence of receptors for PSF on cells in the population . The addition of pure PSF to populations highly enriched in either resident or elicited adherent peritoneal macrophages resulted in stimulation of macrophages with morphological changes, including increases in size, spreading, vacuolation, and the number of cytoplasmic processes, together with stimulation of proliferation and the phagocytosis of opsonized yeast . PSF also stimulated the incorporation of {3H}thymidine by bone marrow-derived adherent macrophages . Addition of pure PSF to cultures that contained only a single macrophage resulted in enhanced survival and proliferation of these isolated cells, demonstrating that the effect of PSF on macrophages was direct . These results indicate that PSF can stimulate well-differentiated functional macrophages and raise the possibility that the effects of PSF on macrophages may play a regulatory role in immune responses.

J Virol, 1985 Oct, 56(1), 183 - 93
Phosphorylation in vitro of Escherichia coli-produced 235R and 266R tumor antigens encoded by human adenovirus type 12 early transformation region 1A; Lucher LA et al.; The tumor (T) antigens encoded by the human adenovirus early transforming region 1A (E1A) are gene regulatory proteins whose functions can immortalize cells . We have recently described the synthesis in Escherichia coli and the purification of the complete T antigens encoded by the adenovirus type 12 (Ad12) E1A 12S mRNA (235-residue {235R} T antigen) and 13S mRNA (266R T antigen) . In this study, we show that the Ad12 E1A T antigens are extensively phosphorylated in Ad12-infected mammalian cells but are not phosphorylated in E . coli . Inasmuch as posttranslational phosphorylation at specific amino acid sites may be important for biological activity, we have studied the phosphorylation of the E . coli-produced T antigens in vitro by using a kinase activity isolated from cultured human KB cells . The kinase was purified about 300-fold and appears to be a cyclic AMP-independent, Ca2+-independent protein kinase requiring only ATP and Mg2+ for activity . To determine which amino acids are phosphorylated and whether phosphorylation in vitro occurs at the same amino acid sites that are phosphorylated in vivo, the Ad12 E1A T-antigen species synthesized by infected cells were metabolically labeled with 32Pi and compared with the E . coli-produced E1A T antigens labeled in vitro with {gamma-32P}ATP by using the partially purified kinase . Partial V8 proteolysis analysis gave similar patterns for in vivo- and in vitro-phosphorylated T antigen . Two-dimensional maps of tryptic phosphopeptides and of chymotryptic phosphopeptides suggested that mainly the same amino acid sites are phosphorylated in vitro and in vivo and that phosphorylation occurred at multiple sites distributed throughout the T-antigen molecule . Serine was the only amino acid that was phosphorylated both in vivo and in vitro, and, surprisingly, most serines appeared to be phosphorylated . The feasibility of faithfully phosphorylating T antigens in vitro suggests that the E . coli-produced Ad12 E1A 235R and 266R T antigens may prove useful for molecular studies on T-antigen function.

J Immunol, 1985 Oct, 135(4), 2378 - 84
Identification of ribosomal protein autoantigens; Francoeur AM et al.; Approximately 20% of patients with systemic lupus erythematosus and with anti-Sm autoantibodies synthesize autoantibodies, called anti-rRNP, to components of the ribosome . We found that anti-rRNP sera reacted predominantly with three ribosomal phosphoproteins of approximate Mr = 38,000, 16,000 and 15,000, both by immunoprecipitation and by immunoblotting . The human autoantibodies cross-reacted with similar antigens present in rodent, brine shrimp, and yeast cells but reacted weakly if at all with proteins of bacteria . Thus the human autoantibodies recognize epitopes that are widely conserved in evolution . Purified ribosomal proteins together with specific rabbit antisera were used to identify the two smaller rRNP antigens as the acidic phosphoproteins of the large ribosomal subunit, designated P1/P2(L40/L41) (rat), eL7/eL12 (Artemia, brine shrimp), and A1/A2 (yeast) . These proteins function in the elongation step of protein synthesis in an analogous fashion to the L7/L12 ribosomal proteins of E . coli . The 38,000-dalton rRNP antigen corresponds to a nonacidic protein also associated with the large ribosomal subunit . The human autoantibodies appear to have a specificity similar to that of a previously described mouse monoclonal antibody obtained from mice injected with heterologous (chick) ribosomes, suggesting that both the human polyclonal autoantibodies and the mouse monoclonal recognize a class of epitope(s) that is common in all three ribosomal proteins . In addition, we found that many of the anti-ribosomal sera contained a further class of autoantibodies reactive with naked RNA . These may be similar to the anti-RNA antibodies previously described in both humans and mice with autoimmune disease.

