Escherichia coli

Nucleic Acids Res, 1989 Jul 11, 17(13), 5057 - 69
Cosmid-derived map of E . coli strain BHB2600 in comparison to the map of strain W3110; Birkenbihl RP et al.; A physical map for the genome of E . coli K12 strain BHB2600 was constructed by use of 570 cloned DNA elements (CDEs) withdrawn from a cosmid library . Dot blot hybridisation was applied to establish contig interrelations with subsequent fine mapping achieved by analysis of EcoR1 restriction patterns on Southern blots . The derived map covers nearly 95% of the E . coli genome resulting in 12 minor gaps . It may be compared to the almost complete map for strain W3110 of Kohara et al . (1) . Except for one tiny gap (lpp,36.5') remaining gaps in BHB2600 do not coincide with those in W3110 so that both maps complement each other establishing an essentially complete clone represented map . Besides numerous minute differences (site and fragment gains and losses) both strains harbour at differing positions extended rearrangements flanked by mutually inverted repetitive elements, in our case insertion elements (IS1 and IS5).

Nucleic Acids Res, 1989 Jul 11, 17(13), 4947 - 56
Uranyl mediated photofootprinting reveals strong E . coli RNA polymerase--DNA backbone contacts in the +10 region of the DeoP1 promoter open complex; Jeppesen C et al.; Employing a newly developed uranyl photofootprinting technique (Nielsen et al . (1988) FEBS Lett . 235, 122), we have analyzed the structure of the E . coli RNA polymerase deoP1 promoter open complex . The results show strong polymerase DNA backbone contacts in the -40, -10, and most notably in the +10 region . These results suggest that unwinding of the -12 to +3 region of the promoter in the open complex is mediated through polymerase DNA backbone contacts on both sides of this region . The pattern of bases that are hyperreactive towards KMnO4 or uranyl within the -12 to +3 region furthermore argues against a model in which this region is simply unwound and/or single stranded . The results indicate specific protein contacts and/or a fixed DNA conformation within the -12 to +3 region.

FEBS Lett, 1989 Jul 3, 250(2), 201 - 4
Diacylglycerol breakdown in plasma membrane of rat intestinal epithelial cells . Effect of E . coli heat-stable toxin; Banik ND et al.; Rat intestinal epithelial cells were isolated and the activity of the enzyme diacyglycerol lipase (DG lipase, EC 3.1.1.3) was investigated . When cells were treated with Escherichia coli heat-stable toxin (ST) liberation of endogenous glycerol and fatty acids was observed . The enzyme responsible for this effect could be demonstrated to be a DG lipase by using specific substrates . It was found that the activity of DG lipase was increased 5-6-fold with the substrates diolein and 1,2-dioleyl-rac-glycerol and triolein being neutral lipid insensitive to DG lipase . ST had no direct effect on the DG lipase . The enzyme DG lipase was activated via a chain reaction due to the hydrolysis of phosphatidylinositol (PI) by the enzyme PI-specific phospholipase C stimulated by ST.

Am J Physiol, 1989 Jul, 257(1 Pt 1), G118 - 23
Differences in jejunal and ileal response to E . coli enterotoxin: possible mechanisms; Cohen MB et al.; Escherichia coli, which produce heat-stable enterotoxin (STa), cause intestinal fluid secretion as a mechanism of diarrhea . To determine the factors that modulate the intestinal secretory response, we first compared the time course of the STa-induced secretion in ligated in situ loops of rat jejunum and ileum . We found that the jejunal secretory response was brief (less than or equal to 30 min) while the ileal response to STa was sustained (greater than or equal to 3 h) . We then compared the modification of purified STa that occurred in jejunum and ileum and found a close correlation between the continued presence of unmodified, authentic STa and continued fluid secretion in the ligated-loop model . At both sites alteration of STa was demonstrated by high-performance liquid chromatography profile and brush-border membrane binding activity . However, in the jejunum, the modification of STa was qualitatively different and quantitatively much greater . We conclude that the degree to which STa is inactivated or removed from the intestine correlates with the secretory response observed . Inactivation of STa may be a mechanism by which the host limits its secretory response.

Biochem Int, 1989 Jul, 19(1), 163 - 72
Expression of the kinase domain of mouse protein kinase C in E . coli; Dietrich A et al.; The kinase domain of mouse protein kinase C type alpha has been expressed at high levels in E . coli . The protein has been purified 500-fold taking advantage of the fact that highly expressed fusion proteins precipitate out in the bacterial cell and can be solubilized in 7 M urea . The purified protein can be detected with an antibody generated against a PKC alpha derived synthetic peptide . The purified kinase domain exhibits no measurable kinase activity in a protamin phosphorylation assay . This could be an indication that post-translational modifications of the protein kinase domain which do not happen in bacteria are a requirement for functional enzyme activity or that the regulatory domain of protein kinase C is indispensable for kinase function.

Comput Appl Biosci, 1989 Jul, 5(3), 211 - 8
An extension of the graph theoretical approach to predict the secondary structure of large RNAs: the complex of 16S and 23S rRNAs from E . coli as a case study; Thanaraj TA et al.; An algorithm using the graph theoretical approach to predict secondary structures of large nucleic acids is discussed . Reliability of prediction can be improved by incorporating available experimental data and sequence homology information . As a case study, this algorithm is applied to predict the secondary structure of the 16S-23S rRNA complex from E . coli . It was found that several structures of the complex can coexist . The computer program developed to predict the secondary structure of large RNAs can be run on IBM PC/AT compatible systems.

Nucleic Acids Res, 1989 Jun 26, 17(12), 4799 - 815
A novel method for promoter search enhanced by function-specific subgrouping of promoters--developed and tested on E.coli system; Rozkot F et al.; A new method for evaluating some complex characteristics of the primary structure of E.coli promoters is proposed . The method, of nonparametric statistical significance, selects important conserved single-base positions in combination with 2-base coupling relations of identity and complementarity . The extended consensus of promoter characteristics thus obtained was used to scan unknown sequences for similarity with E.coli promoters . In terms of this method, a complete set of 244 E.coli promoters was shown to be structurally inconsistent . The set was then broken down into functionally homogeneous subsets of promoters to enhance the selectivity of the search for E.coli-specific promoter sequences, with a high significance level being attained.

Cell, 1989 Jun 2, 57(5), 869 - 80
The interaction of E . coli IHF protein with its specific binding sites; Yang CC et al.; We have used two kinds of footprinting techniques, dimethylsulfate interference and hydroxyl radical protection, to explore the way that IHF recognizes its specific target sequences . Our results lead us to conclude that IHF recognizes DNA primarily through contacts with the minor groove, an unprecedented mode for a sequence-specific binding protein . We have also determined that, although IHF is a small protein that protects a large region of DNA, only a single IHF protomer is present at each binding site . IHF bends the DNA to which it binds . We have combined this fact plus our footprinting and stoichiometry data together with the crystal structure of a related protein, the nonspecific DNA binding protein HU, to propose a model for the way in which IHF binds to its DNA target.

J Biomol Struct Dyn, 1989 Jun, 6(6), 1071 - 6
Preliminary crystallographic analysis of a proteolytically modified form of E . coli single stranded DNA binding protein; Ng JD et al.; A proteolytically modified form of the Escherichia coli single-stranded DNA-Binding protein (SSB) has been crystallized from 15% saturated sodium citrate . Crystals as large as 1.0 mm x 0.3 mm x 0.2 mm were obtained and these diffract beyond 3A resolution . X-ray photographic analysis demonstrated a rhombohedral unit cell of space group R3 with an equivalent triple centered hexagonal unit cell having dimensions of a = b = 62.9A and c = 264.3A . These crystals were judged to be adequate for a three dimensional structure determination.

Mol Gen Mikrobiol Virusol, 1989 Jun, (6), 16 - 20
{Biosynthesis of the membrane protein of the acquired immunodeficiency syndrome virus (HIV) with a deleted hydrophobic region in E . coli cells}; Starov AI et al.; Effect of the structure of cloned env gene of HIV virus on the level of its expression has been studied . The deletion of the hydrophobic region from the env gene has been shown to increase considerably the biosynthesis of antigen-specific proteins in Escherichia coli cells . At the same time, the low level of expression of the gene fragment the transcription of which is controlled by a powerful lac-promoter suggests the presence of toxic regions of nonhydrophobic nature in the synthesized antigen.

Wei Sheng Wu Xue Bao, 1989 Jun, 29(3), 228 - 31
{Restriction map of E . coli shuttle plasmid (p# GTE5) with secretive function}; Chen YX et al.; The shuttle plasmid (p# GTE5) DNA with secretive function was extracted by the alkali lysozyme method from E . coli RRI strain . Its molecular weight is 4.5 Md and DNA size is 6.9 Kb . Restriction fragments of plasmid was obtained by single and double enzymes complete digestion using five different restriction endonucleases . The restriction map of shuttle plasmid (p# GTE5) was established for the enzymes EcoRI, BglII, pstI, PvuII, and TaqI.

FEMS Microbiol Lett, 1989 Jun, 50(3), 345 - 9
Detection and identification of pathotypes of verocytotoxigenic Escherichia coli isolated from weaned piglets using gene probes for seven E . coli toxins; Mainil J et al.; Seventy verocytotoxigenic (VTEC) and sixty-three non VTEC haemolytic Escherichia coli isolated from recently weaned piglets were examined by the colony hybridization assay using gene probes for three verocytotoxins: Edema disease principle (EDP) and Shiga-like toxins I and II (SLTI and SLTII) . The results with the EDP and SLTII probes were identical . All VTEC hybridized with these two probes, while non VTEC did not . All 133 E . coli were negative for the SLTI probe . Hybridization of the plasmid content of 14 VTEC did not show any evidence for plasmid localization of the genes coding for the EDP . The 70 VTEC were also assayed with gene probes for heat-stable (STaP, STb) and heat-labile (LT, LTIIa) enterotoxins . Only the STb probe was hybridized by 36 of them . Most STb-positive isolates belonged to serotype O141: K85 biotypes 9 and 13 PC.

Zentralbl Hyg Umweltmed, 1989 Jun, 188(3-4), 380 - 4
UV disinfecting experiments with E.coli and actinometric determination of the irradiation intensity; Zemke V et al.; The UV disinfection of E . coli was carried out and additionally an actinometric determination of the irradiation intensity was made . This kind of proceeding renders it possible--in regard to the time of exposure--to set up a dose-effect-relation for the UV disinfection of water.

Mutat Res, 1989 Jun, 212(2), 269 - 74
Modifying action of gamma-radiation in mutagenesis of E . coli WU36-10 induced by ethylene oxide, ethyl methanesulfonate and methyl methanesulfonate; Kolman A et al.; The influence of gamma-radiation pre-exposure on ethylene oxide, ethyl methanesulfonate and methyl methanesulfonate mutagenesis in Escherichia coli WU36-10 was studied . Pretreatment with gamma-radiation resulted, in the case of subsequent treatment with ethylene oxide and ethyl methanesulfonate, in a decrease of the frequency of leu+ revertants, and in the case of subsequent treatment with methyl methanesulfonate, in an increase of this mutation frequency.

Brain Res Mol Brain Res, 1989 Jun, 5(4), 271 - 8
Grafting genetically modified cells into the rat brain: characteristics of E . coli beta-galactosidase as a reporter gene; Shimohama S et al.; The utility of grafting genetically modified cells to the mammalian brain was examined using the E . coli beta-galactosidase gene (lacZ) as a reporter gene in retroviral infection . Following implantation of the infected cells to the brain, lacZ continued to be expressed in vivo and could be detected easily with enzyme histochemistry . However, beta-galactosidase-positive cells were also observed in control grafts which had not been infected with the virus . This false-positive staining was found to be endogenous lysosomal activity associated with macrophage infiltration presumably induced by the damage associated with grafting . The E . coli gene product was distinguished from cellular lysosomal beta-galactosidase by using immunohistochemical staining with an antibody specific for E . coli beta-galactosidase . With this antibody, retrovirus-infected cells could be distinguished in the brain, and no false positives were observed in non-infected cells . We conclude that E . coli beta-galactosidase is a useful reporter gene for determining the fate of implanted cells to the brain if appropriate caution is taken to distinguish it from cellular beta-galactosidase by immunocytochemical procedures.

Nucleic Acids Res, 1989 May 25, 17(10), 3855 - 63
Effect of the sequence-dependent structure of the 17 bp AT spacer on the strength of consensuslike E.coli promoters in vivo; Lozinski T et al.; Three E.coli promoters with the consensus sequences in the -35 and -10 regions and the 17 bp spacer made of random, heteronomous, and of both these classes of AT DNA simultaneously were constructed and cloned into plasmid pDS3 . Electrophoretic gel mobilities of restriction fragments containing these promoters indicated that bending of the latter was proportional to the number of heteronomous AT DNA tracts . The strength of these promoters in vivo measured in relation to an internal transcriptional standard was shown to correlate well with gel mobilities of the respective restriction fragments and to decrease with the number of potential DNA bending sites encoded in the promoter structure.

Zhonghua Min Guo Wei Sheng Wu Ji Mian Yi Xue Za Zhi, 1989 May, 22(2), 145 - 9
{Detection of heat-labile enterotoxin produced by E . coli with reversed passive latex agglutination}; Wang FI et al.; From June 1987 to March 1988, 103 E . coli strains were collected from non-foodborne samples, foodborne samples, FDA (USA) etc . and analyzed for heat-labile E . coli enterotoxin (LT) by reversed passive latex agglutination (RPLA) . The results showed that 84 strains were LT-positive, 19 strains were LT-negative . Forty six strains of enteropathogenic E . coli isolated from foodborne samples showed that 34 strains were LT-positive, 12 strains were negative . Four enterotoxigenic E . coli strains from FDA (USA) used as positive controls, can produce heat-labile E . coli enterotoxin and give positive results.

Nippon Seikeigeka Gakkai Zasshi, 1989 May, 63(5), 569 - 79
{Production of rheumatoid factor-like substance and arthritic findings in knees of mice immunized with E . coli 0:14}; Itoh J; Four strains of mice (A . SW, B10 . D2, C57BL/6, DBA/1J) were immunized by subcutaneous injections of heat-killed E . coli 0: 14 . After single or multiple immunizations, histologic sections of knee joints and a serum rheumatoid factor-like substance (RFLS) at different time intervals were studied . Immunized A . SW . mice developed hyperplasia of synovial lining cells (25 out of 49 knees) and pannus (9 out of 49 knees) which was significantly higher rate than control . A serum rheumatoid factor-like substance (RFLS) was detected in B10 . D2 and C57BL/6 mice by using RAHA test and enzyme-linked immunosorbent assay . Serum IgM-RFLS was positive in all B10 . D2 mice (11 of 11 mice) immunized for 24 weeks . The incidence of serum IgM-RFLS positive mice in C57BL/6 immunized for 40 weeks (3 of 4 mice) was significantly higher than in control (p less than 0.05) . Production of serum RFLS was not recognized in a single-immunized group of B10 . D2 mice on statistical evaluation . These findings provided evidence that arthritis was developed in multiple-immunized groups of A . SW mice and RFLS was produced in multiple-immunized groups of B10 . D2 and C57BL/6 mice . In addition, these findings suggested that multiple-immunization was necessary for the production of RFLS.

Trends Genet, 1989 May, 5(5), 139 - 43
The great GATC: DNA methylation in E . coli; Barras F et al.; In Escherichia coli the methylation of the adenine in the sequence 5'-GATC-3' is catalysed by the dam gene product, a DNA adenine methylase . We review the proposed roles for this methylation, and the sequence it modifies, in mismatch repair, DNA-protein interaction, gene expression, the initiation of chromosome replication, chromosome segregation, chromosome structure and the occurrence of mutational hotspots.

Antibiot Khimioter, 1989 May, 34(5), 370 - 4
{Evolutionary changes in the structural and regulatory regions of aminoglycoside-3'-phosphotransferase type I gene of E . coli}; Vakulenko SB et al.; To investigate the evolutionary relationships between the aph(3') genes from different plasmids, the nucleotide sequence of the aph(3') gene from the E . coli R plasmid was determined and compared with the known aph(3') genes of Tn903 and Tn4352 . Three point mutations in the structural part of the cloned aph(3') gene caused amino acid changes in the enzyme molecule at positions 19, 27 and 48 beginning from the start codon . The structural part of the gene was followed by two stop codons and a long DNA region containing no nucleotide sequences homologous to the sequences of Tn903 or Tn4352 . Both the cloned aph(3') gene and Tn4352 were limited on the left by the spacer sequence and the insertion sequence IS176 . Twenty one base pairs deletion abolished the -35 sequence of the promoter suggested for the aph(3') gene of Tn4352 and resulted in formation of a fusion promoter utilizing the -35 box of IS176 and the -10 box of the aph(3') gene . The distance between the -35 and -10 sequences changed from 18 to 17 bp . Changes in the cloned aph(3') gene and the flanking DNA regions resulted in formation of a new promoter and loss of the right IS176 element.

Biofizika, 1989 May-Jun, 34(3), 464 - 7
{The effect of dimethylsulfoxide and thiourea on the water diffusion permeability of the E . coli membrane}; Sakharov BV et al.; It is shown that diffusive water permeability of E . coli cell membranes at 4-24 degrees C is in the range from 16.6 to 35.0 microM.s-1, that is close to erythrocyte membrane water permeability, but higher than that of lipid bilayers . Cryoprotectants (DMSO and thiourea) at the concentration of 1.0 M considerably decrease water bacterial membrane permeability . 1.5- and 2-fold, respectively . The obtained results are discussed in relation to two possible water transport ways through pores of the protein nature or lipid bilayer damages.

Immunol Lett, 1989 May, 21(2), 157 - 63
Expression of Shiga toxin epitopes in E . coli immunological characterization; McEwen J et al.; Synthetic oligodeoxynucleotides encoding for two peptides corresponding to residues 9-21 and 19-31 in the amino acid sequence of the B subunit of Shiga toxin were prepared and inserted into pTOZ plasmid, in phase with the lacZ gene . The resultant vectors were used for transfection of E . coli . The bacteria containing the recombinant DNA expressed the respective peptides in the form of fusion proteins with beta-galactosidase . The N-terminal region of the Shiga toxin B subunit, containing the two peptides, was previously identified as a relevant epitope of the toxin, leading to the induction of neutralizing antibodies . The present study demonstrates that the bacterial extracts containing the fusion proteins as the products of the recombinant vectors, when used for immunization of rabbits, elicited a humoral immune response which was specific toward the respective peptides . Furthermore, the antibodies cross-reacted with the intact Shiga toxin.

Biokhimiia, 1989 May, 54(5), 870 - 6
{Synthesis in E . coli cells of short RNA encoded in plasmids}; Dontsova OA et al.; The synthesis of 5S rRNA and 4.5S RNA in E . coli HB 101 cells harbouring plasmids pKK 5-1 and pKK 247-2 was studied . The plasmids were derived from pBK 322 and contained genes coding for 5S rRNA and 4.5S RNA with regulatory elements of an rRNA transcription operon rrn B . When the cells were grown on enriched or minimal media (2 and 0.3 duplications per hour), the synthesis of both 5S rRNA and 4.5S RNA was proportional to the gene dosage and was greater in the plasmid than in the host strain . Such RNA accumulation did not change the cell growth parameters and was thus not toxic for the cells . At high growth rates, the RNA synthesis in the cells became excessive, and the processing system was upset with the accumulation of RNA precursors . The fact confirms the hypothesis, according to which the whole rRNA operon is essential for its own feedback regulation.

Nucleic Acids Res, 1989 Apr 25, 17(8), 3179 - 97
Effect of nucleotide sequences directly downstream from the AUG on the expression of bovine somatotropin in E . coli; Tomich CS et al.; We have studied the expression of bovine somatotropin (BSt) to gain more understanding of various factors affecting translation in E . coli . The unmodified cDNA coding for mature bovine somatotropin does not produce significant amounts of BSt in E . coli using a pBR322-derived vector . However, a translation fusion with 16 codons from trpLE in front of BSt cDNA results in greater than 20% of total cell protein as the fusion product . Analysis of transcription by measuring the rate and integrity of the mRNA confirms that a post-transcriptional event is responsible for the poor expression of the BSt cDNA . There are two potential stem-loop structures in the 5' region of the mRNA which may interfere with translation . To study their effect on translation, lacZ fusions and oligonucleotide mutagenesis were carried out . The results demonstrate that the secondary structure involving the initiation codon blocks translation initiation . Removal of this stem-loop results in a 100-fold increase in BSt expression . However, the expression level is still low, amounting to only 0.5-1% of total cell protein . High level expression can be obtained by replacement of the beginning sequence of BSt cDNA with trpLE codons . These results suggest that in addition to the secondary structure, the nucleotide sequence or amino acid context within the beginning of BSt is incompatible with one of the steps in translation initiation.

Nucleic Acids Res, 1989 Apr 25, 17(8), 2933 - 45
In vivo gene expression directed by synthetic promoter constructions restricted to the -10 and -35 consensus hexamers of E . coli; Jacquet MA et al.; Two synthetic DNA sequences, carrying no other known E . coli promoter element than the consensus hexamers (CH) TTGACA (CH-35) and TATAAT CH(-10), spaced by 17 bp, were inserted in pBR329, in a position enabling transcription of the complete Cmr gene . The region upstream of the Cmr transcription start was carefully cleared of w.t . promoter elements (full deletion of the wild type (w.t.) Cmr promoter upstream +2 and large portion of an upstream coding sequence) . Both synthetic promoters, which differ only by the sequences of the spacers (non consensus, constrained in AT or GC) support in vivo high level Cmr gene expression . The GC rich spacer is associated with transcription start at the usual +1 position, but with the AT rich spacer, transcription starts at several places, mainly in CH(-10) . Rearranged promoter sequences derived from the synthetic ones upon transformation with partly ligated plasmids, yield new insights on the role of the standard CH pair, the size of the spacer and the sequence downstream of CH(-10).

FEBS Lett, 1989 Apr 24, 247(2), 361 - 6
Structural consequences of a one atom mutation on aspartate transcarbamylase from E . coli; Cherfils J et al.; Tyr-240 of the catalytic chain of aspartate transcarbamylase from E . coli has been substituted by Phe using site-directed mutagenesis . The regulatory mechanisms of the mutant enzyme have been shown to be slightly less effective than the wild-type enzyme . A study of the structural consequences of the mutation using solution X-ray scattering and computer simulations is reported here . No significant change from the wild-type enzyme is detectable in the quaternary structure . Simulations suggest that the only effect of the mutation is an increased mobility of the mutated side chain.

J Biomol Struct Dyn, 1989 Apr, 6(5), 971 - 84
Use of lead(II) to probe the structure of large RNA's . Conformation of the 3' terminal domain of E . coli 16S rRNA and its involvement in building the tRNA binding sites; Gornicki P et al.; The present work shows that lead(II) can be used as a convenient structure probe to map the conformation of large RNA's and to follow discrete conformational changes at different functional states . We have investigated the conformation of the 3' domain of the E . coli 16S rRNA (nucleotides 1295-1542) in its naked form, in the 30S subunit and in the 70S ribosome . Our study clearly shows a preferential affinity of Pb(II) for interhelical and loop regions and suggests a high sensitivity for dynamic and flexible regions . Within 30S subunits, some cleavages are strongly decreased as the result of protein-induced protection, while others are enhanced suggesting local conformational adjustments . These rearrangements occur at functionally strategic regions of the RNA centered around nucleotides 1337, 1400, 1500 and near the 3' end of the RNA . The association of 30S and 50S subunits causes further protections at several nucleotides and some enhanced reactivities that can be interpreted in terms of subunits interface and allosteric transitions . The binding of E . coli tRNA-Phe to the 70S ribosome results in message-independent (positions 1337 and 1397) and message-dependent (1399-1400, 1491-1492 and 1505) protections . A third class of protection (1344-1345, 1393-1395, 1403-1409, 1412-1414, 1504, 1506-1507 and 1517-1519) is observed in message-directed 30S subunits, which are induced by both tRNA binding and 50S subunit association . This extensive reduction of reactivity most probably reflects an allosteric transition rather than a direct shielding.

Wei Sheng Wu Xue Bao, 1989 Apr, 29(2), 113 - 6
{Subcloning of K88ac antigen gene of enterotoigenic E . coli and the restriction map of recombinant plasmid}; Zhang LY et al.; We had reported a recombinant E . coli RR1(pNZ8801) which was obtained from a wild strain E . coli 79-1454 . The recombinant plasmid was digested by EcoRI and generated three segments, medium segment (3.2Md) was removed, the largest and the smallest segment was ligased, then the mixture was transformed into E . coli RRI, screening Ap(r) Tc(s) clones, one of recombinants was named E . coli RR1(pNZ8802) . The recombinant plasmid molecular weight is smaller, but expression of K88ac antigen is higher than first cloning . Subcloning can adhere to mucosae of piglet's intesting . Therefore, the recombinant can be use for oval living vaccine.

Bioorg Khim, 1989 Apr, 15(4), 562 - 5
{Synthesis of oligoribonucleotides with the use of RNA polymerases of E . coli and immobilized synthetic DNA-templates}; Denisova LIa et al.; A new approach to synthesis of oligoribonucleotides is suggested, based on transcription by E . coli RNA polymerase of synthetic immobilized DNA-templates with AUG as primer . The approach has been experimentally verified by synthesis of two oligonucleotides, viz., a RNA fragment of the fr phage (16 nucleotides long) and a RNA fragment of the tickborne encephalitis virus (18 nucleotides long) . Fraction of the synthesized RNA fragments in the whole nucleotide material is about 20% . The templates can be used repeatedly . Sequences of the oligoribonucleotides were confirmed . Advantages of this approach and its usefulness for SP6 DNA-dependent RNA polymerase are discussed.

EMBO J, 1989 Apr, 8(4), 1271 - 7
Autoregulation and multiple DNA interactions by a transcriptional regulatory protein in E . coli pili biogenesis; Forsman K et al.; An operon mediating biogenesis of digalactoside-binding pilus-adhesin of serotype F13 in uropathogenic Escherichia coli includes the regulatory gene papB . The papB gene product was found to act as transcriptional activator of an operon which includes the papB gene and several pap cistrons encoding the proteins of the pilus polymer . Studies of how pap gene expression was affected by increasing amounts of PapB protein in the cells showed that high levels did not stimulate transcription but caused repression . Results from in vitro studies demonstrated that the PapB protein was a sequence-specific DNA-binding protein . Binding studies using gel mobility shift assays and DNase I protection (footprinting) showed that PapB protein binds to three separate sites . A sequence greater than 200 bp upstream of the promoter, and directly adjacent to a binding site for the cAMP receptor protein-cAMP complex, appeared as a preferential PapB binding site . A second site was localized to sequences overlapping the -10 region of the promoter and a third binding site was found within the coding sequence of the papB gene itself . The data suggest that the PapB protein has a dual function as activator/repressor of pilus-adhesin transcription and that its autoregulatory mode of action involves differential binding to separate sites.

Int J Radiat Biol, 1989 Apr, 55(4), 593 - 604
Radiosensitization of E . coli B/r by arylhydrazonopropanedinitriles; Crump PW et al.; Several arylhydrazonopropanedinitriles and an arylhydrazonopropane-diethyl ester (derivatives of well-known uncouplers of oxidative phosphorylation) have been studied with respect to their ability to radiosensitize E . coli B/r under oxic and hypoxic conditions . Of the compounds studied, 2-carboxyphenylhydrazono-propanedinitrile and 2-carboxyphenylhydrazonopropanediethylester were found to be the most efficient radiosensitizers under hypoxia, whilst the former compound was also found to provide radiosensitization under oxic conditions . Increased radiosensitization by 4-carboxyphenylhydrazonopropanedinitrile was observed on decreasing the pH of the irradiation incubation medium . The results are discussed with respect to the physicochemical properties of these compounds and their reactivity with thiols, for which data are presented.