Cell Immunol, 1985 Oct 1, 95(1), 146 - 56
Functional comparison of recombinant interleukin 2 (IL-2) with IL-2-containing preparations derived from cultured cells; Roifman CM et al.; Due to its purity and potential availability in large amounts, human recombinant interleukin 2 (IL-2) expressed in Escherichia coli is an important source of IL-2 for experimentation and possible therapy . To date, very few comparisons between the activity of recombinant IL-2 and conventional cell-derived preparations of IL-2 have been made . This is particularly important since the use of recombinant IL-2 may have some specific limitations . For example, recombinant IL-2 is not post-translationally modified as are cell-derived preparations . Lack of modifications such as glycosylation of threonine 3 may alter efficacy or stability . Comparative studies are necessary to demonstrate the efficacy, species specificity, and stability of recombinant IL-2 . By comparing IL-2 activity of recombinant IL-2 to that of cell-derived IL-2, we have demonstrated that each of the preparations are equally active in several murine and human IL-2 proliferation assays and that IL-2 is the active moiety in these assays . In contrast to previous reports, we also show that recombinant IL-2 is sufficient to establish and maintain long-term cell lines . Additionally, by using "synthetic" recombinant IL-2 of defined protein sequence, we have demonstrated that this amino acid-defined structure is indeed responsible for the functions attributed to IL-2.

Virologie, 1985 Oct-Dec, 36(4), 279 - 84
Preliminary data on the encapsulation of biologically active materials in liposomes; Mihalache O et al.; Multilamellar and unilamellar phospholipid liposomes were prepared and investigated as regards their properties and the capacity of encapsulating biologically active materials, such as CrO4-(2-)ions, basic dyes, proteins, DNA, as well as Sendai virus particles . The efficiency of encapsulation ranged from 10% for CrO4-(2-)ions to about 80% for toluidine blue; it was found to depend not only on the type of encapsulated material, but also on the method used for liposome preparation and on liposome composition.

Bioorg Khim, 1985 Oct, 11(10), 1356 - 60
{New plasmid vectors for cloning and the expression of genes}; Gurevich AI et al.; Multicopy plasmids of pMCR series have been constructed from pRRN2 (a pBR322 derivative) including plasmids carrying transcription terminators downstream the tet gene and those with opposite orientation of the tet and RNAI genes . The plasmids permit cloning and high expression of genes with strong promoters.

Microbiologica, 1985 Oct, 8(4), 303 - 12
Cloning and expression of the Escherichia coli rho gene in a plasmid vector; Gulletta E et al.; In order to further elucidate the role of Rho protein on transcription termination and cells growth control, we have subcloned by two steps the rho+ structural gene of Escherichia coli from Lambda rho+524 into a plasmid vector . The resulting plasmid pEG25 contains a 2.9 kbp insert which is able to complement several different rho mutations and to express a functional Rho protein in U.V . irradiated maxicells.

Thorax, 1985 Oct, 40(10), 774 - 7
Angiotensin converting enzyme and endotoxin induced lung damage in the mouse; Cookson WO et al.; Acute pulmonary oedema can be induced by intraperitoneal injection of Escherichia coli endotoxin in the mouse . A fall in serum angiotensin converting enzyme activity is found in mice given endotoxin and in patients with septic adult respiratory distress syndrome, and has been proposed as an indicator of lung microvascular injury . Protein concentration and angiotensin converting enzyme activity in serum, lung, and bronchoalveolar lavage fluid were determined in male mice up to eight hours after injection of endotoxin . By six hours the serum protein concentration had increased and the bronchoalveolar lavage fluid protein concentration had fallen, suggesting fluid shift into the lung . Angiotensin converting enzyme activity fell in serum and lung but increased in bronchoalveolar lavage fluid . As these changes in enzyme activity were not paralleled by changes in protein concentration they are unlikely to be a result of fluid shift or protein leak, and may indicate an active role of the enzyme in the response to sepsis.

Genetics, 1985 Oct, 111(2), 219 - 31
Joint distribution of insertion elements IS4 and IS5 in natural isolates of Escherichia coli; Dykhuizen DE et al.; A reference collection of natural isolates of Escherichia coli has been studied in order to determine the distribution, abundance and joint occurrence of DNA insertion elements IS4 and IS5 . Among these isolates, 36% were found to contain IS4 and 30% were found to contain IS5 . Among strains containing IS4 the mean number of copies per strain was 4.4 +/- 0.8; the comparable figure for IS5 was 3.7 +/- 1.0 . Although the presence of the elements among the isolates was independent, among those isolates containing both IS4 and IS5, there was a significant negative correlation in the number of copies of the elements . The reference collection was also studied for the presence of the DNA sequences flanking the single copy of IS4 in the chromosome of E . coli K12 . Homologous sequences were found in only 26% of the isolates . The sequences flanking the IS4 invariably occur together, and their presence is significantly correlated with the presence of IS4 . In eight of the strains that carry these flanking sequences, an IS4 is located between them, and the sequences are present at the homologous position as in the K12 strain . We suggest that IS4 and its flanking sequences share a common mechanism of dissemination, such as plasmids, and we present evidence that they are included in a much larger transposable element.

Cell, 1985 Oct, 42(3), 859 - 68
Sequence-specific DNA binding of the Epstein-Barr virus nuclear antigen (EBNA-1) to clustered sites in the plasmid maintenance region; Rawlins DR et al.; Latently infected B lymphocytes continuously express an Epstein-Barr Virus nuclear antigen (EBNA-1) required in trans for maintenance of the plasmid state of the EBV genome . Filter binding assays and DNAase I footprinting analyses revealed that the carboxy-terminal domain of EBNA-1 protects binding sites at three different loci in the 172,000 bp EBV genome . Two of these loci correspond to essential elements within an 1800 bp segment defined as the minimal region required for plasmid maintenance (ori-P) . Binding to each of 20 X 30 bp tandem repeats in the "sink" locus protects 25 bp centered over a 12 bp palindromic consensus sequence TAGCATATGCTA . The nearby dyad symmetry "origin" locus contains two 46 bp protected regions each encompassing two paired core binding sites . The demonstration of sequence-specific binding at multiple loci suggests that EBNA-1 has pleiotropic functions, which may include control of copy number and segregation of the EBV plasmids as well as initiation of replication.

Am J Pathol, 1985 Oct, 121(1), 123 - 7
Aortic endothelial cell death and replication in normal and lipopolysaccharide-treated rats; Hansson GK et al.; Endothelial cell death and replication was analyzed in the rat aorta with the use of combined IgG immunocytochemistry and 3H-thymidine autoradiography . Dead and replicating cells were clustered in the aortic endothelium of normal rats with a low level of spontaneous cell death and replication . Death and replication were also clustered in rats treated with endotoxin (Escherichia coli lipopolysaccharide {LPS}), a substance which is cytotoxic to endothelial cells in culture . LPS caused a dramatic increase both in endothelial cell death and replication, while no endothelial denudation could be discerned with the use of the 111In-labeled platelet technique . We conclude that endothelial cell death and replication are topographically clustered phenomena, which may imply that cell death leads to a local stimulation of cell replication . Furthermore, LPS induces a massive cell death, which is paralleled by cell replication, yet does not cause any significant increase in denudation . This suggests that the primary effect of LPS on the vessel wall is its cytotoxicity for endothelial cells . Endothelial cell death appears to provide the stimulus for cell replication both in normal and in LPS-treated rats.

Proc Natl Acad Sci U S A, 1985 Oct, 82(20), 6990 - 4
Generation of single base-pair deletions, insertions, and substitutions by a site-specific recombination system; Leong JM et al.; The sequence analysis of both products of individual phi 80 site-specific recombination events in vivo shows that recombination with a secondary attachment (att) site generates several different novel joints at the mismatched position: one recombination event resulted in a single base-pair deletion and two other recombination events resulted in two different single base-pair substitutions . The characterized products of recombination can be straightforwardly interpreted as the outcome of strand exchange involving staggered nicks bracketing the heterology within an overlap region of five to nine base pairs . In comparison, more complex segregation patterns have been observed in previous studies of lambda recombination between nonidentical att sites; the nature of the overlap region heterology may have a significant effect on the segregation patterns . To recover both products of a single recombination event, we used a plasmid that carries the phi 80 int and xis genes and both att sites . Because the two att sites are situated in opposite orientation, intramolecular recombination between them inverts rather than deletes the intervening segment of DNA . Although subsequent reinversion restores the original gross genetic arrangement, single base-pair insertions, deletions, and substitutions are introduced at the sites of recombination . One of the mutations improves the recombination efficiency of the secondary att site and thereby converts a formerly "stable" sequence to an efficient target for rearrangement, and other mutations are predicted to alter the specificity of recombination . These pathways may also provide useful models for the efficient generation of localized sequence diversity on a development (as well as evolutionary) time scale.