Biochimie, 1989 Apr, 71(4), 411 - 6
Structure of cDNA of cytosolic aspartate aminotransferase of chicken and its expression in E . coli; Mattes U et al.; The structure of the mRNA of chicken cytosolic aspartate aminotransferase has been determined by analysis of cDNA and genomic clones . Two transcripts of different length were found that appear to arise from the alternate use of 2 polyadenylation signals in the 3' untranslated region . The expression product of the full-length construct in E . coli proved to be catalytically active and possessed the expected molecular weight.

Bioorg Khim, 1989 Apr, 15(4), 492 - 8
{Expression of synthetic human angiogenin gene in E . coli in cleaved beta-galactosidase-angiogenin protein}; Kovalenko SP et al.; A synthetic gene coding for human angiogenin was cloned in pUR290 plasmid in frame with beta-galactosidase, both parts of the resultant fused protein being joined through an Asp-Pro sequence . The fused protein, synthesised in E . coli cells upon IPTG induction and isolated as inclusion bodies, possessed angiogenic activity on the chick chorioallantois membrane and was cleaved upon acid treatment to yield free angiogenin.

Biochim Biophys Acta, 1989 Mar 16, 995(1), 54 - 8
Kinetic consequences of site-specific mutation of Glu-239----Gln in E . coli aspartate transcarbamylase: comparison with catalytic subunits and Phe-240 mutant enzyme; Hsuanyu Y et al.; The kinetic characteristics of E . coli aspartate transcarbamylase, altered by site-specific mutagenesis of Glu-239----Gln, have been determined by equilibrium isotope-exchange kinetics and compared to the wild-type system . In wild-type enzyme, residue Glu-239 helps to stabilize the T-state structure by multiple bonding interactions with Tyr-165 and Lys-164 across the c1-c4 subunit interface; upon conversion to the R-state, these bonds are re-formed within c-chains . Catalysis of both the {14C}Asp in equilibrium C-Asp and {32P}ATP in equilibrium Pi exchanges by mutant enzyme occurs at rates comparable to those for wild-type enzyme . Saturation with different reactant/product pairs produced kinetic patterns consistent with strongly preferred order binding of carbamyl-P prior to Asp and carbamyl-Asp release before Pi . The kinetics for the Gln-239 mutant enzyme resemble those observed for catalytic subunits (c3), namely a R-state enzyme (Hill coefficient nH = 1.0) and Km (Asp) approximately equal to 6 mM . The Glu-239----Gln mutation appears to destablize both the T- and R-states, whereas the Tyr-240----Phe mutation destablizes only the T-state.

Cell, 1989 Mar 10, 56(5), 891 - 904
Reverse transcriptase-dependent synthesis of a covalently linked, branched DNA-RNA compound in E . coli B; Lim D et al.; We have found a branched DNA-RNA compound in E . coli B, that is similar in its secondary structure, but not its nucleotide sequence, to the previously described branched DNA-RNA compounds in myxobacteria . This compound is not produced in E . coli K12 . We have cloned a 3.5 kb chromosomal segment of E . coli B, which, when transferred into E . coli K12, leads to the production of the DNA-RNA compound . We describe the isolation of the DNA-RNA compound, the determination of its nucleotide sequence, and the nucleotide sequence of the genes required for its formation . The sequence contains the coding regions for the DNA component, the RNA component, and an open reading frame encoding a reverse transcriptase . This reverse transcriptase is shown to be required for the formation of the DNA-RNA compound in vivo and in vitro.

Electrophoresis, 1989 Mar, 10(3), 165 - 72
Free flow electrophoresis as a method for the purification of enzymes from E . coli cell extract; Kuhn R et al.; The application of the four techniques of free flow electrophoresis (zone electrophoresis, isotachophoresis, isoelectric focusing and field step electrophoresis) for the purification of proteins from a complex protein mixture was investigated . For this purpose alpha-amylase (EC 3.2.1.1) from Aspergillus oryzae was added and reisolated from E . coli cell extract . The chosen enzyme and the biological extract are models for many industrial separation problems . In optimized experiments purity, purification factor, yield, throughput and efficiency were calculated . The best results were obtained with field step electrophoresis in combination with zone electrophoresis . High purity (0.82 mg enzyme/mg total protein) and high throughput (111 mL sample/h) were achieved using this technique . Field step electrophoresis gave the best throughput (330 mL sample/h), but low purity (0.63 mg enzyme/mg total protein) . This technique can also be used for a simple concentration of the sample . With zone electrophoresis a purity of more than 0.95 mg enzyme/mg total protein was obtained, which was the best of all techniques . However, the enzyme concentration was decreased due to dilution with buffer solution after the separation . Isotachophoresis was the most difficult technique, combined with a relatively low recovery of 31% of the enzyme activity . In a purification scheme, free flow electrophoresis is able to substitute one or even several chromatography steps with a negligible loss of biological activity.

Immunology, 1989 Mar, 66(3), 394 - 7
Comparison of concentration and avidity of specific antibodies to E . coli in breast milk and serum; Sennhauser FH et al.; To investigate the relationship between mucosal and systemic immunity we analysed the specific anti-Escherichia coli antibody concentration and avidity of IgA in colostrum and IgG in paired blood samples from 47 mothers giving birth to premature neonates . The avidity of each sample, expressed as an avidity index, was determined using a novel enzyme immunoassay (EIA)-based procedure, while specific antibody determinations were performed by means of conventional sandwich EIA techniques . All subjects had detectable antibody to E . coli in serum and breast milk . The median avidity index for specific IgA antibody in breast milk (3.53 M NH4SCN, range 2.77-4.90) was significantly higher (P less than 0.0001) than that for specific IgG antibody in serum (median 2.03 M NH4SCN, range 1.15-3.65) . Using Spearman correlation analysis, a weak but significant association was found between the avidity of colostral IgA antibody and the avidity of systemic IgG antibody to pooled E . coli polysaccharides (rs = 0.29, P = 0.02) . There was also a weak correlation between the concentrations of specific serum IgG antibody and of specific colostral IgA antibody (rs = 0.36, P = 0.006) . There was no correlation between the concentration of IgA anti-E . coli antibody in colostrum and the avidity of colostral IgA antibody (rs = 0.14, P less than 0.05) . Similarly, there was no correlation between the concentration and the avidity of serum IgG anti-E . coli antibody (rs = 0.23, P less than 0.05) . The findings of this study suggest independent regulation of concentration and avidity of specific IgA antibody in preterm breast milk . Similar results were seen for specific IgG antibody in serum . The correlations between systemic and mucosal antibody with respect to both concentration and avidity were significant, but are relatively weak and therefore suggest that there also may be independent factors which afford differential regulation of systemic and mucosal antibody responses.

Biofizika, 1989 Mar-Apr, 34(2), 322 - 3
{N,N'-dicyclohexylcarbodiimide-sensitive K+-dependent H+ secretion from anaerobically grown E . coli cells}; Trchunian AA et al.; Dependence of N,N'-dicyclohexylcarbodiimide (DCC)-sensitive H+ secretion of K+ activity was discovered . This dependence took place only in anaerobically grown bacteria, and only at the structural intact DCC-sensitive H+-ATPase complex and K+-ionophore Trk.

Biochimie, 1989 Mar, 71(3), 307 - 12
Structural analysis of human skeletal beta-tropomyosin produced in E . coli; Ferraz C et al.; We have cloned the cDNA coding the beta-tropomyosin of human muscle in an expression vector whose expression depends upon a promotor that can be induced by isopropyl-beta-thiogalactopyranoside . We show that a new protein was synthesized by bacteria containing the engineered plasmid . This protein was heat stable and reacted with antibodies against tropomyosin . We have purified this protein and further identified it by determining its amino acid composition and sequencing the NH2 terminal . Unlike the native muscle tropomyosin, the NH2 terminal is not acetylated and contains a methionine . The circular dichroism spectrum is compatible with 100% alpha-helices . These results show that the protein synthesized in E . coli possesses a native structure.

Mutat Res, 1989 Mar, 217(2), 123 - 34
KIN, a mammalian nuclear protein immunologically related to E . coli RecA protein; Angulo JF et al.; A polypeptide of about 120 kDa, called KIN, has been identified in rat FR 3T3 cells by immunoblotting using affinity-purified antibodies against the RecA protein of Escherichia coli (38 kDa) . The KIN protein as shown by fluorescent light microscopy and electron microscopy is essentially concentrated in the nucleus . Its level is higher in proliferating than in quiescent cells . Cell treatment with mitomycin C increases the level of the KIN protein . We sought similar proteins in other mammalian cells . Proteins with the same electrophoretic mobility were detected in mouse, monkey and human cell lines as well as in rat and mouse embryos.

Mol Microbiol, 1989 Mar, 3(3), 295 - 302
Molecular cloning and expression in Escherichia coli K-12 of the O101 rfb region from E . coli B41 (O101:K99/F41) and the genetic relationship to other O101 rfb loci; Heuzenroeder MW et al.; The gene cluster (rfb region) which determines the synthesis of O101 lipopolysaccharide (LPS) O-antigen was cloned from the Escherichia coli O101:K99:F41 reference strain B41 to give plasmid pPM1301 . The smallest subclones represented by pPM1305 and pPM1330 expressed O-antigen in E . coli K-12 similar to (but not identical to) B41, as judged by immunogold electron microscopy and silver staining of LPS separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) . At least six proteins were detected by minicell analysis of proteins encoded by pPM1305, which suggests that O-antigen synthesis is genetically complex . Restriction and deletion analysis demonstrated that a minimum of 8.9 kb and a maximum of 11.8 kb are required for O101 O-antigen biosynthesis in E . coli K-12 . Examination of LPS banding patterns of other O101 isolates by SDS-PAGE suggested heterogeneity of LPS structure . Southern DNA hybridization analysis using radiolabelled subclones of pPM1305 demonstrated that there was close relationship among the O101 ETEC isolates.

Acta Chir Scand, 1989 Mar, 155(3), 155 - 7
Factors influencing the outcome of E . coli peritonitis in rats; Andersson R et al.; Substances that may influence the course and outcome of intra-abdominal sepsis were investigated in an experimental model of Escherichia coli peritonitis in rats . All rats received an intraperitoneal injection of E . coli . In the first set of experiments, substances commonly contaminating the abdominal cavity after trauma were intraperitoneally injected, and the following mortality rates were found: saline solution (controls) 27%, hemoglobin solution 80% (p less than 0.01), whole blood 20% (p greater than 0.05), whole blood together with bile 93% (p less than 0.001) and bile 87% (p less than 0.01) . In the second set of experiments, intravenous injection of commonly used solutions gave mortality rates of 20% (controls) for saline solution, 80% for dextran (p less than 0.01) and 47% for Intralipid (p greater than 0.05) . E . coli peritonitis in rats thus was aggravated by intraperitoneal hemoglobin, bile or whole blood plus bile, and also by intravenous dextran.

Acta Chir Scand, 1989 Mar, 155(3), 151 - 4
Influence of dextran on blood clearance and organ distribution of radiolabelled E . coli and on survival in experimental E . coli septicemia; Andersson R et al.; Previous reports indicated impaired function of the reticuloendothelial system following administration of dextran . In the present experimental study, intravenous injection of Escherichia coli resulted in four deaths among 15 rats pretreated with i.v . dextran 70, but none in 15 controls (p less than 0.05) . To evaluate the influence of dextran on bacterial clearance from blood and distribution in organs, 125I-labelled, heat-killed E . coli was injected 1 or 24 hours following i.v . injection of 1.0 ml sterile saline solution or 1.0 ml dextran 70 . After both of these intervals, dextran resulted in significant (p less than 0.05) decrease of blood clearance compared with the controls . The organ distribution in counts/minute (cpm)/g tissue was compared in liver, spleen and lungs . Splenic uptake was reduced at both times following dextran administration, while there was increase in pulmonary uptake at 1 hour and in hepatic uptake at 24 hours post-dextran (p less than 0.005 and p less than 0.02, respectively) . Evaluation of cpm/total organ weight showed no change after dextran injection, except for increased (p less than 0.05) pulmonary uptake after 1 hour.

Biochim Biophys Acta, 1989 Mar 1, 1007(2), 158 - 66
The pnd gene in E . coli plasmid R16: nucleotide sequence and gene expression leading to cell Mg2+ release and stable RNA degradation; Sakikawa T et al.; The pnd gene promotes the degradation of stable RNA in the presence of rifampicin at 42 degrees C, but is repressed during normal growth (Ohnishi, Y . and Akimoto, S . (1980) J . Bacteriol . 144, 833-835) . We have determined the sequence of a third srnB-pnd-type gene, and have analyzed the effects of its expression from an inducible promoter . The nucleotide sequence of the pnd gene of the R16 plasmid exhibits an open reading frame for a polypeptide with 50 amino-acid residues, with high sequence homology to the pnd gene of a plasmid (R483) of a different incompatibility group . A possible base-paired stem and loop structure, which may participate in the regulation of gene expression, was detected between the promoter and the initiation codon, analogous to that in two comparable genes, srnB in the F and pnd in the R483 plasmid . When bacterial cells containing a lac-pnd fusion plasmid were incubated with a lac inducer at 30 degrees C, magnesium was released from the cells in bulk, and spheroplasts of the cells lysed even in hypertonic solution . Furthermore, when Mg2+ efflux was inhibited in the medium containing 5 mM Mg2+ or in Tris-HCl buffer, the degradation of stable RNA at 42 degrees C was inhibited . These results suggest that expression of the pnd gene effects a release of cellular magnesium by a membrane alterations, resulting in the stable RNA degradation at a higher temperature.

Biochem Biophys Res Commun, 1989 Feb 28, 159(1), 87 - 94
Chemical synthesis and expression of the HIV-1 protease gene in E . coli; Louis JM et al.; The 297bp HIV-1 protease gene was constructed from five discrete synthetic fragments and expressed in E . coli . A soluble protein product of 11.5 Kd was detected by immunoblotting using protease specific antisera . A quantitative assay system, utilizing a synthetic nonapeptide spanning the cleavage site between p17-p24 in the gag polyprotein, was used to measure the specific protease activity in crude extracts . The protease hydrolyzed tyrosyl-proline bonds with an approximate specific activity of 43 pmoles/min/micrograms of total protein . The chemical synthesis of the protease gene and it's expression provides a feasible method for rapid mutant analysis, important for structure-function studies and rational design of potential inhibitors.

Nucleic Acids Res, 1989 Feb 25, 17(4), 1475 - 91
New RNA-protein crosslinks in domains 1 and 2 of E . coli 30S ribosomal subunits obtained by means of an intrinsic photoaffinity probe; Hajnsdorf E et al.; Functionally active 70S ribosomes containing 4-thiouridine (s4U) in place of uridine were prepared by a formerly described in vivo substitution method . Proteins were crosslinked to RNA by 366 nm photoactivation of s4U . We observe the systematic and characteristic formation of 30S dimers; they were eliminated for analysis of RNA-protein crosslinks . M13 probes containing rDNA inserts complementary to domains 1 and 2 of 16S RNA from the 5'end up to nucleotide 868 were used to select contiguous or overlapping RNA sections . The proteins covalently crosslinked to each RNA section were identified as S3, S4, S5, S7, S9, S18, S20 and S21 . Several crosslinks are compatible with previously published sites for proteins S5, S18, S20 and S21; others for proteins S3, S4, S7, S9, S18 correspond necessarily to new sites.

Cell, 1989 Feb 24, 56(4), 641 - 9
A division inhibitor and a topological specificity factor coded for by the minicell locus determine proper placement of the division septum in E . coli; de Boer PA et al.; The E . coli minicell locus (minB) was shown to code for three gene products (MinC, MinD, and MinE) whose coordinate action is required for proper placement of the division spetum . Studies of the phenotypic effects of expression of the three genes, alone and in all possible combinations, indicated the following: cell poles contain potential division sites that will support additional septation events unless specifically inactivated; the minC and minD gene products act in concert to form a nonspecific inhibitor of septation that is capable of blocking cell division at all potential division sites; and the minE gene codes for a topological specificity factor that, in wild-type cells, prevents the division inhibitor from acting at internal division sites while permitting it to block septation at polar sites.

Nucleic Acids Res, 1989 Feb 11, 17(3), 1139 - 57
Terminal sequences do not contain the rate-limiting decay determinants of E . coli cat mRNA; DeFranco C et al.; The mechanism of E . coli chloramphenicol acetyltransferase (cat) mRNA decay was investigated . Alteration of the 5' untranslated terminus does not appear to have an effect on the turnover rate of the mRNA . Similarly, changes at the 3' terminus of the message, including the addition of a stable stem and loop structure, do not affect the half-life of the message . The data suggest that 5' and 3' terminal untranslated sequences do not contain the rate-limiting determinants for cat message decay . Decay rates for various segments of the cat mRNA were measured and indicate that all regions of the message have similar stabilities . The current model of cat mRNA degradation involves a rate-limiting endonucleolytic decay event that occurs internal to the message followed by degradation of the cleavage products.

Sci China B, 1989 Feb, 32(2), 186 - 92
The expression of enterotoxin A-B+ gene of V . cholerae in E . coli; Ma QJ et al.; The cloning in E . coli of a cholerae toxin gene that is A-B+ has been successfully constructed by using DNA recombinant techniques . E . coli cells carrying the recombinant plasmid pMM-CTB have been shown to produce a large amount of CTB subunits which are secreted as extracellular proteins.

EMBO J, 1989 Feb, 8(2), 569 - 76
Isolation of point mutations that affect the folding of the H chain of human ferritin in E.coli; Luzzago A et al.; We have approached the problem of folding and assembly of the heavy (H) chain of human ferritin by isolating point mutations that affect this process . Apoferritin is an ideal model system to approach the problem of protein folding and assembly into multimeric structures . We have developed a recombinant hybrid molecule that allows us to select for ferritin mutants in which the folding-assembly process is altered or completely impaired . The selection procedure is based on a recombinant protein which consists of a fusion between the H chain of human ferritin and the alpha-peptide of beta-galactosidase . In the wild type situation, the alpha-peptide domain is segregated inside the apoferritin shell upon assembly and is unable to interact with the substrate and perform its enzymic function . We show that by selecting for mutations that restore beta-galactosidase activity we are able to identify ferritin mutations that affect the folding-assembly process . The selective procedure was applied to the analysis of the amino acid side chains that are important for the attainment of the correct conformation of the carboxy-terminal E helix in the 4-fold axis.

Klin Wochenschr, 1989 Feb 1, 67(3), 113 - 8
A rapid phospholipase A2 bioassay using 14C-oleate-labelled E . coli bacterias; Meyer T et al.; Two methods of phospholipase A2 determination using 14C-labelled E . coli bacterias as substrate were compared . One method works with a filter membrane for separation of cleaved 14C-oleate from remaining phospholipids, the other uses the well-known thin-layer chromatography for lipid analysis . Some features of human serum phospholipase A2 regarding pH and Ca2+ dependency were investigated . Possible sources of errors were discussed . It was shown that either method can differentiate between normal and pathologically elevated phospholipase A2 levels, but that the filter method is superior in terms of sensitivity and workload.

Eur J Biochem, 1989 Feb 1, 179(2), 399 - 404
Modulation of the affinity of the single-stranded DNA-binding protein of Escherichia coli (E . coli SSB) to poly(dT) by site-directed mutagenesis; Bayer I et al.; A vector for site-directed mutagenesis and overproduction of the Escherichia coli single-stranded-DNA-binding protein (E . coli SSB) was constructed . An E . coli strain carrying this vector produces up to 400 mg pure protein from 25 g wet cells . The vector was used to mutate specifically the Phe60 residue of E . coli SSB . Phe60 had been proposed to be located near the single-stranded-DNA-binding site . Substitution of the Phe60 residue by Val, Ser, Leu, His, Tyr and Trp gave proteins with no or only minor conformational changes, as detected by NMR spectroscopy . The affinity of the mutant E . coli SSB proteins for single-stranded DNA decreased in the order Trp greater than Phe (wild-type) greater than Tyr greater than Leu greater than His greater than Val greater than Ser, leading to the conclusion that position 60 is a site of hydrophobic interaction of the protein with DNA.

FEBS Lett, 1989 Jan 30, 243(2), 299 - 302
Nucleotide residues of tRNA, directly interacting with proteins within the complex of the 30 S subunit of E . coli ribosome with poly(U) and NAcPhe-tRNA(Phe); Abdurashidova GG et al.; With the aid of photoinduced tRNA-protein cross-linking, nucleotide residues A21, U45 and U60 were shown to interact directly with proteins S5, S7 and S9 respectively, in the complex of the 30 S subunit of E . coli ribosome with poly(U) and NAcPhe-tRNA(Phe) . These nucleotide residues are located in the central part of the L-form in the tertiary structure of RNA.

FEBS Lett, 1989 Jan 30, 243(2), 293 - 8
Identification of the magnesium, europium and lead binding sites in E . coli and lupine tRNAPhe by specific metal ion-induced cleavages; Marciniec T et al.; The Pb, Eu and Mg-induced cleavages in E . coli and lupine tRNAPhe have been characterized and compared with those found in yeast tRNAPhe . The pattern of lupine tRNAPhe hydrolysis closely resembles that of yeast tRNAPhe, while several major differences occur in the specificity and efficiency of the E . coli tRNAPhe hydrolysis . The latter tRNA is cleaved with much lower yield in the D-loop, and interestingly, cleavage is also detected in the variable region, that is highly resistant to hydrolysis in eukaryotic tRNAs . The possible location of tight Pb, Eu and Mg binding sites in E . coli tRNAPhe is discussed on the basis of the specific hydrolysis data.

FEBS Lett, 1989 Jan 30, 243(2), 186 - 92
Synthesis of a (desSer1 Ile29 Leu89) chicken cystatin gene, expression in E . coli as fusion protein and its isolation; Auerswald EA et al.; A synthetic gene coding for the cysteine proteinase inhibitor (desSer1 Ile29 Leu89) chicken cystatin was cloned and expressed in E . coli . The gene was assembled from 12 oligonucleotides and inserted into vector pUC 8 . Expression as fusion protein was performed in a temperature-inducible E . coli system . The expression product was synthesized as 20% of total E . coli protein . The fusion protein was purified, the chicken cystatin homologue was split off with CNBr and the N-terminal sequence confirmed up to position 37 . The properties of the purified material correspond to those of natural chicken cystatin . The recombinant cystatin variant binds anti-chicken cystatin IgG, is inhibitorily active and displays Ki values with papain and with cathepsin B similar to those determined for natural chicken cystatin.

FEMS Microbiol Lett, 1989 Jan 15, 48(2), 129 - 32
Detection of a homologue to an E . coli glutamate synthase gene in a cyanobacterium; Lightfoot DA et al.; The gltBY locus of E . coli, which codes for the two subunits of pyridine nucleotide dependent glutamate synthase, was used as a probe to detect homologues in genomic DNA of Synechococcus PCC6301, a unicellular cyanobacterium . Non-overlapping probes from gltB detected a single homologue with extensive homology, however gltY probes do not detect a strong homologue . The possibility that the gltB homologue encodes a ferredoxin-dependent glutamate synthase of the type found in cyanobacteria, algae and plants is discussed.

Nucleic Acids Res, 1989 Jan 11, 17(1), 317 - 34
The interaction of E . coli integration host factor and lambda cos DNA: multiple complex formation and protein-induced bending; Kosturko LD et al.; The interaction of E . coli's integration Host Factor (IHF) with fragments of lambda DNA containing the cos site has been studied by gel-mobility retardation and electron microscopy . The cos fragment used in the mobility assays is 398 bp and spans a region from 48,298 to 194 on the lambda chromosome . Several different complexes of IHF with this fragment can be distinguished by their differential mobility on polyacrylamide gels . Relative band intensities indicate that the formation of a complex between IHF and this DNA fragment has an equilibrium binding constant of the same magnitude as DNA fragments containing lambda's attP site . Gel-mobility retardation and electron microscopy have been employed to show that IHF sharply bends DNA near cos and to map the bending site . The protein-induced bend is near an intrinsic bend due to DNA sequence . The position of the bend suggests that IHF's role in lambda DNA packaging may be the enhancement of terminase binding/cos cutting by manipulating DNA structure.

Free Radic Res Commun, 1989, 8(1), 1 - 9
The molecular genetics of superoxide dismutase in E . coli . An approach to understanding the biological role and regulation of SODS in relation to other elements of the defence system against oxygen toxicity; Touati D; Superoxide-mediated oxidative stress initiates a chain of events resulting in numerous cellular injuries . We have used genetics and E . coli to investigate the role and regulation of superoxide dismutase (SOD) and its relationship with the other constituents of the oxygen toxicity defence system . Structural SOD genes have been cloned and sequenced, permitting us to refine structural analysis and to isolate SOD-deficient mutants . The conditional oxygen sensitivity of these mutants, together with their increased mutation rate, demonstrated the essential biological role of SOD . Furthermore the complementation of SOD-lacking E . coli deficiencies by introducing a plasmid containing the gene encoding the human SOD supported the proposal that superoxide dismutation is the physiological function of SOD . Regulation of the MnSOD, through which the global SOD level in E . coli is modulated, has been studied using operon and protein fusions with the lactose operon, and led to the conclusion of a multicontrol of MnSOD . Isolation and characterization of regulation mutants are in progress, with the aim of identifying effectors and targets involved in the response to superoxide-mediated oxidative stress.

Oncogene Res, 1989, 5(1), 61 - 6
Expression of human and viral ets genes in E . coli production of human ets-2-specific monoclonal antibodies; Seth A et al.; Full-length and deletion variants of the ets proto-oncogene and v-ets oncogene of E26 virus were expressed in E . coli using the previously described expression vectors pANH1, pJL6 and pJLA16 . The recombinant proteins were expressed at greater than 5% of the total cellular proteins and were characterized by Western blot analysis using ets-specific antipeptide antibodies . A number of monoclonal antibodies were raised against p35, the expressed product of the partial human ets-2 proto-oncogene construct . These monoclonal antibodies are highly specific and recognize the p56 ets-2 product from several human cell lines.

Ann Ist Super Sanita, 1989, 25(1), 163 - 9
Molecular analysis of mutagenesis in E . coli; Fuchs RP; In this paper we present a general strategy that is suitable for the analysis of the forward mutation spectrum induced by any physical or chemical mutagen or carcinogen . The assay is based upon the inactivation of the tetracycline resistance gene located on plasmid pBR322 . Plasmid DNA is treated in vitro with the mutagen and transformed into the host bacteria of choice . Mutant clones are selected and analysed by sequencing . We present also two techniques that allow the determination of the DNA modification spectrum . The comparison for a given mutagen of the modification spectrum and the induced mutation spectrum permits the identification of hot spot sequences . Using different chemical mutagens (derivatives of the carcinogenic aromatic amide N-2-acetylaminofluorene, cis-platinum, etc.) this assay was found to be able to detect the different classes of mutagenic events: base substitutions, frameshifts, insertions and deletions . The advantages and limitations of this assay are discussed.

Bioorg Khim, 1989 Jan, 15(1), 78 - 89
{Klenow fragment of DNA-polymerase I from E . coli . III . The role of internucleotide phosphate groups of the matrix in its binding with the enzyme}; Volchkova VA et al.; The modification of Klenow fragment of DNA polymerase I E . coli was investigated by the affinity reagents d(Tp)2C{Pt2+(NH3)2OH}(pT)7 and d(pT)2pC{Pt2+(NH3)2OH}(pT)7 . The template binding site of the enzyme was modified by these reagents in the presence of NaF (5 mM), which inhibits selectively the 3'----5'-exonuclease activity of the enzyme and therefore prevents the reagent from degradation . NaCN destroyed covalent bonds between reagents and enzyme, restoring activity of the Klenow fragment . The affinity of different ligands (inorganic phosphate, nucleoside monophosphates, oligonucleotides) to the template binding site of Klenow fragment was estimated . Minimal ligands capable to bind with the template site were shown to be triethylphosphate (Kd 290 microM) and phosphate (Kd 26 microM) . Ligand affinity increases by the factor 1.76 per an added (monomer unit from phosphate to d(pT) and then for oligonucleotides d(Tp)nT (n 1 to 19-20) . At n greater than 19-20, the ligand affinity remained constant . The complete ethylation of phosphodiester groups lowers affinity of the oligothymidylates to the enzyme by approximately 10 times, and comparable decrease of Pt2+-oligonucleotide affinity to polymerase is caused by the absence of Mn2+-ions . The data obtained led to suggestion that one Me2+-dependent electrostatic contact of the template phosphodiester group with the enzyme takes place (delta G = -1.45...-1.75 kcal/mole) . Formation of a hydrogen bond with the oxygen atom of P = O group of the same template phosphate is also assumed (delta G = -4.8...-4.9 kcal/mole) . Other template internucleotide phosphates do not interact with the enzyme but the bases of oligonucleotides take part in hydrophobic interactions with the template binding site . Gibbs energy changes by -0.34 kcal/mole when the template is lengthened by one unit.

Folia Biol (Praha), 1989, 35(2), 65 - 77
Cloning of Japanese quail ovalbumin cDNA in E . coli; Klaudiny J et al.; Japanese quail oviduct total cDNA was synthesized from total mRNA by the classical method, using AMV reverse transcriptase, the Klenow fragment of DNA polymerase I and S1 nuclease . The results of the synthesis of total ss-cDNA partially differed from those published for the synthesis of chicken total ss-cDNA . The presumed causes of the differences in the complete reverse transcription of mRNAcon and incomplete reverse transcription of mRNAlys and a large part of mRNAov in our case are discussed . An atypical strategy was used for cloning full-length cDNAov . The total cDNA was dC-tailed, then fractionated by analytical agarose electrophoresis and 4 cDNA fractions of different lengths were isolated from the gel using DEAE cellulose membranes . A cDNA fraction about 1500-2500 bp long containing full-length cDNAov was annealed with dG-tailed PstI-linearized plasmid pBR322 and cloned into competent E . coli DHl cells . Seventy-two clones were screened for the presence of full-length cDNAov, initially by insert size and then by means of hybrid-arrested translation . Four clones containing 1900-1980 bp cDNAov were obtained . The cDNA ends in one of these clones were sequenced . Comparison of these sequences with those of chicken mRNAov indicated that almost full-length cDNAov's had been cloned . They lacked a small number of nucleotides at their 5' ends, which had probably been split off during the degradation of the hairpin loop by S1 nuclease . A sequence of 134 bases from the 5' end of mRNAov is presented and compared with the known sequence of chicken mRNAov . The advantages of the cloning strategy employed, in particular, its cloning efficiency and the possibility of simultaneously identifying clones of also other oviduct cDNA species (in this work: cDNAY and, tentatively, cDNAcon), are discussed.

Arch Esp Urol, 1989 Jan-Feb, 42(1), 11 - 7
{Morphopathologic study of renal glomeruli in Wistar rats in shock experimentally induced with E . coli}; Mendez Sanchez A et al.; The present study analyzed the structural and ultrastructural changes observed in the kidneys of Wistar rats inoculated with E . coli bacteria suspended in saline solution . For the study, we divided 64 Wistar rats into 2 groups . Two ml . of the suspension containing 9.5 X 10(10) E . coli 0 group 26 CECT, no . 351 were given to the rats in group A via the oral route . Rats in group B were inoculated with 1 ml . of the same suspension . Structurally, we observed an increased glomerular area caused by the increased number and activation of mesangial cells . These showed developed organoids, podocytes with dilated organoids of the cytoplasmic vacuolar system, and very fine, disorganized pedicels . The parietal cells revealed vacuolized cytoplasm, and the basement membranes of the glomerular capillaries were thickened and arranged in layers . In the lumen of the glomerular, vessels, we observed histiocytic elements on endothelial walls, with large amounts of lysosomal elements and residual bodies . Inoculation of the E . coli suspension causes renal shock, which is more intense when innoculation is via the intraperitoneal route, causing severe changes in organ function.

Radiobiologiia, 1989 Jan-Feb, 29(1), 23 - 9
{The mutagenic effect of accelerated heavy ions on E . coli cells}; Amirtaev KG et al.; In studying E . coli mutation rate as a function of dose of different types of ionizing radiation it was found that mutagenic efficiency of helium ions (LET-22, 54 and 72 keV/microns) was higher than that of gamma-rays . As LET increased the mutagenic efficiency decreased . The mutation rate for all types of radiation under study was both a power function and a linear-quadratic function of dose.

J Steroid Biochem, 1989 Jan, 32(1A), 5 - 11
DNA binding of glucocorticoid receptor protein A fusion proteins expressed in E . coli; Bonifer C et al.; In an effort to obtain large quantities of glucocorticoid receptor (GR) protein for functional and structural studies, several truncated versions of the human glucocorticoid receptor (hGR) have been expressed in E . coli as C-terminal fusion proteins with protein A . The amount of expressed protein was between 5 and 25 mg/l in the culture . South-Western blotting was initially used to demonstrate the DNA binding capacity of fusion proteins containing the DNA binding domain of GR . The hybrid proteins were highly enriched in the insoluble fraction after cell lysis . For further purification and characterization the fusion proteins were solubilized in 8 M urea . The concentration of denaturing agent was reduced by dilution and the fusion proteins were allowed to refold . The renatured GR protein A fusion proteins bound to DNA in a nitrocellulose filter binding assay . We also show that it is possible to purify the renatured fusion protein to apparent homogeneity using a single chromatographic step on DNA-cellulose.

Mutat Res, 1989 Jan, 210(1), 93 - 102
On the possible role of cytosine deamination in delayed photoreversal mutagenesis targeted at thymine-cytosine dimers in E . coli; Ruiz-Rubio M et al.; While delayed photoreversal (PR) mutagenesis has been interpreted as a measure of misincorporation step in targeted mutagenesis, the specificity to produce glutamine tRNA suppressor mutations (C to T transitions) at sites in DNA where a thymine-cytosine dimer (T = C) may target mutation suggests a deamination model: deamination T = C to T = U and trans-U DNA replication after PR . We describe here two enquires that did not support the latter model: (a) Uracil DNA glycosylase activity as estimated from the restricted plating efficiency of phage T5 containing uracil-substituted DNA showed no variation that might allow an exceptional opportunity for mutation at U in DNA, and (b) . The kinetics of delayed PR mutagenesis were unaltered if UV-irradiated cells were held in buffer suspension for 2 h at 41 degrees C (a procedure known to allow deamination T = C to T = U) and then assayed . Other results with cells containing both umuC and ung (uracil DNA glycosylase) defects showed the magnitude of T = C deamination sufficient to provide T = U at the critical site of mutation to an extent greater than the mutation frequencies produced by delayed PR mutagenesis, and considerations of the kinetics led to the suggestion that the deamination model could apply if there were an optimum period 30-130 min post-UV for efficient recovery of DNA replication after PR . The results underscored the feasibility of delayed PR mutagenesis by deamination and trans-U replication, but a selection between the two models could not be determined.

Free Radic Res Commun, 1989, 8(1), 47 - 53
Effects of E . coli 0111.B4 lipopolysaccharide on spin-labelled murine macrophage and hepatocyte membranes; Jackson SK et al.; Macrophages and hepatocytes from normal and BCG-primed mice have been spin-labelled in their membranes with 5- and 16-doxyl stearic acid . Incubation of spin-labelled cells from BCG-primed animals with lipopolysaccharide from E . coli 0111.B4 produced a detectable and transient disturbance in the cell membranes as reflected by an increase in the order parameter measured from the electron spin resonance spectra of 5-doxyl-stearate . This membrane disturbance was maximal at 3-4 hours of incubation and was only detected with cells from mice primed with BCG . Spectra obtained from the 16-doxyl-stearate-labelled cells showed no change in order parameter on incubation with lipopolysaccharide.

Mol Gen Mikrobiol Virusol, 1989 Jan, (1), 19 - 24
{Transposition of insertional sequence IS21 in E . coli cells activated by an exogenous promotor}; Dubeikovskii AN; Transpositional activity of the IS21-element was found to be activated during its cloning in the pUC18 vector plasmid . The activated IS-element restores ability to transposition of the deficient IS21-element present in the same cell . Simultaneously with the activated transposition the increase in frequencies of precise exclusion of IS-element is registered . The activation of IS-element is concluded to be directed by exogenous promoter.

C R Acad Sci III, 1989, 308(14), 401 - 6
{Neutralising properties for HIV virus of hybrid protein MalE-CD4 expressed in E . coli and purified in 1 step}; Clement JM et al.; Genetic fusions allowing the expression in E . coli of hybrid proteins between a bacterial periplasmic maltose binding protein (MalE) and the CD4 molecule (the receptor of the HIV virus) have been constructed . One of them has kept most of the properties of each constituent: it is exported, can be purified in one step on an affinity column, interacts with anti-MalE and anti-CD4 antibodies, binds HIV gp 120 protein and inactivates HIV virus in an in vitro test.

Folia Biol (Praha), 1989, 35(1), 35 - 41
Expression of a bovine leukaemia virus envelope fusion protein in E . coli; Zajac V et al.; An expression plasmid containing 1.224 bp of the bovine leukaemia virus (BLV) env gene was constructed . The polypeptides encoded by six recombinant plasmids were analysed by electrophoretic transfer blot analysis . Two new proteins of 150-160 kDa and 60 kDa, respectively, were found in whole cellular extracts using sera of naturally infected cattle and/or by use of mouse monoclonal antibodies against gp51.

EMBO J, 1989 Jan, 8(1), 83 - 90
Expression of active hormone and DNA-binding domains of the chicken progesterone receptor in E . coli; Eul J et al.; Bacterially-expressed fusion proteins containing the DNA-(region C) or hormone-binding (region E) domains of the chicken progesterone receptor (cPR) fused to the C terminus of Escherichia coli beta-galactosidase were analysed for the specificity of interaction with natural and synthetic hormone-responsive elements (HREs) and progestins, respectively . The purified fusion protein containing the progestin-binding domain bound progesterone with an apparent Kd of 1.0-1.5 nM and was specifically photocross-linked with the synthetic progestin R5020 in crude bacterial lysates . Labelling of intact bacterial cells with {3H}R5020 revealed that the majority, if not all, of the bacterially produced hormone-binding domain was active . No differences in the binding to a synthetic palindromic glucocorticoid/progestin-responsive element (GRE/PRE) were found when the bacterially produced cPR DNA-binding domain was compared in methylation interference assays with the full-length chicken progesterone receptor form A expressed in eukaryotic cells . The study of dissociation kinetics, however, revealed differences in the half-life of the complexes formed between the palindromic GRE/PRE and either the receptor form A or the fusion protein containing the cPR DNA-binding domain . DNase I protection experiments demonstrated that the bacterially produced region C of the cPR generated specific 'footprints' on the mouse mammary tumour virus long terminal repeat (MMTV-LTR) which were nearly identical to those previously reported for the rat glucocorticoid receptor.

Radiobiologiia, 1989 Jan-Feb, 29(1), 30 - 5
{The effect of genotype on the induction of prophage lambda in E . coli cells by ionizing radiation of various LET}; Bonev MN et al.; A study was made of the influence of the repair genotype on lambda prophage induction by ionizing radiation of different LET in lysogenic E . coli cells . Bacterial strains W3110, P3478, GC244, and 30SO were exposed to gamma-rays and helium ions of 22 keV/microns . Induction of the prophage in GC244 and 30SO strains deficient by lexA and recA genes was either inhibited (GC244) or lacking (30SO) . Inducibility of P3478 carrying polA mutation was 12 and 5 times as high as that of the wild type strain after exposure to gamma-radiation and helium ions, respectively.

Acta Leprol, 1989, 7 Suppl 1, 212 - 6
Transformation of Mycobacterium smegmatis with E . coli plasmids; Zainuddin ZF et al.; One limiting factor in the studies of tuberculosis and leprosy is the lack of a versatile system for genetic analysis and manipulation of mycobacteria . One strategy used in constructing a plasmid vector for transforming Mycobacterium smegmatis was to insert fragments of a mycobacterial plasmid into an Escherichia coli plasmid . We found that the parental E . coli plasmid is capable of self-replication in M . smegmatis yielding chloramphenicol-resistant colonies . Plasmids from different passages of one M . smegmatis transformant were recovered and characterised by restriction digest analysis . The plasmid from the earlier passage was found to be indistinguishable from the original plasmid by restriction analysis . Plasmids from later preparations, however, were found to have undergone modifications in the M . smegmatis host resulting in an apparent increase in transformation efficiency for M . smegmatis . These plasmids can be used as a shuttle vector for the genetic manipulation of mycobacterial species.

Prikl Biokhim Mikrobiol, 1989 Jan-Feb, 25(1), 3 - 14
{Beta-galactosidase from E . coli as a marker in immunoenzyme analysis}; Markarian AN et al.; In the present review the authors analyze factors influencing sensitivity of the enzyme immunoassay (EIA) . beta-Galactosidase from E . coli was chosen as a marker enzyme . Physico-chemical properties of the enzyme, detection methods and various ways of obtaining chemical conjugates with antigens and antibodies are discussed . Some examples of using beta-galactosidase as a label in homogeneous and heterogeneous EIA are given which enables different compounds to be analyzed with a high sensitivity . New approaches employing gene engineering for constructing "fusions" between beta-galactosidase and antigens (alternative to chemical conjugates) are discussed that can be used in the near future.

Acta Biochim Pol, 1989, 36(3-4), 195 - 203
Crystallographic studies on E . coli Trp aporepressor; Joachimiak A et al.; Two crystal forms of Trp aporepressor, an inactive, unliganded form of Trp repressor have been obtained which are suitable for high resolution X-ray diffraction analysis . Trp aporepressor crystallizes in two forms: orthorhombic, P 2(1)2(1)2 and tetragonal P 4(1) (or P 4(3} which diffract to 1.8 A and 2.4 A, respectively . The orthorhombic crystals contain one monomer in the asymmetric unit, therefore the twofold axis relates two subunits of the dimer as in the case of the previously described Trp repressor (R . Schevitz et al., 1985, Nature, 317, 782-786) and Trp pseudorepressor (C . Lawson & P . B . Sigler, 1988, Nature, 333, 869-871) . The tetragonal crystals have two dimers in the asymmetric unit and are nearly isomorphous with the tetragonal crystals of Trp repressor and Trp pseudorepressor grown under similar conditions but in the presence of an activator or inhibitor, respectively.

Biochimie, 1989 Jan, 71(1), 167 - 74
Involvement of ion channels in the transport of phage DNA through the cytoplasmic membrane of E . coli; Letellier L et al.; Upon infection, phage DNA is transported through the bacterial cytoplasmic membrane . This crossing is accompanied by a transient increase in the permeability of the cytoplasmic membrane toward ions and small solutes . This has led several authors to propose that DNA might cross the cytoplasmic membrane through channels . In the first part of the review we present data that we obtained with phage T4 and that strongly support this proposal . We then present the structural and ionic characteristics of these channels . In the second part, we summarize data obtained by several authors concerning the permeability changes induced by different phages and show that these results are compatible with a model of phage DNA transfer through channels . Finally, we discuss the possible origin of these channels.

Acta Chir Scand, 1989, 155(1), 7 - 13
Influence of aprotinin, a protease inhibitor, on porcine E . coli shock . Studies on coagulation, fibrinolytic and hemodynamic response; Svartholm E et al.; The effect of a protease inhibitor, aprotinin, on hemostasis (a2M, a2AP, AT III, prothrombin-convertin activity, fibrinogen, fibrinogen monomers and fibrinolytic activity on fibrin plates) was investigated in pigs with septic shock . Anesthetized pigs were given live Escherichia coli i.v . (n = 7), or aprotinin (1,000,000 KIE i.v.) 15 min before live E . coli (n = 6), or Ringer's solution only (sham controls, n = 8) . Septic shock developed in all E . coli-infused pigs . Pulmonary vascular resistance increased, platelet and leukocyte counts fell and signs of systemic activation of the coagulation and fibrinolytic systems appeared in the E . coli groups, but aprotinin attenuated the effects on these systems and also on the pulmonary circulation . Five of the six aprotinin-pretreated pigs survived, but none of the seven with septic shock and no pretreatment . Thus the shock induced by infusion of live E . coli and the resultant changes in the coagulation and fibrinolytic systems and in cardiopulmonary hemodynamics were diminished by aprotinin pretreatment.

Biochim Biophys Acta, 1988 Dec 20, 951(2-3), 255 - 60
Nucleotide excision by E . coli DNA polymerase I in proofreading and non-proofreading modes; Lecomte PJ et al.; Escherichia coli DNA polymerase I exists in at least two distinct kinetic forms . When it binds to a template, the proofreading activity is usually switched off . As the enzyme progresses along the template, it becomes more and more competent for excision . This phenomenon introduces a link between fidelity and processivity . Processivity is best studied when the chain-length distributions of synthesized polymers are stationary . Even then, however, one cannot avoid multiple initiations on a given template by the same molecule of the enzyme . When synthesis is initiated with primers of lengths 15 or 20, a strange phenomenon is observed . It seems that the polymerase starts by hydrolyzing the primer down to a length of 7-10 nucleotides and only then starts to add nucleotides . It does so in a low-accuracy mode, suggesting that, while the exonuclease is clearly active, it does not contribute to proofreading . The warm-up of the proofreading function is therefore reinterpreted as a switch between two modes of behaviour: a mode 1 of low accuracy in which the 3'----5' exonuclease, while active, is uncoupled from the polymerase and does not contribute to proofreading, and a mode 2 of high accuracy in which the exonuclease is kinetically linked to the polymerase activity.

Science, 1988 Dec 9, 242(4884), 1415 - 8
Human insulin-degrading enzyme shares structural and functional homologies with E . coli protease III; Affholter JA et al.; A proteinase with high affinity for insulin has been proposed to play a role in the cellular processing of this hormone . A complementary DNA (cDNA) coding for this enzyme has been isolated and sequenced . The deduced amino acid sequence of the enzyme contained the sequences of 13 peptides derived from the isolated protein . The cDNA could be transcribed in vitro to yield a synthetic RNA that in cell-free translations produced a protein that coelectrophoresed with the native proteinase and could be immunoprecipitated with monoclonal antibodies to this enzyme . The deduced sequence of this proteinase did not contain the consensus sequences for any of the known classes of proteinases (that is, metallo, cysteine, aspartic, or serine), but it did show homology to an Escherichia coli proteinase (called protease III), which also cleaves insulin and is present in the periplasmic space . Thus, these two proteins may be members of a family of proteases that are involved in intercellular peptide signaling.

Biochim Biophys Acta, 1988 Dec 2, 957(3), 455 - 8
Kinetic mechanism of catalytic subunits (c3) of E . coli aspartate transcarbamylase at pH 7.0; Hsuanyu Y et al.; In contrast to holo-enzyme (c6r6), catalytic subunits (c3) of Escherichia coli aspartate transcarbamylase (carbamoyl-phosphate:L-aspartate carbamoyltransferase, EC 2.1.3.2) do not exhibit allosteric interactions or inhibition effects that complicate kinetic investigations of substrate binding order . Equilibrium isotope-exchange kinetic probes of c3 at pH 7.0 and 30 degrees C produced kinetic saturation patterns consistent with a strongly preferred order random kinetic mechanism, in which carbamoyl phosphate binds prior to aspartate and carbamoyl aspartate is released before Pi . Weak substrate inhibition effects observed with c6r6 did not occur with c3, possibly due to decreased affinity for ligands at the dianion inhibition site.

Cell, 1988 Dec 2, 55(5), 817 - 26
Transmembrane signaling by bacterial chemoreceptors: E . coli transducers with locked signal output; Ames P et al.; Methyl-accepting chemotaxis proteins (MCPs) function as transmembrane signalers in bacteria . We isolated and characterized mutants of the E . coli Tsr protein that produce output signals in the absence of overt stimuli and that are refractory to sensory adaptation . The properties of these "locked" transducers indicate that MCP molecules are capable of generating signals that actively augment clockwise and counter-clockwise rotation of the flagellar motors . Transitions between MCP signaling states can be influenced by amino acid replacements in many parts of the molecule, including the methylation sites, at least one of the two membrane-spanning segments, and a linker region connecting the receptor and signaling domains . These findings suggest that transmembrane signaling may involve direct propagation of conformational changes between the periplasmic and cytoplasmic portions of the MCP molecule.

Zentralbl Bakteriol Mikrobiol Hyg {B}, 1988 Dec, 187(2), 107 - 11
{The glutamic acid decarboxylase disc test . An approach for the simplification and speeding up of E . coli detection}; Schubert R et al.; Although E . coli is certainly one of the best known and most widely examined bacterial species only few of their characters and properties were found suitable for the distinction of this species from other species especially from the coliform organisms . One of these rare characters is l-glutamic acid decarboxylase . The demonstration of the action of this enzyme, however, has hitherto been difficult and time consuming . A micro-method is described which permits the demonstration of l-glutamic acid decarboxylase--the glutamic acid decarboxylase disc test (gd-disc-test) . The conditions and procedures required are described in detail . The validation of this test was done demonstrating in presence of gamma-aminobutyric acid by paper chromatography.

J Bacteriol, 1988 Dec, 170(12), 5529 - 38
Molecular cloning, expression, and regulation in Escherichia coli K-12 of a chromosome-mediated aerobactin iron transport system from a human invasive isolate of E . coli K1; Valvano MA et al.; We have cloned chromosomal genes determining the aerobactin iron transport system from the Escherichia coli K1 strain VW187 . Mapping and hybridization experiments showed that the VW187 aerobactin region was identical to that of the plasmid ColV-K30 . However, in the E . coli K-12 background, the biosynthesis of both siderophore and ferric aerobactin receptor encoded by the VW187-derived recombinant plasmids was not repressed by iron to the same extent found when a recombinant plasmid derived from pColV-K30 was used . RNA-DNA dot-blot hybridization experiments demonstrated that the aerobactin-specific mRNA synthesized by the VW187-derived clones was not iron regulated in E . coli K-12 . In contrast, the synthesis of aerobactin and its receptor in strain VW187 was completely repressed by iron regardless of whether the recombinant plasmids originated from VW187 or pColV-K30 . Similar results were obtained with gene fusions in which a promoterless lac operon was placed under the control of aerobactin promoter regions of either chromosome- or plasmid-mediated aerobactin systems . DNA sequencing of the chromosomal aerobactin promoter region showed changes in bases located immediately upstream to the -35 region compared with the corresponding region in pColV-K30, which is known to be part of the binding site for the Fur repressor protein.

J Biomol Struct Dyn, 1988 Dec, 6(3), 587 - 92
Prediction of the recognition sites on 16S and 23S rRNAs from E . coli for the formation of 16S-23S rRNA complex; Thanaraj TA et al.; Interactions between RNA molecules have been postulated to play an important role in the assembly of ribosomes . Using the sequence analysis and the search of continuous complementary regions on 16S rRNA and 23S rRNA, the recognition sites involved in the formation of ribosome of E . coli are postulated . The number of postulated sites was narrowed down by taking available experimental data . The suggestive evidence for correct postulation is obtained from sequence comparison studies of 16S and 23S rRNAs from various species . The sites 891-899 and 1195-1203 on 16S rRNA along with the corresponding complementary sites 1904-1912 and 760-768 on 23S rRNA are predicted to be the most probable candidates for the sites of recognition between 16S and 23S rRNAs . The possibility of the involvement of the additional site 630-638 on 16S rRNA with its complementary site 2031-2039 on 23S rRNA cannot be ruled out.

Arzneimittelforschung, 1988 Dec, 38(12), 1856 - 8
Detection of antibodies against pre-S1 proteins in sera of patients with hepatitis B virus infection by ELISA using a pre-S fusion protein expressed in E . coli; Theilmann L et al.; To evaluate the importance of antibodies directed against pre-S1 proteins (anti-pre-S1) of hepatitis B virus (HBV) in human sera, an enzyme-linked immunosorbent assay (ELISA) for their detection in human sera was established, using a bacterially synthesized pre-S1 fusion protein . Using this ELISA, it was found that anti-pre-S1 was present in sera from 10 of 11 patients with acute HBV infection who recovered completely, but only present in 1 of 8 patients where the infection was prolonged . In chronic HBV carriers anti-pre-S1 was present in only 3 out of 8 patients and titers were low . In addition, in 5 out of 17 individuals who had recovered from previous HBV infection, anti-pre-S1 was also detected . Individuals immunized with recombinant HBsAg-vaccine and healthy controls were all negative for anti-pre-S1 . It is suggested that presence of anti-pre-S1 in sera of patients with acute HBV infection correlates with rapid recovery.

Biosci Rep, 1988 Dec, 8(6), 619 - 32
Translational control in E . coli: the case of threonyl-tRNA synthetase; Springer M et al.; Genetic studies have shown that expression of the E . coli threonyl-tRNA synthetase (thrS) gene is negatively auto-regulated at the translational level . A region called the operator, located 110 nucleotides downstream of the 5' end of the mRNA and between 10 and 50 bp upstream of the translational initiation codon in the thrS gene, is directly involved in that control . The conformation of an in vitro RNA fragment extending over the thrS regulatory region has been investigated with chemical and enzymatic probes . The operator locus displays structural similarities to the anti-codon arm of threonyl tRNA . The conformation of 3 constituent mutants containing single base changes in the operator region shows that replacement of a base in the anti-codon-like loop does not induce any conformational change, suggesting that the residue concerned is directly involved in regulation . However mutation in or close to the anti-codon-like stem results in a partial or complete rearrangement of the structure of the operator region . Further experiments indicate that there is a clear correlation between the way the synthetase recognises each operator, causing translational repression, and threonyl-tRNA.

APMIS, 1988 Dec, 96(12), 1061 - 5
Binding of E . coli to human blood leukocytes; Guan YZ et al.; The spontaneous binding of both piliated and non-piliated E . coli to human lymphocytes, monocytes, and polymorphonuclear leukocytes (PMN) was investigated . It was found that the piliated E . coli showed a higher binding percentage to lymphocytes than the nonpiliated bacteria . A specific antiserum raised in rabbit against E . coli strongly inhibited the binding of the homologous E . coli strain to lymphocytes and monocytes . On the other hand there was no difference in the binding of piliated and non-piliated E . coli to PMNs, and the specific E . coli antiserum did not inhibit the binding . The binding of E . coli to human lymphocytes obtained from healthy donors was investigated in a 3-week follow-up experiment . It was shown that there were significant differences between the binding of cells from different individuals, whereas there were no differences in the binding ability of the cells from the same individual in the 3-week follow up.

Mutat Res, 1988 Dec, 203(6), 415 - 26
The E . coli multitest: a set of strains to characterize diverse genotoxic effects; Tenenbaum L et al.; A set of E . coli strains was developed by Toman et al . (1985) to study the effects of chemical and physical agents on forward mutation, homologous recombination and induction of the SOS system . New tester strains have been constructed to improve this test system in order to explore quantitative genotoxicity spectra . Through the use of these strains: (i) SOS induction can be specifically detected without interference from mutagenesis; (ii) SOS-dependent and SOS-independent mutational events can be distinguished; (iii) the sensitivity of the recombination system has been considerably increased.