Proc Natl Acad Sci U S A, 1985 Oct, 82(20), 6845 - 9
The replication initiator protein of plasmid pT181 has sequence-specific endonuclease and topoisomerase-like activities; Koepsel RR et al.; Initiation of pT181 DNA replication specifically requires the plasmid-encoded RepC protein . Here we demonstrate that highly purified RepC protein has sequence-specific endonuclease and topoisomerase-like activities . A maximum sequence of 127 base pairs containing the pT181 origin of replication is required for nicking-closing by RepC protein . RepC introduces a single strand break within the pT181 origin . The nick site has been shown by DNA sequencing to lie between nucleotides 70 and 71 in the bottom strand of the DNA within the origin sequence . This nick site probably corresponds to the start site of pT181 replication . The results presented here suggest that, unlike most other plasmids, pT181 replicates by a rolling circle mechanism.

Proc Natl Acad Sci U S A, 1985 Oct, 82(19), 6614 - 8
UV-induced mutagenesis of phage S13 can occur in the absence of the RecA and UmuC proteins of Escherichia coli; Tessman I; The UV-induced mutagenesis of phage S13 that accompanies Weigle repair is known to require the products of the recA and umuDC genes, as does the UV-induced mutagenesis of the Escherichia coli chromosome . I found that UV-induced mutagenesis of phage S13 occurred in the absence of both the RecA and UmuC functions when the irradiated phage was photoreactivated . Furthermore, UV-induced phage mutations were produced in a recA- umuC- cell even without photoreactivation and in the absence of any other known UV repair mechanism, at a frequency 29% of that found after photoreactivation and 7% of that found after Weigle repair, implying that DNA synthesis can proceed past a dimer at an unexpectedly high frequency even when unaided by the UmuC-RecA SOS repair functions . The unaided DNA synthesis appears capable of producing mutations in the vicinity of a pyrimidine dimer; by aiding synthesis past a dimer, a repair mechanism may disclose a mutation without having any active role in producing it.

J Bacteriol, 1985 Oct, 164(1), 63 - 9
Molecular cloning of the Escherichia coli gene for diadenosine 5',5'''-P1,P4-tetraphosphate pyrophosphohydrolase; Mechulam Y et al.; A clone overproducing diadenosine tetraphosphatase (diadenosine 5', 5'''-P1, P4-tetraphosphate pyrophosphohydrolase) activity was isolated from an Escherichia coli cosmid library . Localization of the DNA region responsible for stimulation of this activity was achieved by deletion mapping and subcloning in various vectors . Maxicell experiments and immunological assays demonstrated that a 3.5-kilobase-pair DNA fragment carried the structural gene apaH encoding the E . coli diadenosine tetraphosphatase . The DNA coding strand was determined by cloning this fragment in both orientations in pUC plasmids . It was also shown that the overproduction of diadenosine tetraphosphatase decreased the dinucleoside tetraphosphate concentration in E . coli by a factor of 10.

J Bacteriol, 1985 Oct, 164(1), 487 - 9
Cell length in a wee dnaA mutant of Escherichia coli; de la Campa AG et al.; The cell length of the short siblings of dividing pairs formed in the absence of replication by two strains of Escherichia coli, OV-25-9 {dnaA46 wee(Am)} and OV-25-10 {dnaA46 wee(AM) supF} was measured . In the presence of Wee, the length of these cells increased to those values expected for newborn wild-type cells growing under similar conditions . In its absence, cell length remained at values near the minimum unit length possible for newborn cells . Our results show that both cell elongation and the action of Wee are independent of DNA replication, being compatible with the role proposed for Wee in coordination between cell elongation and division.

J Bacteriol, 1985 Oct, 164(1), 276 - 81
Analysis of the ruv locus of Escherichia coli K-12 and identification of the gene product; Attfield PV et al.; The ruv gene of Escherichia coli, which is associated with inducible mechanisms of DNA repair and recombination, has been cloned into the low-copy plasmid vector pHSG415 . The recombinant plasmid pPVA101 fully complements the DNA repair-deficient phenotype of ruv mutants . Restriction endonuclease analysis of this plasmid revealed a 10.6-kilobase (kb) HindIII DNA insert which contained a 7.7-kb PstI fragment identified as being from the chromosomal ruv region . Deletion analysis and Tn1000 insertional inactivation of ruv function located the ruv coding region to a 2.2-kb section of the cloned DNA fragment . A comparison of the proteins encoded by ruv wild-type and mutant plasmids identified the gene product as a protein of molecular weight 41,000.