Cell, 1988 Nov 18, 55(4), 683 - 92
SecA protein is required for secretory protein translocation into E . coli membrane vesicles; Cabelli RJ et al.; The soluble and membrane components of an E . coli in vitro protein translocation system prepared from a secA amber mutant, secA13{Am}, contain reduced levels of SecA and are markedly defective in both the cotranslational and posttranslational translocation of OmpA and alkaline phosphatase into membrane vesicles . Moreover, the removal of SecA from soluble components prepared from a wild-type strain by passage through an anti-SecA antibody column similarly abolishes protein translocation . Translocation activity is completely restored by addition of submicrogram amounts of purified SecA protein, implying that the observed defects are solely related to loss of SecA function . Interestingly, the translocation defect can be overcome by reconstitution of SecA into SecA-depleted membranes, suggesting that SecA is an essential, membrane-associated translocation factor.

J Biol Chem, 1988 Nov 15, 263(32), 17084 - 91
Dihydrofolate reductase (mouse) and beta-galactosidase (Escherichia coli) can be translocated across the plasma membrane of E . coli; Freudl R et al.; Hybrid genes were constructed . One, ompA153-dfr, encoded the precursor of the 325 residue Escherichia coli outer membrane protein OmpA up to residue 153 which was fused to the complete 186-residue dihydrofolate reductase of the mouse . The other, ompA219-lacZ, coded for the same precursor up to residue 219 which was fused to 1017 COOH-terminal residues of the 1023-residue subunit of the beta-galactosidase of E . coli . Full expression of the ompA153-dfr gene caused accumulation of its precursor and of that of the chromosomally encoded OmpA protein . When the amount of product was reduced, no pro-OmpA and very little pro-hybrid protein accumulated . The precursor was processed and the mature protein was fully accessible to trypsin in permeabilized cells . Expression of the ompA219-lacZ gene led to the presence of the hybrid protein at only 20-30% of the amount expected . About 20% of it appeared to be incorporated in the outer membrane . All of the hybrid was quantitatively accessible to trypsin in permeabilized cells . When the hybrid gene was overexpressed, the protein was found associated with the plasma membrane in the cytosol . It is concluded that both beta-galactosidase and dihydrofolate reductase could quantitatively traverse the plasma membrane, provided the amounts synthesized were sufficiently small.

Cell, 1988 Nov 4, 55(3), 467 - 75
A consensus sequence of three DNA replication terminus sites on the E . coli chromosome is highly homologous to the terR sites of the R6K plasmid; Hidaka M et al.; Using the "Ter assay" we developed, three separate replication terminus (terC1, terC2, and terC3) sites on the E . coli chromosome were identified . The locations are at 28.3, 35.6, and 33.9 min on the linkage map, respectively . The terC1 site can block the counter-clockwise replication fork only, while the terC2 and terC3 sites inhibit the clockwise fork traveling on the chromosome . DNA sequences of the terC sites required for termination of DNA replication are highly homologous to those of terminus (terR) sites of the R6K plasmid, and the 21 bp consensus DNA sequence of terC is 5'-(A or T) TTAGTTACAACAT (A or C) CT (A or T) (A or T) (A or T) T-3' . In addition, all Ter active pUC-terC plasmids had a low copy number and were unstable in the host cells.

Cell, 1988 Nov 4, 55(3), 459 - 66
Identification of the DNA sequence from the E . coli terminus region that halts replication forks; Hill TM et al.; The terminus region of the E . coli chromosome contains two loci, T1 and T2, that inhibit the progress of replication forks and require the trans-acting factor tus . We have identified a 23 bp terminator signal at T1 and T2 that is within 100 bp of the sites of replication arrest . When an oligodeoxyribonucleotide containing the terminator signal was inserted into a plasmid, replication was halted only in a tus+ strain and when the terminator signal was oriented properly . We also found this terminator sequence in the terminus region of the plasmid R6K and in the origin region of RepFIIA class plasmids . In addition, we found striking similarities between the E . coli terminator signal and the terminator sequence of B . subtilis.

Biofizika, 1988 Nov-Dec, 33(6), 973 - 7
{A study of the stability of liposomes from the total lipid fraction of E . coli by means of fluorescence spectroscopy and a radioisotope probe}; Cherniavskii VA et al.; Stability of liposomes from total fraction of E . coli lipids was studied by their ability to preserve encapsulated substance under different incubation conditions--+4 degrees C and +37 degrees C for some concentrations of CaCl2 . Two types of vesicules were used: small ones obtained by ultrasonic treatment and larger ones obtained after successive treatment of lipid suspension with Ca2+ ions and EDTA . It has been shown by spectrofluorimetry and radioisotope probe that for both studied types of liposomes their stability decreases with an increase of CaCl2 concentration and rise of temperature . The effect similar to Ca2+ was produced by Mg+2 ions, but not by Na+ ions . Large liposomes are significantly less stable formations than the small ones . Ca+2 ions were shown to induce at first liposome aggregation and then their destruction . Possible action mechanism of metal ions of the second group on liposome stability, the pattern of initiated membrane disturbances and application of these structures as agents during transfer of biologically active compounds in the cells are discussed.

Mutat Res, 1988 Nov, 202(1), 19 - 23
Antimutagens against spontaneous and induced reversion of a lacZ frameshift mutation in E . coli K-12 strain ND-160; Clarke CH et al.; Isopropyl-beta-D-thiogalactoside (IPTG), at 0.5 or 1.0 mM, is shown to reduce spontaneous, and to virtually abolish caffeine- or quinacrine-induced reversions of the lacZ frameshift mutation in E . coli ND160 . Guanosine at 200 micrograms/ml and Co2+ at 15 micrograms/ml had an erratic partial antimutagenic effect on spontaneous lac+ reversions . All 3 agents reduce caffeine-induced (500 micrograms/ml) mutagenesis . Spermine (250 micrograms/ml) also reduces quinacrine-induced Lac+ reversion frequencies in this system.

Radiobiologiia, 1988 Nov-Dec, 28(6), 767 - 71
{Induction of prophage lambda during the irradiation of E . coli cells exposed to radiations of various LET}; Bonev M et al.; Induction of lambda prophage in lysogenic E . coli cells exposed to ionizing radiation of different LET was studied as a function of dose I(D) . Activities of pleiotropic RecA protein were shown to contribute to the shape of the I(D) curve . The experimental data were fitted by the function I(D) = alpha D(1-exp(-D0-1.D}exp(-beta D) . Inducibility alpha increased with increasing LET which was related to the increased incidence of DNA lesions being a SOS-system call.

Mol Gen Mikrobiol Virusol, 1988 Nov, (11), 18 - 20
{Localization of insertion into E . coli chromosome of Tn7-like transposons controlling resistance to trimethoprim and streptothricin}; Tietze E et al.; To localize the insertion sites for Tn7-like transposons Tn1824, Tn1825 and Tn1826 the EcoRI-cleaved fragments of E . coli recA+ and recA- strains chromosome carrying the transposons were hybridized . It was shown that transposition of Tn7-like elements takes place in the strictly defined region of E . coli chromosome, like it had been previously described for transposon Tn7.

Mutat Res, 1988 Nov, 194(3), 193 - 205
Repair of the plasmid pBR322 damaged by gamma-irradiation or by restriction endonucleases using different recombination-proficient E . coli strains; Bien M et al.; The plasmid pBR322 was treated with BamHI, PvuII and gamma-irradiation to generate double-strand breaks (dsb) containing differently structured ends . Transformation efficiencies, mutation frequencies and clone analyses of enzymatically damaged DNA are compared with the corresponding results from radiolytically damaged DNA . In E . coli K-12 SFX, the yield of transformants produced by the action of BamHI, PvuII and gamma-irradiation (30 Gy) is 4.3%, 0.14%, and 0.10%, respectively . The survival of open circular DNA (ocDNA) produced by 30 Gy is 1.3% . The transformation efficiencies show only a slight dependence on SOS induction and on the RecA protein . Mutation frequencies to tetracycline sensitivity (tets) per surviving plasmid are 2.6% (BamHI), 11.8% (PvuII) and 0.2% (gamma-irradiated DNA with 30 Gy containing approximately 50% ocDNA and 50% linearized (lin) DNA) . The mutation frequency is low at all radiation doses studied (1-50 Gy) . Only 15% of the DNA of the tets mutants from gamma-irradiated plasmids contained deletions whereas with enzymatically damaged DNA, 30-50% (BamHI) or 90% (PvuII) contained deletions . In all cases, the deletions comprised 500-1700 base pairs (bp) . After SOS induction of the host cells, the mutation frequency of gamma-irradiated plasmids increased by a factor of 4, whereas that of the enzymatically damaged plasmids did not change . For the repair of the enzymatically linearized DNA 2 recombinational pathways are discussed which lead to deletant (pathway I) and non-deletant transformants (pathway II) . In addition, BamHI-linearized plasmids may be repaired by enzyme-induced or spontaneous circular alignment followed by ligation . The high percentage of deletions of the tets mutations for PvuII-linearized DNA with blunt ends is explained by the illegitimate or site-specific recombination pathway I (see text) . The lower percentage of deletions of the tets mutations with BamHI-linearized DNA with short cohesive ends (4 bp) is proposed to be due to a greater contribution of pathway II and/or by circular alignment followed by ligation . The very small yield and the low percentage of deletant mutations of tets mutants from radiolytically damaged DNA is proposed to be due to the large overlapping ends (16-100 bp) of the linDNA which easily leads to circular alignment followed by excision repair . The repair of radiolytically produced ocDNA is predominantly due to excision repair . In agreement with this interpretation is the observation that SOS induction of the host increases the mutation incidence of radiolytically damaged DNA but not of enzymatically damaged DNA.

Biochem Biophys Res Commun, 1988 Oct 31, 156(2), 807 - 13
Identification of unusual replacement of methionine by norleucine in recombinant interleukin-2 produced by E . coli; Lu HS et al.; Moderate amounts of norleucine incorporation into recombinant interleukin-2 (IL-2) produced in E . coli have been detected . Incorporation of norleucine occurs both at the amino terminal and internal methionines as confirmed by the isolation of norleucine-containing tryptic peptides which eluted later than the respective methionine-containing peptides by reverse-phase HPLC . The occurrence of norleucine in intact protein and modified peptides was determined by amino acid analysis and amino acid sequencing including Edman degradation and fast atom bombardment mass spectrometry . In the subsequent paper, we determined that norleucine incorporation is caused by the endogenous synthesis of norleucine in E . coli.

Nucleic Acids Res, 1988 Oct 25, 16(20), 9545 - 55
A specific and efficient photoreaction between E . coli RNA polymerase and T+1 in the lacUV5 or deoP1 promoter; Jeppesen C et al.; Upon irradiation of the RNA polymerase-lacUV5 or deoP1 promoter complex with short wavelength ultraviolet light (lambda less than or equal to 300 nm) the polymerase is covalently crosslinked at an efficiency of greater than 10% to the first transcribed base of the template DNA strand when this is a thymine . The temperature dependence of this RNA polymerase-T+1 photoreaction strongly indicates a relation to the formation of the open complex . It is suggested that open complex formation is preceded or accompanied by a specific contact between the RNA polymerase and the first transcribed base of the DNA template.

FEBS Lett, 1988 Oct 24, 239(1), 41 - 4
Cloning a synthetic gene for human stefin B and its expression in E . coli; Jerala R et al.; A gene coding for human stefin B was synthesized by the solid-phase phosphite method and cloned in the pUC8 cloning vector . The insert with the verified DNA sequence was subcloned into two expression vectors and expressed in E . coli as a fusion protein with beta-galactosidase and as a native protein . The CNBr cleaved fusion protein and the native recombinant stefin B were inhibitory to papain and reacted with antibodies against human stefin B.

Cell, 1988 Oct 21, 55(2), 343 - 50
A novel role for cAMP in the control of the activity of the E . coli chromosome replication initiator protein, DnaA; Hughes P et al.; DnaA protein interacts with cAMP with a KD of 1 microM . This interaction stimulates DnaA protein binding to the chromosome replication origin (oriC) and the mioC promoter region, protects DnaA protein from thermal inactivation, releases ADP but not ATP bound to DnaA protein, and restores normal DNA replication activity and ATPase activity in inactive ADP-DnaA protein preparations . A model is proposed in which cellular cAMP levels govern the replication activity of DnaA protein by promoting the recycling of the inactive ADP-DnaA protein form into the active ATP form.

Cell, 1988 Oct 7, 55(1), 113 - 23
Transcriptional activation of initiation of replication from the E . coli chromosomal origin: an RNA-DNA hybrid near oriC; Baker TA et al.; Transcription by RNA polymerase preceding the initiation of replication from the E . coli chromosomal origin (oriC) in vitro enables dnaA protein to open the DNA duplex under conditions when its action alone is insufficient . The RNA polymerases of phages T7 and T3 are as effective as the E . coli enzyme in activating initiation . The persistent RNA transcript hybridized to the template creates an R-loop that is responsible for activation . The activating RNA need not cross oriC, but must be less then 500 bp away . Transcripts lacking a 3' OH group are effective, proving that priming of DNA synthesis is not involved in the activation . Thus, transcription activates the origin of an otherwise inert plasmid by altering the local DNA structure, facilitating its opening by dnaA protein during the assembly of replication forks.

Bioorg Khim, 1988 Oct, 14(10), 1387 - 92
{Location of the initiating codon AUG in relation to the 5'-end of mRNA mediates the effectiveness of translation in E . coli cells}; Kravchenko VV et al.; A semisynthetic gene for beta-galactosidase (lacZ) and a synthetic DNA fragment containing the "ideal" promoter sequence were used for construction of an artificial operon including translation initiation codon ATG and no SD sequence . Cloning this artificial operon into pBR322 vector resulted in a number of pV plasmids; ATG positions were varied by insertions of synthetic oligonucleotides between lacZ coding sequence and starting point of transcription . It was found that efficiency of beta-galactosidase synthesis in E . coli cells harbouring pV plasmids strongly depended on the relative position of AUG and mRNA 5'-end . High level of the synthesis was provided by translation of mRNA with AUG codon in 5'-terminal position . Amounts of synthesized beta-galactosidase diminished with increase of the distance (2, 4, and 5 nucleotides) between 5'-end of lacZ mRNA and AUG codon.

Curr Genet, 1988 Oct, 14(4), 293 - 302
The 5'-upstream region of the yeast 25S rRNA gene contains a promoter element allowing expression in yeast and E . coli; Strobel G et al.; The 25S rRNA gene of Saccharomyces cerevisiae is preceded by a bona fide TATA sequence which allows the initiation of transcription--presumably by polymerase II--from the same strand as the 25S rRNA gene . When the promoter fragment is cloned in front of a lacZ gene equipped with an initiation codon but lacking a promoter, this element permits formation of beta-galactosidase both in yeast and E . coli . Using RNA from yeast transformed with the fusion plasmid, we mapped by primer elongation a single initiation site 63 bp downstream from the presumed TATA sequence, i.e . about 53 bp 5' of the 25S rRNA gene . A similar signal at about the same position was observed when RNA from untransformed wild-type yeast was used as a template for primer elongation . These results suggest that transcription from this polymerase II promoter-like element occurs in vivo . A regulatory function could not be assigned to this transcript . Its initiation is not significantly influenced by heme or carbon source, although two boxes of high homology with upstream activation sequences (UAS) mediating heme dependent expression of the iso-1-cytochrome c gene (CYC1) precede the promoter at the appropriate distance.

Biochem Biophys Res Commun, 1988 Sep 30, 155(3), 1261 - 5
Release of 7-alkylguanines from haloethylnitrosourea-treated DNA by E . coli 3-methyladenine-DNA glycosylase II; Carter CA et al.; Previous studies have related DNA modification by the haloethylnitrosoureas to their antitumor activity . Repair of this damage, particularly by O6-alkylguanine-DNA alkyltransferase, has been linked to tumor resistance by several previous investigations . We report here that E . coli 3-methyladenine-DNA glycosylase II can also remove several of the DNA modifications caused by the haloethylnitrosoureas . 7-Chloroethylguanine, 7-hydroxyethylguanine, and diguan-7-ylethane are all released into the supernatant from DNA modified by N-{2-chloroethyl-1,2-14C}-N'-cyclohexyl-N-nitrosourea . Release of diguan-7-ylethane is of particular interest since this entity evidently represents a DNA intrastrand cross-link . If a similar activity is present in mammalian cells, it might be an important source of resistance to the therapeutic action of the haloethylnitrosoureas.

FEBS Lett, 1988 Sep 26, 238(1), 116 - 8
Total synthesis of the cystatin alpha gene and its expression in E . coli; Katunuma N et al.; A gene encoding cystatin alpha has been chemically synthesized, cloned and expressed in E . coli . The gene of 318 base pairs was assembled by enzymatic ligation of 19 oligonucleotides and cloned into a pBR322-derived expression plasmid down stream of the tac promoter . The expression product of the synthetic gene has been purified by Sephadex G-50 column chromatography and shown to have the same properties as those of the authentic protein isolated from rat epidermis.

Cell, 1988 Sep 23, 54(7), 1003 - 11
ProOmpA is stabilized for membrane translocation by either purified E . coli trigger factor or canine signal recognition particle; Crooke E et al.; We have isolated large amounts of E . coli outer-membrane protein A precursor (proOmpA) . Purified proOmpA is active in membrane assembly, and this assembly is saturable with respect to the precursor protein . A proOmpA-Sepharose matrix allows affinity isolation of trigger factor, a soluble, 63,000 dalton monomeric protein that stabilizes proOmpA in assembly competent form . Comparison of trigger factor's amino-terminal sequence with those in a computer data bank and with those encoded by sec genes, as well as groEL and heat shock gene dnaK, suggests that trigger factor is encoded by a previously undescribed gene . Trigger factor and proOmpA form a 1:1 complex that can be isolated by gel filtration . Purified canine signal recognition particle (SRP) can also stabilize proOmpA for membrane insertion . This postribosomal activity of SRP suggests a unifying theme in protein translocation mechanisms.

FEBS Lett, 1988 Sep 12, 237(1-2), 187 - 90
Processed enzymatically active protease (p15gag) of avian retrovirus obtained in an E . coli system expressing a recombinant precursor (Pr25lac-delta gag); Sedlacek J et al.; Processing proteases of avian and mammalian retroviruses cut the polyprotein precursors encoded by the retroviral genes into mature functional proteins . Retroviral processing proteases are still a rather poorly characterized group as to their relation to other proteases, specificity, and mechanism of enzymatic action . In avian retroviruses the generation of the processing protease itself comprises a processing cleavage event - the protease p15gag is cut off the carboxy-terminus of a gag polyprotein precursor, Pr76gag . We report here that direct and efficient production of the avian retrovirus processing protease p15gag (required for structure-function studies and rational design of inhibitors) was obtained in an E . coli system, where massive expression of a size-reduced, recombinant precursor (Pr25lac-delta gag) was accompanied by its structurally accurate processing.

Cell, 1988 Sep 9, 54(6), 805 - 12
A novel nucleotide excision repair for the conversion of an A/G mismatch to C/G base pair in E . coli; Lu AL et al.; A protein that binds specifically to A/G mismatches has been detected in E . coli by a gel electrophoresis DNA binding assay . A specific endonuclease is associated with the A/G mismatch-binding protein through two chromatographic steps . The endonuclease is specific for A/G-containing DNA fragments and has no cleavage activity on DNA containing the other seven possible mispairs or homoduplex DNA . The endonuclease simultaneously makes incisions at the first phosphodiester bond 3' to and the second phosphodiester bond 5' to the dA of the A/G mismatch . No incision site was detected on the other strand . These results are consistent with the unidirectional A to C conversion and short repair tract of a novel dam- and mutHLS-independent A/G repair pathway we have recently described . A nucleotide excision repair model is proposed for the conversion of an A/G mismatch to a C/G base pair.

Biol Chem Hoppe Seyler, 1988 Sep, 369(9), 1019 - 30
Chemical synthesis of a gene for human stefin A and its expression in E . coli; Strauss M et al.; A DNA containing the coding sequence for the human cysteine proteinase inhibitor stefin A was obtained by enzymic ligation of chemically synthesized deoxyoligonucleotides, using the Khorana ligation method . The 306-bp synthetic gene carries signals for the initiation and termination of its translation . The gene was expressed in E . coli using a cytoplasmic expression vector and stefin A was secreted under the control of the E . coli alkaline phosphatase signal sequence, respectively . The secreted hybrid protein was shown to exhibit biological properties similar to the native protein isolated from human plasma.

Biofizika, 1988 Sep-Oct, 33(5), 800 - 3
{The effect of surface and membrane potential on 1-anilino-8-naphthalenesulfonate binding with E . coli membrane}; Lebedev VS et al.; Dependence of ANS fluorescence on the surface potential of E . coli under lowered resistance of the bacterial membrane and after application of the positive diffusion potential inside the cell was investigated . It was shown that in the absence of the latter ANS binding in de-energised bacteria occurs mainly at the outside surface . It may be due to the high negative charge of the inner side of the cytoplasmic membrane . According to produced evaluation the potential of this surface is 120 +/- 25 mV . The data obtained suggest that low ANS fluorescence in intact cells is due to the membrane modification on energisation.

Biochem Int, 1988 Sep, 17(3), 545 - 54
Aspartokinase III repression in a thialysine-resistant mutant of E . coli; Di Girolamo M et al.; A thialysine-resistant mutant of the E . coli KL16 strain was isolated . It can grow equally well in the presence and in the absence of thialysine . The properties of the two lysine transport systems, of the lysyl-tRNA synthetase and of the aspartokinase III (AK III) were studied in the mutant and in the parent strain . AK III is the first enzyme of the lysine biosynthetic pathway and its activity is involved in the regulation of lysine biosynthesis by feed-back and repression mechanism . No difference between the two strains was evidenced as regards 1) the affinity of the transport systems for lysine and thialysine 2) the activity of the lysyl-tRNA synthetase 3) the allosteric inhibition of the AK III by lysine and thialysine . A marked difference between the two strains has been evidenced in the AK III repression: in the mutant the enzyme is much less repressed both by lysine and thialysine . The possible correlation between the activity of AK III and the thialysine-resistance is discussed in this paper.

FEBS Lett, 1988 Aug 29, 236(2), 489 - 92
Stimulation of phosphoinositides breakdown by the heat stable E . coli enterotoxin in rat intestinal epithelial cells; Banik N et al.; Rat intestinal epithelial cells were labelled with {32P}Pi and extracted, and the phospholipids were analysed by thin-layer chromatography . 32P-incorporation in phosphatidylinositol (PI) and phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-phosphate (PIP2) were measured in control and heat stable enterotoxin (ST)-treated cells . ST was found to induce rapid degradation of PIP and PIP2 . The degradation of inositol lipids was accompanied by an increase of water soluble inositol phosphate (IP1, IP2, IP3) compounds . There was a two-fold increase of radioactivity in IP2 and IP3 but no significant change was observed in IP1 . Phospholipase C activity was increased tenfold with substrate PIP2 in ST-pretreated cells . The present study indicates that ST triggers another second messenger system by increasing the PIP2 hydrolysis with the enzyme phospholipase C.

Nucleic Acids Res, 1988 Aug 25, 16(16), 7885 - 99
The tRNAGlu2 gene in the rrnB operon of E . coli is a prerequisite for correct RNase III processing in vitro; Szymkowiak C et al.; RNase III cleaves precursor 16S RNA and precursor 23S RNA from the ribosomal RNA transcript . In vitro transcription experiments, using plasmids with the rrnB operon truncated in the 16S RNA and with various deletions in the spacer tRNA region, showed that no matter what size of deletion if the tRNA gene was affected RNase III processing of 16S RNA became incomplete . In comparison to a control plasmid, where only the 16S RNA gene was truncated and that showed normal RNA processing, plasmids where the tRNA gene was deleted partially or totally either the 5' or the 3' end of 16S RNA was processed . This relation between RNase III processing and the 3-dimensional structure of tRNA suggests an interaction between RNase III and a tRNA processing enzyme most probably RNase P.

Nucleic Acids Res, 1988 Aug 25, 16(16), 7843 - 53
Mismatch-containing oligonucleotide duplexes bound by the E . coli mutS-encoded protein; Jiricny J et al.; The binding of the mutS gene product, a protein involved in at least two E . coli mismatch correction pathways, to a series of synthetic DNA duplexes containing mismatches or mismatch analogues of the purine/pyrimidine type was studied in order to establish whether a correlation exists between the recognition of these mispairs and the efficiency of their correction in vivo . Experiments using nitrocellulose filter binding or band-shift assays revealed that duplexes containing a G/T mismatch or its analogues I/T and DI/T were bound by the protein with affinities correlating to the efficiency of their repair in vivo . In contrast, the A/C mismatch, contained within the same sequence, was bound only poorly, despite being efficiently corrected in vivo . The analogues of the A/C mispair, uncorrected in vivo, were not detectably bound under the conditions of these assays.

Nucleic Acids Res, 1988 Aug 11, 16(15), 7673 - 83
Defining the consensus sequences of E.coli promoter elements by random selection; Oliphant AR et al.; The consensus sequence of E.coli promoter elements was determined by the method of random selection . A large collection of hybrid molecules was produced in which random-sequence oligonucleotides were cloned in place of a wild-type promoter element, and functional -10 and -35 E.coli promoter elements were obtained by a genetic selection involving the expression of a structural gene . The DNA sequences and relative levels of function for -10 and -35 elements were determined . The consensus sequences determined by this approach are very similar to those determined by comparing DNA sequences of naturally occurring E.coli promoters . However, no strong correlation is observed between similarity to the consensus and relative level of function . The results are considered in terms of E.coli promoter function and of the general applicability of the random selection method.

Nucleic Acids Res, 1988 Aug 11, 16(15), 7477 - 86
Kinetic analysis of an E.coli phenylalanine-tRNA synthetase mutant; Goodman R et al.; A mutation in the pheS gene, encoding phenylalanyl-tRNA synthetase, in E . coli NP37 confers temperature-sensitivity on the organism . A five-fold increase in tRNA(phe) levels complements the mutation . Analysis of the kinetic properties of the mutant enzyme indicates that the KM is 20-fold higher than the wild-type and the dissociation constant of the tRNA(phe)-synthetase complex for the mutant is at least 10-fold higher . These results indicate that the mutation in E . coli NP37 directly affects the tRNA(phe) binding site on the cognate synthetase.

Science, 1988 Aug 5, 241(4866), 703 - 5
An E . coli promoter that regulates transcription by DNA superhelix-induced cruciform extrusion; Horwitz MS et al.; DNA can form structures other than the Watson-Crick double helix . The potential contributions to gene regulation from one such structure have been investigated by assembling a promoter capable of adopting cruciform base-pairing . Transcription from this promoter by RNA polymerase in vitro was repressed as the cruciform was extruded by increasing negative DNA supercoiling . Transcription in vivo was induced as supercoiling was relaxed by growth in conditions that inhibit DNA gyrase . A DNA conformational change is therefore capable of regulating the initiation of transcription.

Immunol Invest, 1988 Aug-Oct, 17(6-7), 551 - 9
A two step purification of recombinant human interleukin-1 beta expressed in E . coli; Yem AW et al.; Recombinant human interleukin-1 beta has been expressed in high yield using E . coli with a cDNA clone obtained from SKhep1RNA . The rIL-1 beta is purified to apparent homogeneity using freeze-thaw extractions followed by hydrophobic interaction chromatography over phenyl Sepharose . The procedure can provide pure rIL-1 beta (up to 15 mg per liter of E . coli culture) without the use of denaturants and if desired, in the absence of column chromatographic steps . Purity is defined by the presence of a single band on 1-D polyacrylamide gels and a single spot on 2-D polyacrylamide gels . The purified protein exhibits a biological activity of 1 x 10(7) units/mg in a fibroblast proliferation assay and is shown to cross-react with rabbit anti-human IL-1 beta sera.

Circ Shock, 1988 Aug, 25(4), 319 - 23
Oxygen free radical activity during live E . coli septic shock in the dog; Morgan RA et al.; Free radicals generated during purine catabolism or by activated granulocytes cause tissue injury by peroxidation of lipid membranes . In a canine model of sepsis initiated by intravenous live Escherichia coli, fluorescent products of lipid peroxidation (FP) were measured in serum . Four groups of five dogs infused with 10(9)E . coli/kg were analyzed--I: no further treatment; II: prior depletion of granulocytes with a cytotoxic antibody; III: pre-treatment with superoxide dismutase and catalase; and IV: resuscitation after bacterial infusion to maintain cardiac output greater than 80% of pre-bacteremic levels . In Groups I, II, and III, cardiac output fell to less than 50% of baseline within 1 hr and remained there throughout the study . FP in Groups I and II rose to greater than 200% of baseline (P less than .02 and less than .03) . In Groups III and IV, FP did not rise significantly from baseline . The rise in serum FP and the prevention of this rise by-treatment with antioxidants indicate generation of oxygen radicals . Their presence had no effect on hemodynamic parameters . Granulocyte depletion did not alter appearance of FP; however, prevention of low cardiac output blocked FP formation . These data suggest that oxygen free radicals were generated by tissue ischemia, rather than by granulocytes, in this model of septic shock.

Behring Inst Mitt, 1988 Aug, (83), 246 - 9
E . coli derived human granulocyte-macrophage colony-stimulating factor (rh GM-CSF) available for clinical trials; Gronski P et al.; Recombinant human GM-CSF has been expressed as a fusion protein in E . coli in the form of inclusion bodies . Using denaturing agents, acid cleavage and sulfitolysis, the biologically inactive GM-CSF protein could be highly purified and additionally renaturated under suitable reoxidizing conditions . The thorough repair of the two disulfide bridges could be confirmed by sequencing fragments obtained by tryptic digestion . Refolding of the molecule has been studied by CD spectrometry and identity by Western blotting and SDS-PAGE analysis . As could be demonstrated, full biological activity (colony-forming assay with fresh human bone marrow cells) was restored during renaturation of the GM-CSF protein . Further proof of biological equivalence of the E . coli-derived protein with a yeast-derived biologically active rh GM-CSF has been published elsewhere.

Nucleic Acids Res, 1988 Jul 25, 16(14A), 6397 - 410
Rapid, large-scale purification and characterization of 'Ada protein' (O6 methylguanine-DNA methyltransferase) of E . coli; Bhattacharyya D et al.; The E . coli Ada protein (O6-methylguanine-DNA methyltransferase) has been purified using a high-level expression vector with a yield of about 3 mg per liter of E . coli culture . The 39-kDa protein has an extinction coefficient (E280 nm (1%)) of 5.3 . Its isoelectric point of 7.1 is lower than that predicted from the amino acid content . The homogeneous Ada protein is fully active as a methyl acceptor from O6-methylguanine in DNA . Its reaction with O6-methylguanine in a synthetic DNA has a second-order rate constant of 1.1 x 10(9) M-1 min-1 at O degree C . Both the native form and the protein methylated at Cys-69 are monomeric . The CD spectrum suggests a low alpha-helical content and the radius of gyration of 23 A indicates a compact, globular shape . The middle region of the protein is sensitive to a variety of proteases, including an endogenous activity in E . coli, suggesting that the protein is composed of N-terminal and C-terminal domains connected by a hinge region . E . coli B has a higher level of this protease than does K12.

Nucleic Acids Res, 1988 Jul 25, 16(14A), 6309 - 26
Effects of macromolecular crowding on the association of E . coli ribosomal particles; Zimmerman SB et al.; The equilibrium for the binding reaction between the 30 S and 50 S ribosomal subunits of E . coli is shifted towards formation of 70 S ribosomes in the presence of a variety of polymers . The polymers also increase a further interaction between 70 S particles to form the 100 S dimer . The requirement for relatively high concentrations of non-specific polymers indicates that the shifts in equilibria arise from excluded volume effects . Analysis using scaled particle theory is consistent with this mechanism . The effects of high concentrations of polymers on the interactions between ribosomal species may make important changes in the function of ribosomes under the crowded conditions which occur in vivo.

Biochem Biophys Res Commun, 1988 Jul 15, 154(1), 113 - 7
Inorganic pyrophosphate: fructose-6-phosphate 1-phosphotransferase of the potato tuber is related to the major ATP-dependent phosphofructokinase of E . coli; Yuan XH et al.; A procedure was developed for the purification of inorganic pyrophosphate: fructose-6-phosphate 1-phospho-transferase (PPi-PFK) from potato tubers . The enzyme has the structure alpha 4 beta 4 with a subunit of 68 kDa and a beta subunit of 60 kDa . The structural relationship of this enzyme to other PFKs and to fructose bisphosphatase was examined by immunoprecipitation and immunoblotting . Antibodies to the plant enzyme did not react with E . coli PFK . No cross-reaction was seen among the following enzymes or their antibodies: yeast fructose bisphosphatase; rabbit PFKs A, B, or the enzyme from brain; and the two subunits of the potato PPi-PFK . On the other hand, antibody to E . coli PFK-1 strongly cross-reacts with the 60 kDa polypeptide but not 68 kDa peptide.

Nucleic Acids Res, 1988 Jul 11, 16(13), 6127 - 45
High efficiency transformation of E . coli by high voltage electroporation; Dower WJ et al.; E . coli can be transformed to extremely high efficiencies by subjecting a mixture of cells and DNA to brief but intense electrical fields of exponential decay waveform (electroporation) . We have obtained 10(9) to 10(10) transformants/micrograms with strains LE392 and DH5 alpha, and plasmids pUC18 and pBR329 . The process is highly dependent on two characteristics of the electrical pulse: the electric field strength and the pulse length (RC time constant) . The frequency of transformation is a linear function of the DNA concentration over at least six orders of magnitude; and the efficiency of transformation is a function of the cell concentration . Most of the surviving cells are competent with up to 80% transformed at high DNA concentration . The mechanism does not appear to include binding of the DNA to the cells prior to entry . Possible mechanisms are discussed and a simple procedure for the practical use of this technique is presented.

Nucleic Acids Res, 1988 Jul 11, 16(13), 5755 - 70
Photocleavage of DNA and photofootprinting of E . coli RNA polymerase bound to promoter DNA by azido-9-acridinylamines; Jeppesen C et al.; The long-wavelength ultraviolet (lambda approximately 420 nm) radiation induced reaction between 6-azido-2-methoxy-9-acridinylamines and supercoiled plasmid DNA results in single strand scissions and formation of covalent adducts (ratio approximately 1:10) . By treating azidoacridine-photomodified DNA with piperidine at 90 degrees C, additional strand scissions are observed in a complex sequence dependent manner with an overall preference for T greater than or equal to G greater than C much greater than A . The resulting DNA fragments migrate as 5'-phosphates in polyacrylamide gels . Photofootprinting of the binding site of RNA-polymerase on promoter DNA is demonstrated with an azido-9-acridinylamino-octamethylene-9-aminoacridine . Similar experiments using 9-amino-6-azido-2-methoxyacridine indicate that this reagent recognizes changes in the DNA conformation induced by RNA polymerase binding, in relation to open complex formation.

FEBS Lett, 1988 Jul 4, 234(1), 203 - 7
Electro-stimulated transformation of E . coli cells pretreated by EDTA solution; Cymbalyuk ES et al.; The phenomenon of transformation of E . coli cells under electric treatment has been studied . The cells of strains MH 1, HB 101 and DH 1 after EDTA treatment in an isotonic medium were transformed with DNA pBR322 by applying a single exponential pulse (E = 10 kV/cm, T = 1.5 ms) to the suspension . The maximum transformation efficiency obtained was 4 X 10(6) colonies/micrograms DNA . The maximum transformation frequency was 0.4% at a DNA concentration of 15 micrograms/ml.

FEBS Lett, 1988 Jul 4, 234(1), 61 - 4
Over-expression of natural and variant human H-chain ferritins in E . coli; Prozzi D et al.; The natural human H-chain ferritin was expressed in E . coli using a multi-copy expression vector containing the lambda pL promoter . A variant H-ferritin, having an altered N-terminus, was also produced . These proteins are overproduced (greater than 30% of the soluble protein), correctly assembled into its 24-subunit shell, and able to bind iron . The identity of the products was confirmed using an antibody specific for H-ferritin.

Mol Cell Biochem, 1988 Jul-Aug, 82(1-2), 113 - 8
The redox state and the phosphorylation state of the mannitol-specific carrier of the E . coli phosphoenolpyruvate-dependent phosphotransferase system; Robillard GT et al.; This review summarizes the recent developments in identifying the activity-linked cysteine as one of the phosphorylation sites on the mannitol-specific EII of the E . coli phosphoenolpyruvate-dependent mannitol transport system . Two phosphorylation sites have been identified, one being the HPr/P-HPr exchange site, the other being the mannitol/mannitol-P exchange site . The activity-linked cysteine and the second phosphorylation site are located in the same 14 residue peptide . Phosphorylation of the second site and phosphoryl group transfer to mannitol do not occur as long as the activity-linked cysteine is oxidized or alkylated . A kinetic scheme has been developed which accounts for the relationships between the redox state, the phosphorylation state and the activity of the carrier . Kinetics of the individual reactions determine whether the enzyme cycles through an oxidized/reduced state during a cycle of phosphorylation/dephosphorylation.

Bioorg Khim, 1988 Jul, 14(7), 963 - 4
{Amino acid substitutions in the beta-subunit of RNA-polymerase from E . coli compensating for mutation-induced damage of the rho termination factors}; Ogryz'ko EP et al.; Ts-phenotype of the E . coli rho-factor mutant rho 15 is suppressed by two rifampicin-resistance mutations, rhoB1019 resulting in a single amino acid substitution Val146----Phe and rhoB268 resulting in a single substitution Gln513----Leu in beta-subunit of the E . coli RNA polymerase . Rifampicin-resistance mutations rhoB255 (Asp516----Val), rhoB1016 (Asp516----Asn), rhoB1001 (His526----Tyr), rhoB1004 (Ser531----Phe), rhoB1005 (Pro564----Leu), and streptolydigin-resistance' mutation rhoB1018 (double substitution Gly544----Asp and Phe545----Ser) do not suppress the rho15 mutation.

Prikl Biokhim Mikrobiol, 1988 Jul-Aug, 24(4), 504 - 13
{Immobilization of E . coli cells in polyacrylamide-based microporous cryogels}; Lusta KA et al.; E . coli cells were immobilized in polyacrylamide cryogel by three ways: (1) introduction of cells in the reaction mixture followed by cryopolymerization; (2) the filling of the cryogel pores followed by cell fixation with diluted glutaric dialdehyde (GDA), and (3) the filling of the macropores of the polymeric matrix with modified surface . The ultrastructure of the gels and immobilized cells as well as distribution of attachment of the cells immobilized by different techniques were studied . The first type of immobilization was characterized by the highest quantity of the biomass in the gel (by protein) and by a sharp decrease of the cell viability . The second failed to retain the cells in the pores, and the GDA treatment significantly decreased the viability index . The latter technique was the mildest and completely maintained the viability of the population . However, the biomass content was lower as compared to the first type of immobilization, but could be considerably increased by the GDA treatment.

Biokhimiia, 1988 Jul, 53(7), 1103 - 10
{Effect of the protonophore carbonylcyanide-m-chlorophenylhydrazone on the localization of secreted alkaline phosphatase in E . coli}; Tsfasman IM et al.; It was shown that the total amount of synthesized alkaline phosphatase as well as the value of enzymatic activity in E . coli cells decrease in the presence of the protonophore, carbonylcyanide-m-chlorophenylhydrazone . The enzyme content in the periplasm also decreases, while the amount of the enzyme bound to the spheroplasts increases . This effect is enhanced with a rise in the protonophore concentration . An electron cytochemical analysis showed that in the presence of the protonophore, alkaline phosphatase is partly localized in the cytoplasm and on the inner surface of the cytoplasmic membrane, which is unobserved in control cells . It was assumed that carbonylcyanide-m-chlorophenylhydrazone suppresses the translocation of alkaline phosphatase across the cytoplasmic membrane and enzyme biosynthesis, on the whole.

Virus Genes, 1988 Jul, 1(4), 351 - 67
Computer-assisted primary and secondary structure analyses of DNA polymerases of herpes simplex, Epstein-Barr and varicella zoster viruses reveal conserved domains with some homology to DNA-binding domain in E . coli DNA pol I; Becker Y; The primary and secondary structure of herpes simplex virus type 1 (HSV-1), varicella-zoster (VZV) and Epstein-Barr virus (EBV) DNA polymerases was calculated by means of computer programs . The comparison of HSV-1 polymerase (pol) sequence to the known primary and tertiary structure of E . coli DNA pol I revealed five short homologous sequences, one of which coincided with the alpha-helical structure of the DNA-binding domain of E . coli DNA pol . Comparison by primary and secondary structure computer programs of the three DNA polymerases coded by herpesviruses HSV-1, VZV and EBV led to the identification of polypeptide sequences shared by the three DNA pols . In a similar way, the secondary structure of the DNA pol polypeptide in the vicinity of the mutation leading to PAA resistance in HSV-1 DNA pol helped to identify the role of this sequence in the binding of phosphate donated by the nucleoside triphosphate molecule which binds to the DNA pol . Although the computer secondary structure programs are about 60% accurate, it was possible to obtain new information on the properties of certain domains in the DNA polymerases of HSV-1, VZV and EBV.

Cell, 1988 Jul 1, 54(1), 127 - 35
The replicative origin of the E . coli chromosome binds to cell membranes only when hemimethylated; Ogden GB et al.; DNA from the E . coli replicative origin binds with high affinity to outer membrane preparations . Specific binding regions are contained within a 463 bp stretch of origin DNA between positions -46 and +417 on the oriC map . This region of DNA contains an unusually high number of GATC sites, the recognition sequence for the E . coli DNA adenine methylase . We show here that oriC DNA binds to membrane only when it is hemimethylated . The E . coli chromosomal origin is hemimethylated for 8-10 min after initiation of replication, and origin DNA binds to membranes only during this time period . Based on these results, we propose a speculative model for chromosome segregation in E . coli.

Nucleic Acids Res, 1988 Jun 24, 16(12), 5661 - 72
Phosphotriester formation by the haloethylnitrosoureas and repair of these lesions by E . coli BS21 extracts; Carter CA et al.; The alkylation of phosphates in DNA by therapeutically active haloethylnitrosoureas was studied by reacting N-chloroethyl-N-nitrosourea (CNU) with dTpdT, separating the products by HPLC, and identifying them by co-chromatography with authentic markers . Both hydroxyethyl and chloroethyl phosphotriesters of dTpdT were identified; a similar reaction between CNU and dTR yielded 3-hydroxyethyl and 3-chloroethyl dTR as the major products of ring alkylation . A DNA-like substrate for repair studies was synthesized by reacting 14C-labelled N-(2-chloroethyl)-N'-cyclohexyl-N-nitrosourea (14C-CCNU) with poly dT and annealing the product to poly dA . An extract of E . coli strain BS21 selectively transferred a chloroethyl group from one of the chloroethyl phosphotriester isomers in this substrate to the bacterial protein; chemical instability of the hydroxyethyl phosphotriesters precluded definite conclusions about the repair of this product.

Biochem Int, 1988 Jun, 16(6), 1033 - 40
Degradation of thialysine- or selenalysine-containing abnormal proteins in E . coli; Di Girolamo M et al.; Thialysine and selenalysine can be utilized for protein synthesis by lysine-requiring E . coli cells even in the absence of lysine . Protein synthesis has been determined as labeled leucine incorporation into acid-insoluble material, as increase of cell proteins and as protein-lysine substitution by the analog . Either analog can be incorporated into proteins, in the absence of lysine, for a limited time interval after which cells stop to duplicate . Proteins synthesized during this period contain most of their lysine residues substituted by the analog . Moreover, it has been shown that the analog-containing proteins are unstable and rapidly degraded . Their instability would account for the inability of lysine-requiring E . coli cells to utilize the analog as growth factor.

Jpn J Genet, 1988 Jun, 63(3), 251 - 8
Mischarging mutants of Su+2 glutamine tRNA in E . coli . II . Amino acid specificities of the mutant tRNAs; Yamao F et al.; Among the mischarging mutants isolated from strains with Su+2 glutamine tRNA, two double-mutants, A37A29 and A37C38, have been suggested to insert tryptophan at the UAG amber mutation site as determined by the suppression patterns of a set of tester mutants of bacteria and phages (Yamao et al., 1988) . In this paper, we screened temperature sensitive mutants of E . coli in which the mischarging suppression was abolished even at the permissive temperature . Four such mutants were obtained and they were identified as the mutants of a structural gene for tryptophanyl-tRNA synthetase (trpS) . Authentic trpS mutations, such as trpS5 or trpS18, also restricted the mischarging suppression . These results strongly support the previous prediction that the mutant tRNAs of Su+2, A37A29 and A37C38, are capable of interacting with tryptophanyl-tRNA synthetase and being misaminoacylated with tryptophan in vivo . However, in an assay to determine the specificity of the mutant glutamin tRNAs, we detected predominantly glutamine, but not any other amino acid, being inserted at an amber codon in vivo to any significant degree . We conclude that the mutant tRNAs still accept mostly glutamine, but can accept tryptophan in an extent for mischarging suppression . Since the amber suppressors of Su+7 tryptophan tRNA and the mischarging mutants of Su+3 tyrosine tRNA are charged with glutamine, structural similarity among the tRNAs for glutamine, tryptophan and tyrosine is discussed.

Jpn J Genet, 1988 Jun, 63(3), 237 - 49
Mischarging mutants of Su+2 glutamine tRNA in E . coli . I . Mutations near the anticodon cause mischarging; Yamao F et al.; In order to select the mischarging mutants of Su+2 glutamine tRNA, auxotrophic amber mutants of E . coli K12 which cannot be suppressed particularly by Su+2 were screened . By utilizing these mutants, cysam235 and metam3, several tens of mischarging mutants of Su+2 were isolated, as those conferring altered suppression patterns for a set of tester amber mutants of bacteria and phages . Nucleotide sequence analysis revealed that the mutation sites were found to be exclusively at psi 37 residue located at the 3'-end of anticodon loop, changing it to either A37 or C37 . These mutants were obtained as those suppressing cysam235, and not metam3 . From these, secondary mutants were selected . In these mutants suppression patterns were further altered by the additional base substitutions, capable of suppressing metam3 . Such mutants were obtained exclusively from A37 and not from C37 mutant tRNA . Additional mutations to A37 were found to be either A29 or C38, which are located at the lowermost two base pairs in anticodon stem . The mischarging sites in Su+2 glutamine tRNA locate in the newly detected region of tRNA, differing from the previous case of Su+3 tyrosine or Su+7 tryptophan tRNAs . Implication of this finding is discussed on L-shaped tRNA molecule in relation to aminoacyl-tRNA synthetase recognition . Suppression patterns given by the double-mutants, A37A29 and A37C38, were consistent with the observation that the mutant tRNAs interact with tryptophanyl-tRNA synthetase.

Biokhimiia, 1988 Jun, 53(6), 925 - 30
{Aggregation of membrane proteins of E . coli cells after treatment with singlet oxygen}; Igumenov VL et al.; It was found that interaction of 1O2 with bacterial membranes of E . coli cells results in covalent binding and aggregation of membrane proteins . This process was shown to be inhibited by water-soluble free radical scavengers, e . g., 1O2 quenchers (cysteine, sodium azide, histidine), of which the latter afforded the strongest inhibition . No protective effect of the fat-soluble free radical scavenger ionol (BHT) on membrane protein aggregation was observed . It was assumed that the main role in oxidative destruction of bacterial membranes (in contrast to membranes from animal sources) is ascribed to processes which are not coupled to lipid peroxidation.

J Appl Physiol, 1988 Jun, 64(6), 2468 - 73
Indomethacin, but not dazoxiben, reduced lung fluid filtration after E . coli infusion; Winn R et al.; Goats were divided into three groups and given infusions of live Escherichia coli bacteria . Group I received no treatment, group II was treated with indomethacin (a cyclooxygenase inhibitor), and group III with dazoxiben (a thromboxane synthase inhibitor) . Double indicator-dilution extravascular lung water (EVLW) in group I was significantly different from the treated groups . There was an early increase in EVLW in group I and group III but not in group II animals . At 6 h EVLW's in group I, group II, and group III were 100, 45, and 30% above base line, respectively . Lymph flow (QL) and lymph-to-plasma protein ratio (L/P) was not statistically different between groups . Estimated total fluid filtration {QL + d(EVLW)/dt} in group I and III was markedly elevated between 0 and 1.5-2 h after E . coli infusion . Cardiac output (QT) decreased to 40% of base line in group I, and it decreased slightly in group II because of the indomethacin but did not decrease after E . coli . QT decreased in group III but recovered more rapidly than group I . Mean pulmonary arterial pressure increased more rapidly in group I and reached a higher peak than either treated group . At 6 h these groups had similar pulmonary arterial and pulmonary arterial wedge pressures . We conclude that 1) indomethacin but not dazoxiben blocks the early increase in total fluid filtration after bacterial infusion, 2) dazoxiben does not prevent the increased endothelial permeability resulting from infusion of live bacteria, and 3) indomethacin may somewhat ameliorate the endothelial permeability change.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochem Biophys Res Commun, 1988 May 31, 153(1), 301 - 6
Site-directed mutagenic replacement of glu-461 with gln in beta-galactosidase (E . coli): evidence that glu-461 is important for activity; Bader DE et al.; Glutamic acid 461 of beta-galactosidase (E . coli) was replaced by gln using site-directed mutagenesis . Kinetic studies on the purified Q461-beta-galactosidase showed that it had less than 0.4% of the wild-type activity (with ONPG as substrate), confirming other studies which have suggested that the negative charge on glu-461 is important for activity . The Km values did not increase, indicating that binding of the substrate was not decreased by this change . Thermal denaturation studies showed Q461-beta-galactosidase to be somewhat more susceptible to heat denaturation than the wild-type enzyme.

FEBS Lett, 1988 May 23, 232(2), 369 - 71
Effect of structure of the initiator codon on translation in E . coli; Khudyakov YuE et al.; A set of plasmids carrying different initiator codons--either AUG, or GUG, or UUG, or CUG (as a control) in the hybrid gene lacIZ--was constructed by using synthetic oligonucleotides . GUG and UUG codons were demonstrated to be 2-3 times less effective than AUG in translation initiation . Furthermore, the correlation between the efficiencies of different initiator codons in translation initiation proved to vary, depending on the phase of bacterial growth . The rarely occurring usage in nature of the initiator codons GUG and UUG is supposed to be due to the particular role played by the initiator triplets in regulation of gene expression.

Biochem Biophys Res Commun, 1988 May 16, 152(3), 1050 - 5
Site-directed mutagenesis of beta-galactosidase (E . coli) reveals that tyr-503 is essential for activity; Ring M et al.; By using the technique of site-directed mutagenesis we have succeeded in replacing tyr-503 of beta-galactosidase (E . coli) with a phe . A study of the kinetic and stability properties of this mutant enzyme (F-503 beta-galactosidase) showed that the loss in activity upon this change is due to the loss of a catalytic group (rather than a detrimental change in the enzyme's overall structure or a change in the enzyme's binding capacity) . This confirms previous suggestions that this tyr residue is involved in catalysis.

Biochem Biophys Res Commun, 1988 May 16, 152(3), 1045 - 9
Expression of human c-raf-1 oncogene proteins in E . coli; Kolch W et al.; Full length and truncated versions of the human c-raf-1 cDNA were cloned into the inducible E . coli expression vector pJL6 . C-raf proteins of 73 kD, 57 kD and 39 kD were produced upon induction . p73 differs from normal p73 c-raf by deletion of the two first N-terminal amino acids and their replacement by 16 amino acids encoded by the vector . The p57 and p39 represent N-terminal deletions which leave the transforming protein kinase domain intact . These proteins could be readily purified from E . coli lysates by immunoprecipitation with raf-specific antisera.

Nucleic Acids Res, 1988 May 11, 16(9), 4097 - 109
3' end of the malEFG operon in E.coli: localization of the transcription termination site; Francoz E et al.; The nucleotide sequence of a 981 bp's HincII-PvuII DNA fragment containing the 3' end of the malEFG operon in E . coli was determined . This sequence displayed a putative Rho-independent transcription termination site localized 87 bp's after the stop codon of malG . When cloned into plasmid pKG1800, the HincII-PvuII fragment containing this structure acted as a strong transcription termination signal . By S1 mapping, we demonstrated that the 3' end of the malEFG transcript coincided with the putative transcription termination site . One short open reading frames orf1 (123 bp) and and the beginning of another one orf2 were localized after malG . The transcription termination site is localized within orf1 . Consequently malG is the last gene of the malEFG operon . orf2 corresponds exactly to the 5' part of the xylE gene reported independently (Davis & Henderson, 1987) as the gene coding for the XylE protein, the xylose-proton symport of Escherichia coli.

FEBS Lett, 1988 May 9, 232(1), 107 - 10
Application of the small-angle X-ray scattering technique for the study of equilibrium enzyme-substrate interactions of phenylalanyl-tRNA synthetase from E . coli with tRNAPhe; Tuzikov FV et al.; The small-angle X-ray scattering technique (SAXS) is proposed for the investigation of equilibrium macromolecular interactions of the enzyme-substrate type in solution . Experimental procedures and methods of analysing the data obtained from SAXS have been elaborated . The algorithm for the data analysis allows one to determine the stoichiometric, equilibrium and structural parameters of the enzyme-substrate complexes obtained . The thermodynamic characteristics for the formation of complexes of tRNAPhe with phenylalanyl-tRNA synthetase have been determined and demonstrate negative cooperativity for binding of the two tRNAPhe molecules . The structural parameters (Rg, Rc, semi-axes) have been determined for free phenylalanyl-tRNA synthetase and tRNAPhe from E . coli MRE-600 and of enzyme complexes possessing one and two tRNAPhe molecules, indicating structural rearrangements of the enzyme in the interaction with tRNAPhe.

FEBS Lett, 1988 May 9, 232(1), 111 - 4
E . coli F1-ATPase: site-directed mutagenesis of the beta-subunit; Parsonage D et al.; Residues beta Glu-181 and beta Glu-192 of E . coli F1-ATPase (the DCCD-reactive residues) were mutated to Gln . Purified beta Gln-181 F1 showed 7-fold impairment of 'unisite' Pi formation from ATP and a large decrease in affinity for ATP . Thus the beta-181 carboxyl group in normal F1 significantly contributes to catalytic site properties . Also, positive catalytic site cooperativity was attenuated from 5 X 10(4)- to 548-fold in beta Gln-181 F1 . In contrast, purified beta Gln-192 F1 showed only 6-fold reduction in 'multisite' ATPase activity . Residues beta Gly-149 and beta Gly-154 were mutated to Ile singly and in combination . These mutations, affecting residues which are strongly conserved in nucleotide-binding proteins, were chosen to hinder conformational motion in a putative 'flexible loop' in beta-subunit . Impairment of purified F1-ATPase ranged from 5 to 61%, with the double mutant F1 less impaired than either single mutant . F1 preparations containing beta Ile-154 showed 2-fold activation after release from membranes, suggesting association with F0 restrained turnover on F1 in these mutants.

Infect Immun, 1988 May, 56(5), 1382 - 4
Mitogenic activities of synthetic Escherichia coli lipid A and a synthetic partial structure (tripalmitoyl pentapeptide) of E . coli lipoprotein; Brade L et al.; Synthetic Escherichia coli lipid A and synthetic S-{2,3-bis-(palmitoyloxy)propyl}-N-palmitoylpentapeptide (tripalmitoyl pentapeptide {TPP}), representing the mitogenically active principles of bacterial lipopolysaccharide (LPS) and lipoprotein, respectively, were compared for their mitogenic activities on splenocytes of LPS responder (BALB/c) and LPS-low-responder (C3H/HeJ) mice . Whereas lipid A was active only in LPS-responder mice, TPP resulted in mitogenic activation of B lymphocytes from both LPS-responder and LPS-low-responder mice . When the mitogens were added simultaneously, mainly additive effects of both activators were observed . The data suggest that two different B-lymphocyte populations are responding to these two mitogens.