J Bacteriol, 1985 Oct, 164(1), 270 - 5
Novel one-step cloning vector with a transposable element: application to the Myxococcus xanthus genome; Furuichi T et al.; A new strategy was developed for rapid cloning of genes with a transposon mutation library . We constructed a transposon designated TnV that was derived from Tn5 and consists of the gene coding for neomycin phosphotransferase II as well as the replication origin of an Escherichia coli plasmid, pSC101, flanked by Tn5 inverted repeats (IS50L and IS50R) . TnV can transpose to many different sites of DNA in E . coli and Myxococcus xanthus and confers kanamycin resistance (Kmr) to the cells . From the Kmr cells, one-step cloning of a gene which is mutated as a result of TnV insertion can be achieved as follows . Chromosomal DNA isolated from TnV-mutagenized cells is digested with an appropriate restriction enzyme, ligated, and transformed into E . coli cells with selection for Kmr . The plasmids isolated contain TnV in the target gene . The plasmid DNA can then be used as a probe for characterization of the gene and screening of clones from a genomic library . We used this vector to clone DNA fragments containing genes involved in the development of M . xanthus.

J Bacteriol, 1985 Oct, 164(1), 25 - 32
Delineation of two distinct regulatory domains in the 5' region of the nar operon of Escherichia coli; Li S et al.; A detailed restriction site map was determined for an 8.4-kilobase DNA fragment containing the 5' regulatory and promoter region of the nar operon of Escherichia coli . The 5' end of the nar operon was subcloned as a 2.5-kilobase fragment, and an intact nar operon was constructed from this subcloned fragment and an EcoRI fragment containing the remainder of the nar operon . A set of Bal 31 deletions extending into the 5' region of the intact operon was selected, mapped, and characterized . Based on the synthesis of the alpha and beta subunits of nitrate reductase in a nar::Tn5 mutant, three categories of deletions were found: (i) those which permitted normal expression, (ii) those which completely prevented expression, and (iii) those which permitted anaerobic expression of the operon but prevented any additional induction by nitrate . The nucleotide sequence was determined for a segment of the nar promoter region starting at one of the latter deletion end points and extending into the first structural gene of the operon . The position of the deletion end point relative to the translation start site for the first structural gene, narG, was defined by identifying the nucleotide sequence for the first 20 N-terminal amino acid residues of the alpha subunit of nitrate reductase . Deletions terminating 161 base pairs (bp) and approximately 200 bp upstream from the narG translation start site permitted anaerobic formation of nitrate reductase but interfered with the stimulation of nar operon expression by nitrate . A maximum size for the regulatory region was defined by two Tn5 insertions, which mapped approximately 550 bp 5' from the translation start site and did not interfere with the normal expression of nitrate reductase under anaerobic conditions with or without nitrate . We conclude that the nar operon 5' regulatory region is divided into two distinct regions: the 100 to 150 bp immediately 5' to the narG gene include a transcriptional start site and the signals necessary for anaerobic expression of the operon, and an adjacent region of 50 to 400 bp is required for the stimulation of operon expression by nitrate.

J Bacteriol, 1985 Oct, 164(1), 136 - 42
Isolation and characterization of Escherichia coli pantothenate permease (panF) mutants; Vallari DS et al.; Mutants of Escherichia coli K-12 defective in the pantothenate permease (panF) were isolated and characterized . The panF mutation resulted in the complete loss of pantothenate uptake and of the ability to use extracellular vitamin for growth . The growth phenotypes of panF panD, panF panB, and panF panC double mutants showed that the cytoplasmic membrane was impermeable to external pantothenate . Analysis of the intracellular and extracellular metabolites from strain DV1 (panF panD) labeled with beta-{3-3H}alanine demonstrated that a carrier-mediated mechanism for efficient pantothenate efflux remained in the panF mutant . Genetic mapping of this nonselectable allele was facilitated by the isolation of three independent Tn10 insertions close to panF . Two- and three-factor crosses located panF at minute 72 of the E . coli chromosome and established the gene order fabE panF aroE.

J Virol, 1985 Oct, 56(1), 144 - 52
Nucleotide sequence and biochemical activities of the Moloney murine sarcoma virus strain HT-1 mos gene; Seth A et al.; The nucleotide sequence of the Moloney murine sarcoma virus strain HT-1 (HT1MSV) mos gene differs from that of the cellular mos gene in three positions, but these are silent changes, and the amino acid sequence of the v-mos and c-mos open reading frames are identical . We have overproduced the mos HT1MSV (equivalent to c-mos) in Escherichia coli under the control of phage lambda promoter (pL) . The E . coli p40mos protein thus obtained was partially purified and examined for several biochemical activities . We show that the p40mos binds ATP analog p-fluorosulfonylbenzoyladenosine and exhibits ATPase activity.

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