Klin Padiatr, 1988 May-Jun, 200(3), 184 - 9
{Clinical experiences with polyethylene glycol-bound E . coli L-asparaginase in patients with multiple recurrences of acute lymphoblastic leukemia}; Jurgens H et al.; The efficiency and toxicity of E . coli-L-Asparaginase coupled to polyethyleneglycol (PEG-ASP) was investigated in 5 patients with second relapse of acute lymphoblastic leukemia . PEG-ASP was administered at a dose of 2000 U/m2 as infusion over 2 hrs . every 2 weeks . Following an initial single agent phase, PEG-ASP was combined with prednisone, vincristine, adriamycin and methotrexate i.t . Following induction, all 5 patients were in third remission . The remissions lasted from 3 to 9 months, median 4 months . The toxicity was transient and mild . Also in patients sensitized against native L-Asparaginase no anaphylactic reactions were observed.

Biol Chem Hoppe Seyler, 1988 May, 369 Suppl, 209 - 18
Chemical synthesis of a gene for human cystatin C and its expression in E . coli; Strauss M et al.; A DNA containing the coding sequence for the human cysteine proteinase inhibitor protein cystatin C has been obtained by enzymatic ligation of chemically synthesized deoxyoligonucleotides, using the Khorana ligation method . The 375 bp synthetic gene carries signals for the translation initiation and termination and was expressed in E . coli as a beta-galactosidase fusion protein as well as a secreted protein under the control of the E . coli alkaline phosphatase signal sequence . The secreted hybrid protein was shown to have similar biological properties as the authentic protein isolated from human plasma.

Acta Chem Scand B, 1988 May, 42(5), 314 - 8
Production of active human carbonic anhydrase II in E . coli; Forsman C et al.; cDNA encoding human carbonic anhydrase II has been isolated and its nucleotide sequence determined . Expression of the isolated carbonic anhydrase gene in Escherichia coli from a plasmid containing the tac promoter yielded an active enzyme at a level of about 1% of total protein.

Prikl Biokhim Mikrobiol, 1988 May-Jun, 24(3), 319 - 22
{The use of monoclonal antibodies for purification of ribonuclease H from E . coli}; Smolianinov VV et al.; Monoclonal antibodies to E . coli ribonuclease H were obtained from two hybrid clones . Using the monoclonal antibodies two immunosorbents were synthesized for RNase H which have a slight difference in the capacity and do not differ in the conditions of antigen elution . A homogeneous (according to electrophoresis in PAAG) preparation of the enzyme was obtained using the synthesized immunosorbents.

Mol Gen Mikrobiol Virusol, 1988 May, (5), 36 - 41
{Effect of recB- and recA-mutations on phage restriction in various modification-restriction plasmid systems of E . coli}; Torosian MV et al.; The effect of recB and recA mutations on lambda vir and P1 vir restriction by different restriction-modification plasmid systems of E . coli was studied . It was shown that effect of R1 plasmid coded restriction-modification in E . coli K12 and E . coli B strains and pJA4620 plasmid coded restriction in E . coli K12 is observed only in RecB+ strain . Phenomenon of restriction-modification determined by R124, R245 plasmids does not depend of recB mutation . Effect of recA mutation has not been found in cultures harbouring R1, R245, R124 pJA4620 plasmids.

Biochem Biophys Res Commun, 1988 Apr 29, 152(2), 940 - 7
Characterization of a functional recombinant rat liver aldehyde dehydrogenase: expression as a non-fusion protein in E . coli; Harper K et al.; A cDNA encoding a rat liver inducible aldehyde dehydrogenase carried in a pUC8 plasmid is expressed in E . coli as a dimeric enzyme molecule functionally and physically identical to the authentic rat enzyme . The cDNA appears to be transcribed using the lac promoter, but is translated from an initiator codon 174 base pairs from the 5' end of the cDNA . The aldehyde dehydrogenase polypeptide is not produced as a fusion protein . This is the first example of the production by E . coli of a catalytically active, multimeric eukaryotic protein which is not a fusion protein.

Nucleic Acids Res, 1988 Apr 25, 16(8), 3313 - 26
Integration host factor (IHF) represses a Chlamydomonas chloroplast promoter in E . coli; Thompson RJ et al.; We show that in E . coli, a Chlamydomonas chloroplast promoter, PA, is repressed by Integration Host Factor (IHF) . The himA 42 mutation, altering the alpha-subunit of E . coli IHF, leads to over-accumulation of PA transcripts in vivo . This effect requires upstream chloroplast DNA sequences . DNAase I and methylation protection experiments show that IHF binds in vitro to a site within PA and band-retardation shows that IHF inhibits formation of PA-E . coli RNA polymerase open complexes . We interpret these results, together with our previous deletion analyses, to mean that in E . coli, repression of PA by IHF minimally requires both binding of IHF to a site overlapping PA and binding of one or more additional proteins, perhaps including IHF itself, to sequences upstream of PA.

Cell, 1988 Apr 22, 53(2), 273 - 83
The antifolding activity of SecB promotes the export of the E . coli maltose-binding protein; Collier DN et al.; Evidence is presented that the E . coli secB gene encodes a soluble protein that interacts with the mature region of the precursor maltose-binding protein (MBP), and promotes MBP export by preventing premature folding of the newly synthesized polypeptide into an export-incompetent form . The interaction of SecB with MBP was indicated by the finding that synthesis of various export-defective MBP species interfered with normal protein export by limiting SecB availability . The antifolding activity of SecB was demonstrated by the following: the defect in MBP export in SecB- cells was suppressed by mutational alterations affecting MBP folding; export of a mutant MBP that is accomplished in a strictly posttranslational mode was totally blocked in SecB- cells; and the rate of folding of wild-type MBP synthesized in vitro was found to be accelerated when SecB was absent and greatly retarded when excess SecB was present.

Nucleic Acids Res, 1988 Apr 11, 16(7), 3013 - 24
The AGG codon is translated slowly in E . coli even at very low expression levels; Bonekamp F et al.; Data are presented which indicate that AGG codons for arginine are translated significantly more slowly than the CGU codons for the same amino acid even when their expression level from the probe is very low . The two types of codons were inserted (three in tandem) on a multicopy plasmid in an artificial leader peptide gene in front of the pyrE attenuator where the frequency of transcription termination is regulated by the degree of coupling between transcription and translation . Transcription of the operon is initiated from the lac-promoter dependent on the concentration of the lac-operon inducer IPTG . At all induction levels it was found that the frequency of transcription past the pyrE attenuator was approximately nine times lower when the AGG codons were present in the leader than with CGT codons present . This shows that AGG codons decouple translation from transcription in the pyrE attenuator region even when the concentration of this codon is not increased significantly relative to that in the unperturbed wild type strain . Thus the results indicate that AGG codons are always slowly translated in Escherichia coli.

Nucleic Acids Res, 1988 Apr 11, 16(7), 2825 - 39
The E . coli 16S rRNA binding site of ribosomal protein S15: higher-order structure in the absence and in the presence of the protein; Mougel M et al.; We have investigated in detail the secondary and tertiary structures of E . coli 16S rRNA binding site of protein S15 using a variety of enzymatic and chemical probes . RNase T1 and nuclease S1 were used to probe unpaired nucleotides and RNase V1 to monitor base-paired or stacked nucleotides . Bases were probed with dimethylsulfate (at A(N-1), C(N-3) and G(N-7)), with 1-cyclohexyl-3 (2-(1-methylmorpholino)-ethyl)-carboiimide-p- toluenesulfonate (at U(N-3) and G(N-1)) and with diethylpyrocarbonate (at A(N-7)) . The RNA region corresponding to nucleotides 652 to 753 was tested within: (1) the complete 16S rRNA molecule; (2) a 16S rRNA fragment corresponding to nucleotides 578 to 756 obtained by transcription in vitro; (3) the S15-16S rRNA complex; (4) the S15-fragment complex . Cleavage and modification sites were detected by primer extension with reverse transcriptase . Our results show that: (1) The synthetized fragment folds into the same overall secondary structure as in the complete 16S rRNA, with the exception of the large asymmetrical internal loop (nucleotides 673-676/714-733) which is fully accessible in the fragment while it appears conformationally heterogeneous in the 16S rRNA; (2) the reactivity patterns of the S15-16S rRNA and S15-fragment complexes are identical; (3) the protein protects defined RNA regions, located in the large interior loop and in the 3'-end strand of helix {655-672}-{734-751}; (4) the protein also causes enhanced chemical reactivity and enzyme accessibility interpreted as resulting from a local conformational rearrangement, induced by S15 binding.

Immun Infekt, 1988 Apr, 16(2), 41 - 8
{Mechanism of cell damage by E . coli hemolysin}; Bhakdi S et al.; Many strains of E . coli elaborate a hemolysin which is responsible for the zone of beta-hemolysis surrounding bacterial colonies on blood agar . The significance of this cytolysin as a determinant of bacterial pathogenicity has been established in animal models with the use of genetically engineered, isogenic bacterial strains . An analogous role in human infections has been inferred from the high association of hemolysin production with disease . Studies at a molecular genetical level have defined 4 genes that are required for the synthesis, post-translational modification and secretion of the hemolysin . The structural gene hlyA encodes for a 107-110,000 polypeptide which must be modified in an unknown manner to its active form by the product of the neighboring hlyC gene . Genes hlyB and hlyD encode for proteins that export the molecule to the extracellular medium . The signal for secretion is contained in the C-terminal portion of the toxin molecule . The secreted hemolysin attacks plasma membranes of target mammalian cells by inserting as a monomer into the bilayer and generating a hydrophilic transmembrane pore of approximately 2 nm effective diameter . The pore displays a marked selectivity for cations over anions and pore-opening is dependent on the presence of a correct transmembrane potential . Binding to a membrane target does not require the presence of a specific receptor, and pores may be generated in planar lipid membranes consisting solely of phosphatidylcholine . Pore formation in nucleated cells can trigger secondary reactions such as stimulation of arachidonate metabolism with release of lipid mediators, probably initiated by passive influx of extracellular Ca2+.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochem Int, 1988 Apr, 16(4), 727 - 35
The cloning and over-expression of PABA synthase in E . coli; McLeish MJ et al.; Both the genes encoding E . coli p-aminobenzoic acid synthase have been cloned and an overproducing strain has been obtained . The partial purification of the large subunit is described . The kinetic properties of the cloned enzyme, while similar to those reported for the B . subtilis enzyme, show some differences to those reported for the S . griseus enzyme.

Bioorg Khim, 1988 Apr, 14(4), 478 - 83
{Plasmids containing 2 new variants of the E.coli lacZ gene}; Khudiakov IuE et al.; New plasmids containing partially deleted lacZ genes were obtained . These genes determine high-level synthesis of polypeptides of molecular mass 43-45 and 49-51 kD under the control of the lambda phage PR-promoter; inspite of the deletion, E . coli cells carrying new plasmids were found to possess beta-galactosidase activity . Use of these plasmids as new expression vectors is suggested.

Biochim Biophys Acta, 1988 Mar 31, 949(3), 311 - 7
Supercoiling response of E . coli promoters with different spacer lengths; Aoyama T et al.; The effect of negative supercoiling on a series of synthetic Escherichia coli promoters has been investigated . These promoters carry perfect consensus sequences at the -35 and -10 regions, but with different spacer lengths (Aoyama, T . et al . (1983) Nucleic Acids Res . 11, 5855-5864) . Topoisomeric plasmids carrying these synthetic promoters were constructed, and their activities were compared by detecting in vitro transcripts with the probe-hybridization method . In the relaxed state, the one with 17 basepairs (bp) spacing showed the highest activity, and the activity steeply decreased both sides of the optimal spacing . Similar results have been observed by run-off transcription . By introducing negative superhelicity, the 17 bp spacing promoter showed a relatively little response to supercoiling . In contrast, the activities of those with 16 and 18 bp spacings were markedly stimulated by supercoiling, with the mean, negative superhelical density (-sigma) which gave the maximum activity being about the same for the 16-18 bp spacing promoters (-sigma = 0.03 to 0.04) . The promoter with 19 bp spacing, which showed no activity in the relaxed state, exhibited a significant activity at higher superhelicities (-sigma = 0.06) . Even the 20 bp spacing promoter showed some activity by increasing superhelicity, while the 15 bp spacing promoter did not . On the basis of these observations, possible mechanisms by which negative supercoiling of DNA stimulates the protomer activity are discussed.

Cell, 1988 Mar 25, 52(6), 893 - 901
Site-specific endonucleolytic cleavages and the regulation of stability of E . coli ompA mRNA; Melefors O et al.; The stability of ompA mRNA is growth-rate dependent . We show that the 5' noncoding region of this mRNA provides a target for site-specific endonucleases . The rate of degradation of ompA mRNA parallels the rate of these endonucleolytic cleavages, implying that endonucleolytic rather than exonucleolytic attack is the initial step in ompA mRNA degradation . Thus the 5' noncoding region appears to be a determinant of mRNA stability, and endonucleolytic cleavages in the 5' noncoding region may well regulate expression of the ompA gene.

Nucleic Acids Res, 1988 Mar 25, 16(6), 2601 - 12
Randomly picked cosmid clones overlap the pyrB and oriC gap in the physical map of the E . coli chromosome; Knott V et al.; Sets of overlapping cosmid clones generated by random sampling and fingerprinting methods complement data at pyrB (96.5') and oriC (84') in the published physical map of E . coli . A new cloning strategy using sheared DNA, and a low copy, inducible cosmid vector were used in order to reduce bias in libraries, in conjunction with micro-methods for preparing cosmid DNA from a large number of clones . Our results are relevant to the design of the best approach to the physical mapping of large genomes.

Nucleic Acids Res, 1988 Mar 25, 16(5), 2031 - 44
Far upstream sequences of the bla promoter from TN3 are involved in complexation with E . coli RNA-polymerase; Duval-Valentin G et al.; The structure of the final initiation complex between E . coli RNA polymerase (RNAP) and the bla promoter from the transposon TN3 has been probed by footprinting experiments and base accessibility to dimethyl sulfate at 37 degrees C . At RNAP/promoter molar ratios "standard" for these experiments (greater than or equal to 10), the contacts on bla extend from -100 to +20, i.e . a length exceeding twice the dimension of the RNAP major axis {33} . Since footprinting at about equimolar amounts of RNAP and bla extends to the usual (-55 to +20) promoter domain, it is very likely that at least two RNAP's participate in the complex observed at tenfold higher RNAP/bla ratios . Under the latter conditions, the extended footprint (-100 to +20) is observed above 30 degrees C, whereas at 15 degrees C, only the -55 to +20 promoter area is contacted . Furthermore, gel retardation experiments show the presence of two complexes of different migration rates . We have reported earlier {21} that at the "standard" RNAP/bla ratio, transcription initiation from the bla promoter is inhibited . The correlation of this inhibition with the postulated two RNAP/bla complex suggests a regulation of bla gene expression by RNAP availability controlled for instance by growth rate . These results can be correlated with those reported in {14, 15} for the tyrT promoter . Interestingly, both promoter share significant sequence homologies.

Nucleic Acids Res, 1988 Mar 25, 16(5), 1999 - 2014
Second-strand cDNA synthesis with E . coli DNA polymerase I and RNase H: the fate of information at the mRNA 5' terminus and the effect of E . coli DNA ligase; D'Alessio JM et al.; A simple method for generating cDNA libraries has been described (1) in which RNase H-DNA polymerase I-mediated second-strand cDNA synthesis primes from an RNA oligonucleotide derived from the 5' (capped) end of mRNA . The size of this oligonucleotide and the fate of the information corresponding to the RNA during subsequent cloning have not been established . We show here that the 5'-most RNA primer varies in length from 8 to 21 nucleotides, and that information corresponding to the length of the RNA primer is normally lost during cloning . A modification of the second-strand cDNA synthesis procedure is described which allows cloning of all, or almost all, of the primer sequence information . In addition, we show that the presence of E . coli DNA ligase during second-strand cDNA synthesis can increase the length of the cDNA clones obtained from long RNAs . Cloning by addition of linkers provides the greatest chance of obtaining near full-length cDNA clones from long mRNAs.

J Theor Biol, 1988 Mar 21, 131(2), 235 - 41
What does the homology between E . coli tRNAs and RNAs controlling ColE1 plasmid replication mean?
Yavachev L, Ivanov I.
Nucleotide sequences of E . coli tRNAs and RNA I or RNA II (controlling replication of ColE1 plasmids) were compared using the computer . The homology between some of these molecules is over 60% . The distribution of homologous nucleotides among the functional elements (stems and loops) of either RNA I or RNA II and the tRNAs molecules was studied . It was found that the homologous domains are located mainly in the loop regions of RNA I or RNA II . A consensus sequence, the nonanucleotide AGUUGGUAG, was discovered in loop II of RNA I and in the dihydrouridylic loop of tRNAs showing homology with RNA I . Based on this observation, a hypothesis was drawn for a possible role of the tRNAs in the regulation of plasmid DNA replication.

Nature, 1988 Mar 17, 332(6161), 284 - 6
Zinc-finger motifs expressed in E . coli and folded in vitro direct specific binding to DNA; Nagai K et al.; The short sequence motif named 'zinc finger', first recognized repeated in tandem in the Xenopus transcription factor IIIA (TFIIIA), is also found in the yeast transcriptional activator SWI5 (ref . 3) and many other regulator proteins . Embedded in the 709-amino-acid polypeptide chain of SWI5 are three tandemly repeated zinc-finger motifs . Because the zinc fingers of TFIIIA are known to bind to DNA, it is probable that in the case of SWI5 these finger motifs also play an important, but not necessarily exclusive, role in the sequence-specific binding of the protein to DNA . To test this prediction we have expressed the 89-amino-acid sequence of the domain containing the three zinc fingers of SWI5 in Escherichia coli as a cleavable fusion protein, purified under denaturing conditions and folded in vitro . This experimental approach allows us to study directly both the metal requirement and DNA-binding properties of the isolated polypeptide . We find that zinc is required for specific DNA recognition and, most significantly, DNaseI protection studies show that the isolated three-fingered domain is sufficient for sequence-specific binding to DNA.

Cell, 1988 Mar 11, 52(5), 713 - 22
Stringent regulation of stably integrated chloramphenicol acetyl transferase genes by E . coli lac repressor in monkey cells; Figge J et al.; Monkey cell lines that constitutively synthesize 38.6 kd lac repressor protein and bear stably integrated chloramphenicol acetyl transferase (CAT) genes linked to a lac operator-containing SV40 early promoter-enhancer were generated . When grown in medium containing isopropyl beta-D-thiogalactoside (IPTG), these cells acquired a CAT+ phenotype . In contrast, when grown in parallel in medium lacking IPTG, the cells remained CAT- . Maximum induction of CAT activity occurred after 4 days of IPTG exposure . Three days after removal of IPTG, induced cells had reverted to CAT- . Specific CAT activity increased up to 60-fold after induction, while background activity in uninduced cells was similar to or only slightly above that of parental, CAT- cells . CAT activity increased stepwise over a wide range of IPTG concentrations . Thus lac repressor-operator complexes can form on primate chromosomes and stringently block transcription from an adjoining promoter.

Zentralbl Bakteriol Mikrobiol Hyg {A}, 1988 Mar, 267(4), 576 - 88
Enterohemolysin, a new type of hemolysin produced by some strains of enteropathogenic E . coli (EPEC); Beutin L et al.; 42 Escherichia coli O26 strains which had been isolated at geographically different places and over a long time period were examined for hemolysin synthesis . 17 of these were found to be hemolysin-negative, nine strains were found to produce plasmid encoded alpha-hemolysin and 16 strains were shown to produce a phenotypically different hemolysin . This new type of hemolysin was called enterohemolysin and found to be genetically and immunologically non-related with the already described E . coli alpha-hemolysin . Enterohemolytic E . coli were not found in feces of 200 healthy infants under the age of two years . However, four E . coli O111 strains, one O121:H- and one O25:K5:H- strain, all from infants with diarrhoea were also enterohemolysin producers.

Int J Pept Protein Res, 1988 Mar, 31(3), 255 - 64
Purification and conformation of ribosomal protein L25 from E . coli ribosome; Fox JW et al.; Ribosomal protein L25 from the large subunit of E . coli ribosomes has been purified using a new procedure involving a 2M LiCl extraction followed by phosphocellulose chromatography in 6 M urea elution buffer . The conformation of purified L25 was studied employing circular dichroism and ultraviolet absorption spectroscopy in reconstitution buffer . The analysis of the far u.v . circular dichroism spectrum of L25 indicates L25 contains approximately 16% alpha-helix and approximately 19% beta-structure . The conformation of L25 was also studied using the predictive methods of Chou & Fasman and Maxfield & Scheraga . Both of these methods predict approximately three times the percent alpha-helix present in L25 as compared with that determined from the analysis of the circular dichroism spectrum . A structure for L25 is predicted which contains two positively charged binding domains and is consistent with published binding data on the interaction of 5S RNA and L25 . The large difference in the % alpha-helix as determined from the analysis of the circular dichroism spectrum and the predictive techniques is suggested to result from the denaturing effects of 6 M urea used in the preparation of ribosomal proteins.

Radiobiologiia, 1988 Mar-Apr, 28(2), 171 - 5
{The oxygen effect in cells of E . coli K-12 with different repair genotypes during irradiation with neutrons and gamma rays}; Komova OV et al.; The oxygen enhancement ratio, as estimated after the effect of 137Cs-gamma-quanta, depends on the repair genotype of E . coli K-12 cells and increases in the studied strains in the following order: recA-uvrA(-)----recA(-)----wild type----pol A- . These variations are levelled with the effect of fast neutrons of division spectrum (0.75 MeV); the oxygen enhancement ratio for the strains under study decrease, while the oxygen effect is virtually absent in recA-uvrA--mutant.

Mutat Res, 1988 Mar, 198(1), 53 - 60
Induction of the SOS function sfiA in E . coli by systems which generate triplet ketones; Nassi L et al.; Generation of triplet ketones, either chemically through thermal decomposition of 3-hydroxymethyl-3,4,4-trimethyl-1,2-dioxetane and 3-{N-(pyridino)carbamoyl}methyl-3,4,4-trimethyl-1,2-dioxetane++ + or enzymatically via the aerobic oxidation of isobutyraldehyde trimethylsilyl enol ether catalyzed by horse-radish peroxidase, triggers the SOS function sfiA in E . coli . Although the observed effects are relatively weak and the triplet ketone scavenger tryptophan was ineffective in this system, our results provide evidence for the involvement of triplet ketones in this type of DNA damage . Possible mechanisms are discussed.

J Trauma, 1988 Mar, 28(3), 379 - 82
The effect of splenectomy on antibody response to lipopolysaccharide (E . coli) immunization; Ohshio G et al.; The influence of splenectomy on the antibody response to lipopolysaccharide (LPS: E . coli 0128:B12) was investigated in mice . Splenectomy had little effect on the primary response to the LPS . However, the level of IgG anti-LPS antibodies of splenectomized mice was significantly lower than that of sham-operated mice when the mice were immunized 1, 3, and 7 days after the operation and reimmunized 7 days after the first immunization . There was no significant difference in those immunized 30 days after the operation and reimmunized 7 days later . In mice immunized before splenectomy and reimmunized 30 days after splenectomy, the level of IgG anti-LPS antibodies was low, even in the mice splenectomized 30 days after primary immunization . Our results indicate that splenectomy impairs the antibody response to lipopolysaccharides.

Mutat Res, 1988 Mar, 198(1), 45 - 51
Gene expression in E . coli after treatment with streptozotocin; Fram RJ et al.; Gene induction by the methylating agents streptozotocin (STZ), N-methyl-N-nitrosourea (MNU), and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) was evaluated in E . coli fusion mutants . These mutants have fusions of the lac operon to genes induced by treatment with sublethal levels of alkylating agents and were previously selected from random insertions of the Mu-dl (Apr lac) phage by screening for induction of beta-galactosidase activity in the presence of methyl methanesulfonate or MNNG . The results demonstrate that STZ differs from MNNG and MNU in failing to induce aidC expression . Further, expression of aidC after exposure to MNU and MNNG occurs only in nonaerated cultures; aeration blocks the induction . Induction of aidD, alkA, aidB, and sfiA expression occurs with all 3 agents although at markedly lower concentrations of MNNG and STZ compared to MNU . alkA and to a lesser extent aidD mutants of E . coli strains were more sensitive to these agents, while no differences were evident between wild-type and aidB or aidC fusion mutants.

Mol Gen Mikrobiol Virusol, 1988 Mar, (3), 33 - 9
{Mutation fruB in the fructose regulon affecting beta-galactosidase synthesis and adenylate cyclase activity of E . coli K12}; Bol'shakova TN et al.; Escherichia coli mutant devoid of fructosespecific factor III (factor IIIfru) of phosphoenolpyruvate (PEP) carbohydrate phosphotransferase system was isolated . The mutation fruB was localized on 46 min of chromosomal map of Escherichia coli in the fru-operon region . The mutant bacteria are unable to accumulate fructose . PEP-dependent phosphorylation of this carbohydrate in cellular extracts was considerably decreased . The mutational damage of factor IIIfru results in the suppression of beta-galactosidase synthesis . The impaired synthesis of the enzyme was partially resistant to glucose catabolite repression . However, the deficiency was suppressed by addition of exogenous cyclic AMP . The adenylatecyclase activity in fruB mutant was found to be 50% lower as compared with the one in the parent strain.

Zentralbl Bakteriol Mikrobiol Hyg {A}, 1988 Mar, 267(4), 485 - 94
Haemolytic activity and characteristics of plasmid and chromosomally borne hly genes isolated from E . coli of different origin; Grunig HM et al.; 20 hly plasmids of different origin and source and 8 recombinant plasmids containing either chromosomally or plasmid encoded hly determinants were characterized with emphasis on their haemolytic activity . Remarkable differences among the hly genes tested were found in their haemolytic activity and in the regulation of the haemolytic activity by the extracellular iron concentration . Under iron-limiting growth conditions the haemolysin secretion encoded by 6 hly plasmids, but not by the remaining 14 hly plasmids and the chromosomal hly genes, was significantly induced . The 6 plasmids all derived from human isolates, belonged to the incompatibility groups FIV and FVI and showed similar restriction patterns . Two hly determinants, encoding an inducible haemolysin secretion, cloned from plasmids isolated in Berne and Paris, respectively, showed identical restriction maps, indicating a common origin . Compared to the hly determinant of a plasmid encoding a noninducible haemolysin secretion, distinct differences, mainly in the flanking regions, were observed.

Virus Genes, 1988 Mar, 1(2), 221 - 33
An MuLV transmission vector system designed to permit recovery in E . coli of proviral and cellular flanking sequences; Jorgensen P et al.; We have introduced a bacterial suppressor gene (supF) into the long terminal repeat of a molecular clone of the murine leukemia virus (MuLV) SL3-3 . A panel of replication competent virus was derived that replicates to high titers in NIH3T3 cells in culture . The tRNA gene is stably carried in the provirus . The supF and viral sequences are present in equimolar amounts in the RNA genome of the expressed recombinant virus . The proviral sequences containing supF can be recovered by cloning into a lambda vector carrying amber mutations . The DNA sequences in the recovered lambda recombinants show a high degree of stability . The presented system should facilitate the study of the interaction between proviral and cellular sequences flanking the integration site.

Biochem Biophys Res Commun, 1988 Feb 29, 151(1), 598 - 607
Modulation of expression of the human gamma interferon gene in E . coli by site-directed mutagenesis; Lee SG et al.; Plasmids expressing 2 forms of human immune interferon (IFN-gamma) in E . coli have been constructed: 1) pIFNTacI which expresses IFN-gamma with an N-terminal amino acid sequence of met-cys-tyr-cys-gln-, and 2) pIFNTacII which is a derivative of pIFNTacI from which the 9 base pairs (bp) coding for the cys-tyr-cys have been deleted . Quantitation of Western blots showed that approximately 10-fold more IFN-gamma was produced in cells harboring pIFNTacII (7.5% of total cellular protein) as compared to pIFNTacI . The IFN-gamma expressed in E . coli pIFNTacII is biologically active and routinely recoverable at 10(9) units per liter . When examined microscopically, IPTG induced E . coli harboring either plasmid construction contains prominent cytoplasmic inclusion bodies.

Biochim Biophys Acta, 1988 Feb 28, 949(2), 233 - 9
Preparation of raf-oncogene-specific antiserum with raf protein produced in E . coli; Kolch W et al.; In this report we describe the expression of v-raf protein in E . coli using a tryptophan-promoter-driven expression vector and its immunological characterization by anti-peptide sera . The purified recombinant protein was used to produce raf-specific antibodies which are suitable for studying v-raf and c-raf proteins in vitro and in vivo in a variety of species ranging from mouse to man.

Biochim Biophys Acta, 1988 Feb 28, 949(2), 213 - 23
Reactivity of an HIV gag gene polypeptide expressed in E . coli with sera from AIDS patients and monoclonal antibodies to gag; Marcus-Sekura CJ et al.; A segment of the gag gene of the human immunodeficiency virus (HIV) (HTLV-IIIB strain), the virus which causes acquired immunodeficiency syndrome (AIDS), has been cloned into the bacterial expression vector, pCQV2, and mapped to the right-hand portion of the gag gene containing the carboxyl-terminal portion of p24 and the amino-terminal portion of p15 . Nucleic-acid sequencing of the insert-vector junctions further defined the 5'-terminal nucleotide of HIV sequence as nucleotide 997 and the 3'-terminal nucleotide as 1696 . When used in an enzyme-linked immunosorbent assay (ELISA) with sera from HIV-infected patients, the cloned antigen reacted with a subset of sera which were positive on a standard ELISA using whole virus as antigen . Western-blot screening of these sera with whole virus indicated that all p24-positive sera were positive with the clone, suggesting that the carboxyl-terminal portion of p24 contains a highly antigenic epitope(s) . A serum which was p24-negative p15-positive by Western blot analysis was also highly reactive, indicating that a p15 epitope is present in the cloned antigen . Epitope mapping with a series of monoclonal antibodies to gag resulted in positive ELISA with 2 of 3 anti-p24, 0 of 1 anti-p15, and 0 of 1 anti-p17 Western-blot-positive monoclonal antibodies, suggesting that one of the anti-p24 monoclonal antibodies reacts with epitopes amino-terminal to those coded from nucleotide 997, two anti-p24 monoclonals react with epitopes carboxyl-terminal to those coded from nucleotide 997, and the anti-p15 monoclonal reacts with epitopes carboxyl-terminal to those coded from nucleotide 1696.

Cell, 1988 Feb 26, 52(4), 569 - 84
A physiological role for DNA supercoiling in the osmotic regulation of gene expression in S . typhimurium and E . coli; Higgins CF et al.; The proU locus encodes an osmotically inducible glycine betaine transport system that is important in the adaptation to osmotic stress . We present evidence that DNA supercoiling plays a key role in the osmotic induction of proU transcription . An increase in extracellular osmolarity increases in vivo DNA supercoiling, and the expression of proU is highly sensitive to these changes . Furthermore, topA mutations can mimic an increase in osmolarity, facilitating proU expression even in media of low osmolarity in which it is not normally expressed . Selection for trans-acting mutations that affect proU expression has yielded only mutations that alter DNA supercoiling, either in topA or a new genetic locus, osmZ, which strongly influences in vivo supercoiling . Mutations in osmZ are highly pleiotropic, affecting expression of a variety of chromosomal genes including ompF, ompC, fimA, and the bgl operon, as well as increasing the frequency of site-specific DNA inversions that mediate fimbrial phase variation.

Nucleic Acids Res, 1988 Feb 25, 16(4), 1603 - 15
Identification of defined sequences in domain V of E . coli 23S rRNA in the 50S subunit accessible for hybridization with complementary oligodeoxyribonucleotides; Marconi RT et al.; The accessibility of specific sequences in domain V of E . coli 23s rRNA in the 50S subunit to complementary oligodeoxyribonucleotides (cDNA) has been investigated . The apparent percentage of subunits engaged in complex formation was determined by incubation of radiolabeled cDNA probe with 50S subunits, followed by nitrocellulose membrane filtration of the reaction mixtures and measurement of the bound radiolabeled cDNA probes by liquid scintillation counting of the filters . The site(s) of hybridization were determined by digestion of the RNA in the RNA/DNA heteroduplex by RNase H . The results of this study indicated that single-stranded sequences, 2058-2062, 2448-2454, 2467-2483, and 2497-2505 were available for hybridization to cDNA probes . Bases 2489-2496, which have been postulated to be base paired with 2455-2461 were also accessible for hybridization.

Nucleic Acids Res, 1988 Feb 25, 16(4), 1563 - 75
McrA and McrB restriction phenotypes of some E . coli strains and implications for gene cloning; Raleigh EA et al.; The McrA and McrB (modified cytosine restriction) systems of E . coli interfere with incoming DNA containing methylcytosine . DNA from many organisms, including all mammalian and plant DNA, is expected to be sensitive, and this could interfere with cloning experiments . The McrA and B phenotypes of a few strains have been reported previously (1-4) . The Mcr phenotypes of 94 strains, primarily derived from E . coli K12, are tabulated here . We briefly review some evidence suggesting that McrB restriction of mouse-modified DNA does occur in vivo and does in fact interfere with cloning of specific mouse sequences.

Biochem Biophys Res Commun, 1988 Feb 15, 150(3), 917 - 24
Characterization of a photoaffinity analog of UTP, 5-azido-UTP for analysis of the substrate binding site on E . coli RNA polymerase; Woody AY et al.; The substrate binding site on E . coli RNA polymerase was investigated by photoaffinity labeling with a photoaffinity analog of UTP, 5-azido-UTP . We have established that 5-azido-UTP is a substrate for RNA polymerase by specific transcription on 229 bp DNA containing the gene II promoter of M13 phage . Analysis of the initial rate of RNA synthesis gives Km(5-azido-UTP) approximately 80 microM . Photolabeling with varying concentrations of 5-azido-UTP follows a saturation curve with the midpoint occurring at a 5-azido-UTP concentration of 65 microM near to the Km obtained by kinetic analysis . 5-Azido-UTP photolabels the beta', beta, and sigma subunits to about the same extent, both in the presence (33, 31, and 36%) and absence (35, 30 and 35%) of DNA . This labeling pattern is somewhat different from that obtained with 8-azido-ATP (beta' greater than sigma much greater than beta greater than alpha).

Nucleic Acids Res, 1988 Feb 11, 16(3), 1063 - 78
Induction of an abortive and futile DNA repair process in E . coli by the antitumor DNA bifunctional intercalator, ditercalinium: role in polA in death induction; Lambert B et al.; Ditercalinium, an antitumor bifunctional intercalator which forms a high affinity reversible complex with DNA, was found to be specifically cytotoxic for polA and lig7 E . coli strains . In the polA strain, the cytotoxic effect of ditercalinium was suppressed by the uvrA mutation . DNA single strand breaks accumulated in presence of ditercalinium at high temperature in lig7 strains but not in polA strains . Ditercalinium caused no DNA synthesis inhibition although it was able to induce SOS functions . It is proposed that the ditercalinium DNA complex because of its non covalent nature acts as a dummy lesion for the UV repair system in E . coli leading to a futile and abortive repair process . Polymerase I appears to be required to prevent the malfunctioning of a DNA repair process triggered by molecules forming non covalent complex with DNA.

Nucleic Acids Res, 1988 Feb 11, 16(3), 1135 - 41
The mechanisms of action of E . coli endonuclease III and T4 UV endonuclease (endonuclease V) at AP sites; Kim J et al.; Treatment of DNA containing AP sites with either T4 UV endonuclease or with E . coli endonuclease III followed by a human class II AP endonuclease releases a putative beta-elimination product . This result suggests that both the T4 endonuclease and E . coli endonuclease III class I AP endonucleases catalyze phosphodiester bond cleavage via a lyase- rather than a hydrolase mechanism . Indeed, we have not detected a class I AP endonuclease which hydrolytically catalyzes phosphodiester bond cleavage . Whereas these enzymes use a lyase-like rather than a hydrolytic mechanism, they nonetheless catalyze phosphodiester bond cleavage . We suggest that the term endonuclease can be properly applied to them.

FEBS Lett, 1988 Feb 8, 228(1), 1 - 6
Crosslinking of ribosomal protein S18 to 16 S RNA in E.coli ribosomal 30 S subunits by the use of a reversible crosslinking agent: trans-diamminedichloroplatinum(II); Moine H et al.; We have previously developed {(1987) Biochemistry 26, 5200-5208} the use of trans-diamminedichloroplatinum(II) to induce reversible RNA-protein crosslinks in the ribosomal 30 S subunit . Protein S18 and, to a lesser extent, proteins S13/S14, S11, S4 and S3 could be crosslinked to the 16 S rRNA . The aim of the present work was to identify the crosslinking sites of protein S18 . Three sites could be detected: a major one located in region 825-858, and two others located in regions 434-500 and 233-297 . This result is discussed in the light of current knowledge of the topographical localization of S18 in the 30 S subunit and of its relation with function.

Mol Gen Mikrobiol Virusol, 1988 Feb, (2), 39 - 41
{Effect of the molecular weight of DNA on the effectiveness of plasmid transformation of E . coli K12}; Molchanova ES et al.; The level of plasmid transformation and transfection by the high molecular mass DNA was studied for Escherichia coli mutants having increased efficiency of plasmid transformation by low molecular mass DNA . Decreased level of plasmid transformation and transfection registered in some mutants as compared to the one in wild type strain suggests the specificity of Escherichia coli cells penetration for DNA of different molecular mass.

Liver, 1988 Feb, 8(1), 1 - 9
Histological changes in the liver and portal hypertension subsequent to repeated intraportal injections of killed E . coli in the dog; Sugita S et al.; The etiology of idiopathic portal hypertension (IPH) is not known . To obtain clues to the pathogenesis, an attempt was made to produce a hepatic lesion similar to that in IPH by repeated injections of aggregated killed non-pathogenic E . coli directly into the portal vein . In the treated dogs, histology of the liver showed dense fibrosis in the portal tract and an aberrant vasculature around the portal area after 1 month . Portal pressure was elevated and middle-to-small-sized portal branches were decreased in number as studied by portography . These changes closely mimic those seen in human IPH . The possibility is discussed that chronic entrance of an antigen such as bacteria from the intestine to the portal venous system plays an etiologic role in IPH.

Klin Wochenschr, 1988 Feb 1, 66(3), 110 - 6
Expression of viral hemagglutinin on the surface of E . coli; Pistor S et al.; Expression of a foreign protein molecule on the E . coli bacterial surface has been achieved through hybrid plasmid construction of fusion proteins using outer membrane protein ompA as a carrier system . Influenza virus hemagglutinin fusion proteins of this character have been shown to become integrated into the bacterial outer membrane and to expose their hemagglutinin moiety at the exterior surface in a conformation which is at least similar to the authentic viral antigen structure.

Int J Radiat Biol Relat Stud Phys Chem Med, 1988 Feb, 53(2), 301 - 8
Effects of monoenergetic X-rays with resonance energy of bromine K-absorption edge on bromouracil-labelled E . coli cells; Maezawa H et al.; In order to examine enhanced killing that might be induced by Auger cascades in the incorporated atoms in cells, bromouracil(BrU)-labelled E . coli cells were irradiated with monoenergetic X-rays at 13.49 and 12.40keV, just above and below the K-absorption edge of bromine . In both cases BrU-labelled cells were more sensitive for killing than were normal cells . However, when the degree of BrU-sensitization was compared between the two energies of X-rays, the enhanced killing at 13.49 keV was only small, 2 +/- 8 per cent based on the D0 value in saline . By the addition of DMSO, which is believed to suppress radical-mediated effects, killing of BrU-labelled cells was enhanced at 13.49 keV by 8 +/- 4 per cent as compared with 12.40 keV, based on D0 . These results have been examined in terms of absorbed energy in BrU-labelled cells and in terms of the number of induced Auger events.

APMIS, 1988 Feb, 96(2), 177 - 84
The E . coli immunosorbent as used in serodiagnosis of Legionella infections studied by crossed immunoelectrophoresis; Bangsborg JM et al.; In this study we investigated an immunosorbent, E . coli blocking fluid (BF), proposed for use in the Legionella Indirect Fluorescent Antibody Test (IFA) . With crossed immunoelectrophoresis (CIE) of clinically relevant Legionella species, only one heat-stable antigen (no . 1) cross-reacted with the BF preparation . Patients' sera with elevated Legionella IFA titres did not react with this antigen in CIE . Out of 23 IFA positive patients' sera, six had titres lowered significantly to negative, when BF was applied as serum diluent for the titration (IFA BF negative sera) . All six sera were negative in the micro agglutination test (MA) . None of the IFA BF negative sera contained any Legionella precipitins in CIE, whereas nine out of the remaining 17 IFA BF positive sera unchanged by BF contained one or more precipitins . CIE results could not explain the effect of BF in Legionella IFA, and further studies are needed to sufficiently define the use of immunosorbents in diagnostic Legionella serology.

J Surg Res, 1988 Feb, 44(2), 178 - 84
Modification of catecholamine and prostaglandin tissue levels in E . coli endotoxin-treated rats; Garcia-Barreno P et al.; Epinephrine and norepinephrine adrenal levels were depleted in Escherichia coli endotoxin-treated (10 mg/kg) male Wistar rats from 504 +/- 203 to 119 +/- 77 ng/mg in control and from 200 +/- 97 to 123 +/- 66 ng/mg wet wt, respectively . However dopamine increased from 4.2 +/- 1.9 to 14.9 +/- 3.4 ng/mg . After endotoxin administration, norepinephrine content in peripheral organs, heart 1.27 +/- 0.19 ng/mg, spleen 1.52 +/- 0.59 ng/mg, liver 0.15 +/- 0.05 ng/mg, and kidney 0.24 +/- 0.09 ng/mg wet wt . decreased by 34, 36, 47, and 18%, respectively . Indomethacin treatment kept the catecholamine levels constant in endotoxic rats, but naloxone had no effect . PGF2 alpha tissue levels (12.0 +/- 10.1 pg/mg protein in spleen and 1.85 +/- 0.6 pg/mg protein in liver) were increased twofold by endotoxin treatment: PGE2 content in spleen and liver 0.5 +/- 0.2 pg/mg protein and 2.3 +/- 1.9 pg/mg protein, respectively, increased by only 27 and 26% . In the kidneys of endotoxin-treated animals, PGF2 alpha and PGE2 levels were lower than in control . Indomethacin treatment decreased PGF2 alpha and PGE2 and increased the norepinephrine content in the same organs . It is suggested that prostaglandins play a participative role in the control of norepinephrine tissue levels in endotoxemia.

Acta Chir Scand, 1988 Feb, 154(2), 133 - 9
Arachidonic acid cascade metabolites in porcine E . coli shock . Coagulation, fibrinolytic and hemodynamic response; Svartholm E et al.; Cardiopulmonary hemodynamics and changes in various hemostatic factors (alpha 2M, alpha 2AP, AT III, prothrombin-proconvertin activity, fibrinogen concentration, ethanol gelation test and fibrinolytic activity on fibrin plates) were investigated in pigs during shock induced with live Escherichia coli . Anesthetized pigs were treated with indomethacin or with the combined cyclooxygenase/lipoxygenase inhibitor BW755C before the E . coli infusion or were left untreated as septic controls . Septic shock developed in all of these animals . Pretreatment attenuated the early deterioration of pulmonary circulation but did not modify the coagulation/fibrinolytic activation or the disturbed cardiopulmonary hemodynamics seen in the delayed phase of shock . The arachidonic acid cascade metabolites thus seems to mediate the early, but not the delayed cardiopulmonary reaction and to have minor importance for activation of coagulation and fibrinolysis in E . coli-shocked pigs.

Cell, 1988 Jan 29, 52(2), 197 - 206
Processed mRNA with differential stability in the regulation of E . coli pilin gene expression; Baga M et al.; E . coli expressing the papA-I genes produce pili that mediate specific adhesion to mammalian cells . We show that the major pilus subunit gene, papA, is part of a polycistronic transcriptional unit subject to specific posttranscriptional processing . A primary transcript also encoding the papB regulatory gene product is endonucleolytically cleaved, resulting in the rapid decay of the papB-encoding 5' half of the mRNA, whereas the papA-encoding 3' half remains as a quite stable transcript . Processing and differential mRNA stability thereby result in accumulation of mRNAs encoding only the major pilus subunit . A sequence immediately downstream of the papA coding region may serve as a stability determinant for the papA transcript and concomitantly attenuate read-through transcription into the minor pilus subunit gene papH . This suggests that differential expression of genes within an operon may include endo- and exonucleolytic processing of the mRNA.

Science, 1988 Jan 15, 239(4837), 293 - 5
A 13-kilodalton maize mitochondrial protein in E . coli confers sensitivity to Bipolaris maydis toxin; Dewey RE et al.; The Texas male-sterile cytoplasm (cms-T) of maize carries the cytoplasmically inherited trait of male sterility . Mitochondria isolated from cms-T maize are specifically sensitive to a toxin (BmT-toxin) produced by the fungal pathogen Bipolaris maydis, race T, and the carbamate insecticide methomyl . A mitochondrial gene unique to cms-T maize, which produces a 13-kilodalton polypeptide associated with cytoplasmic male sterility, was expressed in Escherichia coli . After addition of BmT-toxin or methomyl, inhibition of whole cell respiration and swelling of spheroplasts were observed in Escherichia coli cultures producing the novel mitochondrial protein; these effects are similar to those observed with isolated cms-T mitochondria . The amino-terminal region of the 13-kilodalton polypeptide appears to be essential for proper interaction with the BmT-toxin and methomyl . These results implicate the 13-kilodalton polypeptide in conferring toxin sensitivity to mitochondria of cms-T maize.

Ultramicroscopy, 1988, 25(1), 13 - 22
Three-dimensional matching of macromolecular structures obtained from electron microscopy: an application to the 70S and 50S E . coli ribosomal particles; Carazo JM et al.; In this work we present a general computational method capable of finding the relative orientation of two structures represented by samples on a three-dimensional grid . It is shown that the three-dimensional shift and the three independent rotations necessary for the correct relative spatial placement of the two volumes can be obtained either from the auto-correlation function of the volumes or from a direct cross-correlation, depending on the specific problem to be solved . This method has been applied to the problem of fitting the 50S ribosomal subunit into the 70S monosome from E . coli, structures that were available as three-dimensional reconstructions from electron microscopical data.

Orig Life Evol Biosph, 1988, 18(1-2), 97 - 105
Evolution of E . coli tRNA(Trp); Staves MP et al.; Earlier studies (1) have shown there are direct correlations between the hydrophobicity ranking of most amino acids and their anticodonic nucleotides . However, four anticodonic assignments, i.e . those for Trp, Tyr, Ile and the XGA anticodons for Ser, did not correlate . It was our proposal that this failure to correlate was due to the fact that these assignments were made late, relative to the bulk of the assignments, in evolution through the mutation of existing tRNAs . We have shown (2) that E . coli tRNA(Ile 1) and tRNA(Ile 2) were likely derived from tRNA(Val 1) and tRNA(Lys) respectively and E . coli tRNA(Tyr) was possibly derived from E . coli 5s rRNA or a common precursor with 5s rRNA (3) . The fact that quite high homologies were observed in these comparisons is consistent with the late evolution of the tRNAs in question . We now examine the evolution of E . coli tRNA(Trp) by comparing its homology with other E . coli tRNAs . The data suggest a possible evolutionary relationship with E . coli tRNA(Gly) or tRNA(Arg) . The data support the idea of the late assignment of anticodons to Trp.

Zentralbl Chir, 1988, 113(1), 59 - 64
{Regeneration of the liver under the influence of E . coli endotoxin}; Bauknecht KJ et al.; Studies were conducted into regeneration of rat liver exposed to the action of E . coli endotoxin, following two-third resection . No accelerated regeneration was found to take place in the wake of two-third hepatectomy and following postoperative intraperitoneal administration of E . coli endotoxin . However, regeneration was accelerated with significance in response to preoperative administration of 0.1 mg/kg body weight of E . coli endotoxin (B 5:55 DIFCO).

Microbiologica, 1988 Jan, 11(1), 13 - 20
Kinetics of phagocytosis and killing of E . coli by murine macrophages in presence of different serum preparations; Adinolfi LE et al.; In this study we evaluated the kinetics of phagocytosis and killing of E . coli by thioglycollate-elicited murine peritoneal macrophages and the role of specific antibodies and complement present in different serum preparations in modulating these processes . In our system phagocytosis of E . coli by macrophage monolayer was exponential for 180 min . The killing activity was high in the first 30-60 min and then virtually ceased . The least phagocytosis and killing occurred in presence of heat-inactivated fetal calf serum (HFCS) . These activities were 2-fold increased in presence of normal mouse serum (NMS) or heat-inactivated newborn calf serum (HNCS) and were highly stimulated in presence of immune mouse serum (IMS) . IMS without complement was less efficient in enhancing phagocytosis and killing by macrophages . However when IMS or HNCS were deprived of specific antibodies their activity was remarkably reduced . When macrophages containing phagocytized bacteria were reincubated with different sera, multiplication of intracellular E . coli occurred with HFCS, NMS or antibody-deprived IMS or HNCS . In contrast, a significant decrease in the survival of intracellular bacteria was seen in presence of IMS, HNCS or complement-deprived IMS . The results indicated that specific bacterial antibodies play a major role in the phagocytic process and in the activation of killing mechanisms . However optimal macrophage activity resulted from the presence of both specific antibodies and complement.

Mutat Res, 1988 Jan, 207(1), 7 - 11
Enhancement and inhibition of benzo{a}pyrene-induced SOS function in E . coli by synthetic antioxidants; Potenberg J et al.; 8 antioxidants were tested in the SOS chromotest for induction of SOS function and for modulation of benzo{a}pyrene-induced SOS function . None of the antioxidants leads to increased beta-galactosidase activity by itself . Butylated hydroxytoluene at concentrations between 10(-5) M and 3 X 10(-4) M enhances benzo{a}pyrene-induced SOS function at benzo{a}pyrene concentrations between 10(-6) M and 3 X 10(-5) M . Butylated hydroxyanisole, ethoxyquin, propyl gallate and octyl gallate also slightly enhance benzo{a}pyrene-induced SOS function at concentrations up to 3 X 10(-4) M though to a lesser degree than butylated hydroxytoluene . Dodecyl gallate, vitamin C and alpha-tocopherol do not increase benzo{a}pyrene action . In concentrations exceeding 3 X 10(-4) M all synthetic antioxidants tested but not vitamin C and alpha-tocopherol decrease beta-galactosidase activity both in the absence and, more extensively, in the presence of benzo{a}pyrene . Preliminary data suggest that the apparent suppression of benzo{a}pyrene-induced SOS function is not due to an effect on the formation of benzo{a}pyrene metabolites by the metabolizing system used.

Res Exp Med (Berl), 1988, 188(5), 357 - 65
Effects of a calcium antagonist (nifedipine) on cats in live E . coli bacteriemic shock; Bosson S et al.; The effects of a calcium antagonist, nifedipine, on cardiovascular reactions and on gastrointestinal mucosal integrity was studied in a standardized feline bacteriemic model . Nifedipine pretreatment delayed the development of cardiovascular derangement and reduced the severity of the intestinal but not the gastric mucosal injury . The effect on the intestinal mucosa could be due to the delayed development of hypotensive shock but also to a protective effect on the superficial mucosal cells.

Physiol Chem Phys Med NMR, 1988, 20(3), 193 - 7
Thialysine- and selenalysine-resistance in a E . coli mutant; Busiello V et al.; A thialysine-resistant mutant of E . coli strain KL16 also shows a lower sensitivity to selenalysine, the lysine analog containing selenium . No difference between the mutant and the parental strain has been shown regarding the affinities of the transport systems and the lysyl-tRNA synthetase for selenalysine, thialysine and lysine as well as the inhibitory effects of these three aminoacids on the activity of the lysine biosynthetic pathway . A marked difference between the two strains has been evidenced in the AK III repression: in the mutant the repression by selenalysine, thialysine and lysine is much lower than in the parental strain.

Physiol Chem Phys Med NMR, 1988, 20(2), 109 - 13
Utilization of lysine analogs by a lysine-requiring E . coli mutant; Busiello V et al.; Thialysine and selenalysine cannot substitute lysine as a growth factor for a lysine-requiring E . coli mutant, but can nevertheless be utilized for protein synthesis in the presence of lysine . In order to have information about the effects of lysine on the utilization of the two analogs, the extent of the incorporation of the three aminoacids into newly synthesized proteins has been determined . The analog starts to be utilized by cells growing in a medium containing either analog and lysine when lysine concentration becomes very low . Of the two analogs, thialysine is more easily utilized . In fact thialysine can be utilized when the lysine/thialysine ratio in the medium is 1/25 . Selenalysine starts to be utilized when the lysine/selenalysine ratio is 1/200.

C R Acad Sci III, 1988, 306(13), 385 - 90
{Comparative topography of the catalytic sites of chromosomal dihydrofolate reductases and that of the enzyme encoded in R 67 plasmid from E . coli}; Tu YX et al.; The trimethoprim-resistant dihydrofolate reductase specified in E . coli by plasmid R 67, when compared, with other enzymes catalyzing the same reaction, to have a dissimilar primary, secondary, tertiary and quaternary structure . In regard to the tertiary structure, we show here that the pteridine binding site, in the plasmid-encoded enzyme, has a geometrical similarity with that of other chromosomal specified reductases.

Adv Exp Med Biol, 1988, 250, 81 - 9
Structural and mechanistic properties of E . coli adenosylmethionine decarboxylase; Anton DL et al.; Adenosylmethionine decarboxylase catalyzes one of the first committed steps in polyamine biosynthesis . It is a member of a small class of decarboxylases that use a pyruvovyl prosthetic group rather than the more common pyridoxal cofactor . We have recently shown that AdoMet decarboxylase from E . coli is composed of stoichiometric amounts of two types of subunits; alpha (Mr = 19,000), and beta (Mr = 14,000) . The NH2-terminal of the alpha subunit is blocked by the pyruvoyl group and can be sequenced only after reductive amination, which converts this to an alanine residue . The beta subunit, on the other hand, has an unblocked NH2-terminal and sequences normally . The molecular weight of the holoenzyme, estimated by gel filtration, is 136,000 suggesting that the enzyme is an alpha 4 beta 4 octamer . AdoMet decarboxylase undergoes a time dependent inactivation during turnover . The mechanism of this inactivation involves a transamination from the product, decarboxylated AdoMet, and the pyruvoyl group generating an NH2-terminal alanine . The nascent product aldehyde then eliminates methylthioadenosine, resulting in the formation of acrolein, which covalently labels the alpha subunit . How this mechanism may explain AdoMet decarboxylase turned over, and how AdoMet decarboxylase inhibitors can affect its half life will be discussed.

Nucleic Acids Symp Ser, 1988, (19), 193 - 7
Computer-aided detection and alignment of weakly homologous amino acid sequences of RNA replicase beta (MS2 phage) and DNA polymerases (T7 phage and E . coli); Ohnishi K; Alignment of the amino acid (aa) sequences of T7 phage DNA polymerase (DPase), E . coli DNA polymerase I (Pol I) and MS2 phage RNA replicase beta subunit (MS2 Repl) were established by computer-aided methods . The results showed that the entire length (aa's 16-704) of T7 DPase is homologous to Pol I aa's 207-928(C-term) with 21.5% aa identity, and that domains I (aa's-1-311) and II (312-451(C-term} were found to be homologous to each other and to N-terminal region of T7 DPase (aa's 1-250) . Thus these enzymes and domains are homologous to one another and must have evolved from a co-ancestral enzyme.

Nucleic Acids Symp Ser, 1988, (19), 179 - 80
Construction of a plasmid for high-level expression of goat alpha-lactalbumin and its derivative in E . coli; Kumagai I et al.; The high-level expression system of goat alpha-lactalbumin (alpha-LA) in E . coli was established by fusing the alpha-LA cDNA to porcine adenylate kinase cDNA and expressing the fused gene under the control of tac promoter . For high-level expression, elimination of 3'-noncoding region of the alpha-LA cDNA was found to be necessary.

Bull Soc Pathol Exot Filiales, 1988, 81(4), 712 - 20
{Role of E . coli enterotoxins in the etiology of infantile diarrhea . Comparison of an African country Yaoundé (Cameroon) and France (Grenoble)}; Foucaud-Gamen J et al.; The detection of heat-labile enterotoxin by two tests (culture on Y1 cells and GM1-ELISA) has been carried out on strains of E . coli isolated in stools of children with diarrheal disease (220 strains isolated in the Pasteur Institute in Yaounde, Cameroon, and 133 stool specimens selected in Grenoble) . This work was undertaken to determine the frequency of enterotoxigenic E . coli in two different populations . In Yaounde the isolation rate (6%) is not very high in comparison with other developing countries . It should be observed that some strains belong to enteropathogenic serogroups . In Grenoble results confirm the very low frequency of enterotoxigenic E . coli in industrialized countries.

Arch Inst Pasteur Tunis, 1988 Jan-Apr, 65(1-2), 59 - 68
{DNA repair and its relation to cell division in E . coli}; Karoui MH; In E . coli, lesions introduced by agents such as UV radiation, chemical agents, thymine starvation, lead to the induction of a series of bacterial fonctions called the "SOS response" (DNA reparation, mutagenesis, filamentation, prophages induction etc...) . Genetics and biochemical study set up the evidences of a regulated mechanism . The central effector of this mechanism is the Rec A protein . When a cell's DNA is damaged or its DNA replication is inhibited, an inducing signal is generated . The inducing signal reversebly activates a specific protease activity of Rec A which allows it to cleave the Lex A repressor . Thus inducing operons repressed by Lex A . These operons are implicated in DNA reparations and also in cellular division . A coordination between reparation and cellular division is thus established.

Environ Mol Mutagen, 1988, 12(2), 155 - 66
Molecular analysis of formaldehyde-induced mutations in human lymphoblasts and E . coli; Crosby RM et al.; The molecular nature of formaldehyde (HCHO)-induced mutations was studied in both human lymphoblasts and E . coli . Thirty HPRT- human lymphoblast colonies induced by eight repetitive 150 microM HCHO treatments were characterized by Southern blot analysis . Fourteen of these mutants (47%) had visible deletions of some or all of the X-linked HPRT bands, indicating that HCHO can induce large losses of DNA in human lymphoblasts . In E . coli, DNA alterations induced by HCHO were characterized with use of the xanthine guanine phosphoribosyl transferase (gpt) gene as the genetic target . Exposure of E . coli to 4 mM HCHO for 1 hr induced large insertions (41%), large deletions (18%), and point mutations (41%) . Dideoxy DNA sequencing revealed that most of the point mutations were transversions at GC base pairs . In contrast, exposure of E . coli to 40 mM HCHO for 1 hr produced 92% point mutations, 62% of which were transitions at a single AT base pair in the gene . Therefore, HCHO is capable of producing different genetic alterations in E . coli at different concentrations, suggesting fundamental differences in the mutagenic mechanisms operating at the two concentrations used . Naked pSV2gpt plasmid DNA was exposed to 3.3 or 10 mM HCHO and transformed into E . coli . Most of the resulting mutations were frameshifts, again suggesting a different mutagenic mechanism.

Gut, 1988 Jan, 29(1), 41 - 3
Hydrophobic adhesin of E coli in ulcerative colitis; Burke DA et al.; Pathogenic E coli have adhesive properties which are mirrored by an increase in their surface hydrophobicity . E coli isolated from patients with ulcerative colitis possess a mannose resistant adhesin similar to that found in pathogenic E coli . In this study 42 E coli isolates from patients with colitis have been compared with 15 from controls to assess hydrophobicity and cellular adherence . The salting out method and the buccal epithelial cell technique were used respectively . E coli isolated from colitics are significantly more hydrophobic than control E coli (p less than 0.001) . The salting out score correlates negatively with the buccal epithelial cell adhesion index . When E coli are grown at 18 degrees C both properties are temporarily reduced suggesting that they are related to each other . The salting out method clearly differentiates between E coli isolated from colitics and controls, and offers a simple method of detecting adhesive E coli in inflammatory bowel disease.

Arch Virol, 1988, 103(1-2), 61 - 72
Purification of the Sendai virus nonstructural C protein expressed in E . coli, and preparation of antiserum against C protein; Omata-Yamada T et al.; An expression plasmid, ptac-C, was constructed by inserting the cDNA of the coding region of the Sendai virus nonstructural C protein downstream of the tac promoter of E . coli expression plasmid ptac12-Bam . A new protein produced in E . coli after induction was purified to near homogeneity . The purified protein was found to be identical with the C protein predicted from the C gene cDNA in molecular weight, isoelectric point, amino acid composition, and the amino acid sequence at the N-terminal of the protein as well as those of several fragments obtained on V8 protease digestion . Antiserum raised against the purified protein specifically reacted with the C protein in infected cells . Using this antiserum, the localization of the C protein in infected cells was examined by immunofluorescence, which revealed that it appeared in the cytoplasm but not in nuclei.

Arch Microbiol, 1988, 150(5), 499 - 503
Differential roles for menaquinone and demethylmenaquinone in anaerobic electron transport of E . coli and their fnr-independent expression; Unden G; Escherichia coli grown with glucose in the absence of added electron acceptors contained 3-4 times more naphthoquinones (menaquinone plus demethylmenaquinone) than in the presence of O2 . Presence of electron acceptors resulted in a slight additional increase of the naphthoquinone content . A strain defective in the fnr gene, which encodes the transcriptional activator of anaerobic respiration, showed the same response . With fumarate or dimethyl sulfoxide present, 94% of the naphthoquinones consisted of menaquinone, while with nitrate up to 78% was demethylmenaquinone . With trimethylamine N-oxid as the acceptor the proportion was intermediate . From the donor substrates of anaerobic respiration only glycerol had a significant influence on the ratio of the contents of the 2 quinones . It is concluded that FNR, the gene product of the fnr gene, is not required for anaerobic derepression of naphthoquinone biosynthesis . Menaquinone appears to be involved specifically in the respiration with fumarate or dimethyl sulfoxide, and demethylmenaquinone in nitrate respiration . Both naphthoquinones appear to serve in trimethylamine N-oxide respiration.

Arch Microbiol, 1988, 149(4), 344 - 9
Characterization of an iron sensitive Mud1 mutant in E . coli lacking the ribonucleotide reductase subunit B2; Hantke K; The mutant, generated by a Mud1 insertion, formed long non-viable filaments in the presence of iron and air . Under anaerobic conditions normal growth in the presence of iron was observed . The mutation was mapped by P1 transductions at 48 min on the genetic map of Escherichia coli . By Southern blotting the insertion point was determined to be in nrdB, the structural gene for the ribonucleotide reductase subunit B2 . The mutation could be complemented by the cloned nrdB gene . Up to now it was assumed that E . coli possesses only one enzyme for the synthesis of deoxyribonucleotides and only conditional lethal (temperature sensitive) mutants were isolated in nrdB . The insertion of Mud1 in nrdB should lead to a complete loss of the essential B2 subunit . Since the strain was able to grow under anaerobic conditions on minimal medium lacking deoxyribonucleotides and additional pathway for the synthesis of deoxyribonucleotides is postulated.

Carcinogenesis, 1988 Jan, 9(1), 81 - 7
Transfection of murine multi-potent haemopoietic stem cells with an E . coli DNA alkyltransferase gene confers resistance to the toxic effects of alkylating agents; Jelinek J et al.; O6-alkylguanine-DNA-alkyltransferase (ATase)-deficient murine haemopoietic stem cells were transfected, following electroporation, with a G418-selectable expression vector containing the protein coding region of the Escherichia coli ATase gene ada . Clones of cells that were resistant to G418 or the chloroethylating agent mitozolomide (Mz) were selected and most were shown to express very high levels of bacterial gene-encoded ATase . In comparison with control cells that were transfected with the parent vector, the ATase-expressing clones were considerably more resistant to the toxic effects of the methylating agents N-methyl-N-nitrosourea and methylmethanesulphonate or the chloroethylating agents Mz or taurine chloroethylnitrosourea, but unchanged in their susceptibility to the bis-chloroethylating agent nitrogen mustard . Thus alkylation damage in DNA that can be repaired by the E . coli ATase constitutes the principal lethal lesion produced by alkylating agents in murine haemopoietic stem cells and the ATase deficiency in these cells can be complemented by electroporation-mediated gene transfection.

Nucleic Acids Res, 1987 Dec 23, 15(24), 10523 - 30
Peptides at the tRNA binding site of the crystallizable monomeric form of E . coli methionyl-tRNA synthetase; Schulman LH et al.; A protein affinity labeling derivative of E . coli tRNA(fMet) carrying lysine-reactive cross-linking groups has been covalently coupled to monomeric trypsin-modified E . coli methionyl-tRNA synthetase . The cross-linked tRNA-synthetase complex has been isolated by gel filtration, digested with trypsin, and the tRNA-bound peptides separated from the bulk of the free tryptic peptides by anion exchange chromatography . The bound peptides were released from the tRNA by cleavage of the disulfide bond of the cross-linker and purified by reverse-phase high-pressure liquid chromatography, yielding three major peptides . These peptides were found to cochromatograph with three peptides of known sequence previously cross-linked to native methionyl-tRNA synthetase through lysine residues 402, 439 and 465 . These results show that identical lysine residues are in close proximity to tRNA(fMet) bound to native dimeric methionyl-tRNA synthetase and to the crystallizable monomeric form of the enzyme, and indicate that cross-linking to the dimeric protein occurs on the occupied subunit of the 1:1 tRNA-synthetase complex.

FEBS Lett, 1987 Dec 10, 225(1-2), 87 - 92
Evidence for a dipyrromethane cofactor at the catalytic site of E . coli porphobilinogen deaminase; Jordan PM et al.; Porphobilinogen deaminase isolated from Escherichia coli is shown to contain a dipyrromethane cofactor (DPMC) linked covalently to the enzyme . The structure of the cofactor is proposed on the basis of its reaction with Ehrlich's reagent and from its chemical properties . The cofactor is involved in the binding of intermediates during the catalytic reaction but is not incorporated into the product preuroporphyrinogen, E . coli strains containing the cloned porphobilinogen deaminase gene (hemC) when grown on 5-amino{14C}-levulinic acid incorporate 14C radioactivity specifically into the dipyrromethane cofactor of porphobilinogen deaminase.

Mol Gen Genet, 1987 Dec, 210(2), 256 - 61
Expression of the Escherichia coli trpE gene in E . coli K12 bacteria: maximum level, rate and time of initiation of anthranilate synthetase production; Hessing HG et al.; We have investigated the effect of alterations in the structure of the plasmid-borne Escherichia coli tryptophan (trp) coding region and other regions of the same replicon on the level, rate and time of initiation of anthranilate synthetase component I (ASase) synthesis in E . coli K12 . The maximum level of ASase produced corresponds to 60%-65% of the total cellular proteins . Adding sequences downstream of the trpE coding region decreases the level but does not affect the time of initiation and rate of trpE expression (ASase synthesis) . The presence of additional protein coding sequences on the plasmid outside the trpE-A region causes ASase production to start earlier and decreases the rate of ASase synthesis . A second copy of the trpE coding sequences, if present within or outside the trpE-A coding region on the same replicon, doubles the rate of synthesis of ASase and slightly increases its final level of production . The initiation of ASase production occurs earlier when the two trpE copies are located within two distinct transcription units.

Int J Radiat Biol Relat Stud Phys Chem Med, 1987 Dec, 52(6), 921 - 33
Radiation sensitization of E . coli B/r by mixtures of oxygen and nitrous oxide; Ewing D; Oxygen (O2) sensitizes bacterial cells in at least two mechanistically different ways, depending on the specific O2 concentration present during irradiation . Based on previous work from this laboratory, it has been proposed that nitrous oxide (N2O) and low concentrations of O2 share a common mechanism for damage . This mechanism, involving the production of superoxide anion radicals (O2-), is different from that which causes damage at high O2 concentrations . Others, however, have presented evidence that N2O and O2 (usually tested only at high concentrations) act in different ways to sensitize bacterial cells . We have now measured the radiation sensitivity in mixtures of N2O and O2 to observe additivity patterns and to determine if these two agents have any common processes for sensitization . We found that some low O2 concentrations do not increase the response in N2O, although they can have significant sensitizing effects in N2 . This lack of additivity is taken as evidence for a common mechanism of damage from N2O and low concentrations of O2 . In contrast, damage from high concentrations of O2 is additive to the damage from N2O . The greatest sensitivity, observed with a gas mixture of about 15 per cent O2/85 per cent N2O, is equivalent to the response in 100 per cent N2 plus the maximum amount of damage O2 can cause plus the maximum amount of damage N2O can cause . This additivity is taken as evidence that N2O and high concentrations of O2 sensitize in different ways . Thus, O2 is known to sensitize these bacteria in at least two different ways; one of these is apparently also the way N2O sensitizes.

Biochem Pharmacol, 1987 Dec 1, 36(23), 4125 - 8
Inhibitor properties of some 5-substituted uracil acyclonucleosides, and 2,2'-anhydrouridines versus uridine phosphorylase from E . coli and mammalian sources; Drabikowska AK et al.; Two series of 5-substituted uracil N(1)-acyclonucleosides, each with a different acyclic chain, were examined as inhibitors of uridine phosphorylase from rat intestinal mucosa, and several against the enzyme from Ehrlich ascites cells . In addition, several 5-substituted analogues of 2,2'-anhydrouridine were tested for their inhibitory effects vs a highly purified uridine phosphorylase from Escherichia coli . The results are compared with previously published data for inhibition of the E . coli enzyme by the acyclonucleosides, and of the rat enzyme by the anhydrouridines . In all instances, the inhibitors were active only vs the uridine, but not thymidine, phosphorylase from E . coli, and inhibition was competitive with respect to uridine as substrate . In general, with one or two exceptions, inhibitory effects were more pronounced against the enzyme from mammalian sources . Amongst the acyclonucleoside analogues, the most effective inhibitor of the enzyme from the rat and Ehrlich ascites cells exhibited a Ki = 0.1 microM, comparable to that reported with the Sarcoma-180 enzyme, whereas the Ki for inhibition of the E . coli enzyme was 0.7 microM . By contrast, another effective inhibitor of the bacterial enzyme was 7-fold less potent against the mammalian enzyme . The 2,2'-anhydrouridines were 10- to 30-fold more effective against the rat, as compared to the E . coli, enzyme . The overall quantitative data provide a reasonably good basis for the further design of potent inhibitors for possible use in chemotherapy.

Mutat Res, 1987 Dec, 181(2), 235 - 42
Carcinogenic potency in rodents versus genotoxic potency in E . coli: a correlation analysis for bifunctional alkylating agents; Quinto I et al.; The mutagenic (M), recombinagenic (R) and SOS inducing (I) potencies of 6 bifunctional directly acting alkylating agents (mitomycin C, thiotepa, chlorambucil, nitrogen mustard, bis(2-chloroethyl)ether and bis(2-chloroethyl)nitrosourea) were measured in an E . coli test system (E . coli multitest) as the integral under the yield-dose curve obtained for each event . This potency corresponds to the cumulative yield of the affected cell population over the entire effective dose range of the chemical treatment . A weak mutagenic activity was detected only for mitomycin C and thiotepa . Except for bis(2-chloroethyl)ether, all agents were recombinagenic and SOS inducing . When the 3 genotoxic potencies (M, R and I) of these bifunctional alkylating agents were correlated, separately or in combination, with the respective carcinogenic potencies in rodents, a highly significant correlation was obtained with both the recombinagenic and SOS inducing potencies.

Mutat Res, 1987 Dec, 181(2), 219 - 26
Current understanding of UV-induced base pair substitution mutation in E . coli with particular reference to the DNA polymerase III complex; Bridges BA et al.; UV mutagenesis in E . coli is believed to occur in two discrete steps . The second step involves continued DNA synthesis beyond a blocking lesion in the template strand . This bypass step requires induced levels of umuD and umuC gene products and activated recA protein . DNA polymerase III may be involved since a dnaE mutator strain (believed to have defective base selection) is associated with enhanced UV mutagenesis in conjunction with a genetic background permitting the bypass step . In non-UV-mutable umu and lexA strains, UV mutagenesis can be demonstrated if delayed photoreversal is given . This is interpreted as indicating that an earlier misincorporation step can occur in such strains but the resulting mutations do not survive because the bypass step is blocked . The misincorporation step does not require any induced SOS gene products and can occur either at the replication fork or during repair replication following excision of a DNA lesion . Neither a dnaE mutator gene (leading to a defective alpha subunit of DNA polymerase III holoenzyme) nor a mutD5 mutator gene (leading to a defective epsilon proofreading subunit) had any effect on the misincorporation step . Although this is consistent with DNA polymerase III holoenzyme not being involved in the misincorporation step, other interpretations involving the inhibition of epsilon proofreading activity by recA protein are possible . In vitro studies are reported in which sites of termination of synthesis by DNA polymerase III holoenzyme on UV-irradiated M13 mp8 DNA were examined in the presence of inhibitors of the 3'-5' proofreading exonuclease (including recA protein) . No evidence was found for incorporation of bases opposite photoproducts suggesting that either inhibition is more complete in the cell and/or that other factors are involved in the misincorporation step.

Mol Gen Mikrobiol Virusol, 1987 Dec, (12), 34 - 40
{Weakening of type I restriction in E . coli: the effect of 2-aminopurine and 5-bromouracil}; Belogurov AA et al.; 2-aminopurine (2-AP) and 5-bromouracil, strong mutagens of base analog type, were found to induce efficiently the alleviation of I type restriction in Escherichia coli . 2-AP induced restriction alleviation occurs in recA, lexA and mut- mutants, but no additional relief of restriction is registered in dam-bacteria in the presence of sublethal 2-AP concentrations . 2-AP specifically alleviates I type restriction in Escherichia coli (EcoA, EcoB, EcoD, and EcoK) and does not affect restriction systems of II (EcoRI) and III (EcoP1) types . We suggest that 2-AP-induced mismatches and other replication errors may be signals inducing restriction alleviation in Escherichia coli.

Sci Sin {B}, 1987 Dec, 30(12), 1298 - 304
Affinity labelling of E . coli leucyl-tRNA synthetase with 3'-oxidized tRNA(Leu); Huang ST et al.; The E . coli leucyl-tRNA synthetase (E.C . 6.1.1.4) was specifically labelled with 3'-oxidized tRNA(Leu) (tRNA(oxLeu)) . The procedure involves a Schiff's base formation and its subsequent reduction by sodium cyanoborohydride . Stoichiometric inactivation of aminoacylation was achieved with the incorporation of 1 mol of tRNA(oxLeu) per mol LeuRS . On the other hand, the amino acid activation activity of LeuRS-tRNA(ox) complex was partially inhibited . After extensive digestion of the complex by pancreatic ribonuclease, the amino acid activation activity was fully recovered, while the aminoacylation activity was not restored at all.

Nucleic Acids Res, 1987 Nov 25, 15(22), 9177 - 93
Characterisation and nucleotide sequence of ogt, the O6-alkylguanine-DNA-alkyltransferase gene of E . coli; Potter PM et al.; The plasmid pO61 that was isolated from an E . coli genomic DNA library and codes for O6-alkylguanine (O6AG) DNA alkyltransferase (ATase) activity (1) has been further characterised . Subclones of the 9 Kb insert of pO61 showed that the ATase activity was encoded in a 2Kb Pst1 fragment but a partial restriction endonuclease map of this was different to that of the E . coli ada gene that codes for O6-AG and alkylphosphotriester dual ATase protein . Fluorographic analyses confirmed that the molecular weight of the pO61-encoded ATase was 19KDa i.e . similar to that of the O6AG ATase function that is cleaved from the 39KDa ada protein but rabbit polyclonal antibodies to the latter reacted only very weakly with the pO61-encoded protein . A different set of hybridisation signals was produced when E . coli DNA, which had been digested with a variety of restriction endonucleases was probed with 2Kb Pst 1 fragment or the ada gene . These results provided evidence for the existence of a second ATase gene in E . coli . The 2Kb Pst-1 fragment of pO61 was therefore sequenced and an open reading frame (ORF) that would give rise to a 19KDa protein was identified . The derived amino acid sequence of this showed a 93 residue region with 49% homology with the O6AG ATase region of the ada protein and had a pentamer and a heptamer of identical sequence separated by 34 amino acids in both proteins . The pentamer included the alkyl accepting cysteine residue of the ada O6AG ATase . The hydrophobic domains were similarly distributed in both proteins . Shine-Dalgarno, -10 and -35 sequences were identified and the origin of transcription was located by primer extension and S1 nuclease mapping . The amino-terminal amino acid sequence of the protein was as predicted from the ORF.

Biochem Biophys Res Commun, 1987 Nov 13, 148(3), 1490 - 5
Effect of GDP on the interactions between chloroplast EF-Ts and chloroplast and E . coli EF-Tu; Spremulli GH et al.; The effects of varying concentrations of GDP on the stability of homologous and heterologous EF-Tu:EF-Ts complexes formed with the elongation factors from the chloroplast of Euglena gracilis and from E . coli have been investigated . The complexes formed with chloroplast EF-Ts were significantly more stable to GDP-induced dissociation than those formed with E . coli EF-Ts . The complex between chloroplast EF-Tu and chloroplast EF-Ts required nearly 1,000-fold higher concentrations of GDP for dissociation than the complex between chloroplast EF-Tu and E . coli EF-Ts . The E . coli EF-Tu:chloroplast EF-Ts complex required nearly 100-fold higher levels of GDP for dissociation than the E . coli EF-Tu:E . coli EF-Ts complex.

Sci Sin {B}, 1987 Nov, 30(11), 1190 - 8
Structure in the precore region of hepatitis B core gene affecting its expression in E . coli; Ma XK et al.; Restriction fragments of HBV-DNA, cleaved by endonuclease HhaI, containing HBcAg gene were trimmed by BAL-31 exonuclease to remove different lengths of the precore sequence . They were inserted into plasmid pUR222 at EcoRI site through synthetic linker ligation . Transformants in E . coli BMH7118 showing different levels of HBcAg gene expression were screened and analyzed for their nucleotide sequences in the junction region both by Maxam and Gilbert's chemical degradation method and by M13 chain termination method . Results of sequence analysis of different transformants revealed a partial palindromic (loop and stem) structure, at -7 to -35 nucleotide with regard to ATG of the HBcAg gene as position +1, which has dramatic effect on the level of expression of the inserted gene using the same promoter, SD sequence and identical N-terminus . The amount of HBcAg synthesized differed from 9% in the high expressing plasmid to less than 0.01% of the total cell proteins in the low expressing transformants . The findings were compared to results obtained by other workers in studies of HBcAg expression in procaryotes and their significance in the expression of eucaryotic genes in procaryotic cells were discussed.

Nucleic Acids Res, 1987 Oct 26, 15(20), 8235 - 48
Deletions in the tL structure upstream to the rRNA genes in the E . coli rrnB operon cause transcription polarity; Zacharias M et al.; A number of deletions have been constructed within the leader region of the rrnB operon from E . coli . The deletions remove a potential transcription terminator structure downstream from an antitermination recognition sequence (Box A), which precedes the structural gene for the 16S RNA . Cells harbouring plasmids, where the terminator structure was deleted, partially or totally, showed a reduction in growth rate under minimal growth conditions . Measurement of the ribosomal RNA synthesis rates of such cells determined by pulselabeling and hybridisation to appropriate DNA probes, showed that the amount of the more distally located 23S RNA was reduced compared to the promoter-proximal 16S RNA . This polarity in transcription, resulting in a non-stoichiometric synthesis of the ribosomal RNAs, is most likely the result of a defective antitermination . The reduction in the amount of 23S RNA in such cells is compensated for by an increase in the overall ribosomal RNA synthesis, in concordance with the ribosomal RNA feedback regulation model . The accumulation of transcripts of the tRNAGlu2 gene, coded in the spacer region between the 16S and 23S RNA genes, in cells with an altered rRNA stoichiometry supports this interpretation.

Biochim Biophys Acta, 1987 Oct 9, 910(1), 11 - 20
E . coli minichromosome replication in vitro and in vivo: comparative analyses of replication intermediates; Munson BR et al.; The process of replication of Escherichia coli minichromosomes was examined by following the intermediates formed in vitro and in vivo . Replication initiated on a supercoiled closed circular (CC) monomer, proceeded rapidly to a late but incomplete stage in polymerization (the LC form) in both systems, passed more slowly through a series of open and closed circular catenated dimers with varying extents of intertwining between the monomer units, and then yielded, after decatenation, the supercoiled CC monomer . The replication patterns of two different minichromosomes were similar, although the LC form and the multiply intertwined dimers were much more evident in the smaller pAL4 than in pAL2 . The same basic replication scheme was seen in vitro and in vivo but completion of polymerization and processing of the dimers were slower in vitro . Some radioactivity was detected in OC monomer early during replication, consistent with occasional decatenation of LC structures to produce OC molecules which then completed replication to form CC molecules . However, progression to CC catenated dimers prior to formation of CC monomers represented the major replication pathway.

Protein Eng, 1987 Oct-Nov, 1(5), 425 - 31
Cloning and expression in E . coli of a synthetic gene for the bacteriocidal protein caltrin/seminalplasmin; Heaphy S et al.; A synthetic gene coding for the bacteriocidal protein caltrin/seminalplasmin was constructed and expressed in Escherichia coli as a fusion with beta-galactosidase . The gene was designed with a recognition site for the plasma protease, Factor Xa, coded for immediately prior to the N-terminus of caltrin . The beta-galactosidase-caltrin fusion protein was cleaved with Factor Xa to give caltrin, which was identified by its size on SDS-PAGE, its ability to react with an antiserum raised to the N-terminal nonapeptide of caltrin and its N-terminal amino acid sequence . After partial purification, synthetic caltrin was found to be active in an assay involving inhibition of growth of E.coli.

J Biochem (Tokyo), 1987 Oct, 102(4), 715 - 24
Expression of murine interleukin-2 cDNA in E . coli and biological activities of recombinant murine interleukin-2; Kashima N et al.; Murine interleukin-2 (MIL-2) cDNA was inserted into an expression vector carrying an Escherichia coli tryptophan promoter and was expressed in E . coli . Recombinant MIL-2 produced by E . coli supported the growth of murine CTLL-2 cells, but not that of human T-cell blasts . Recombinant MIL-2 strongly inhibited the binding of recombinant human IL-2 (HIL-2) to murine responder cells, but only very weakly inhibited the binding to human responder cells . Moreover, recombinant MIL-2 induced secondary alloantigen specific cytotoxic T lymphocytes (2 degrees CTL) from memory CTL and activated natural killer (NK) cells in murine systems in the same manner as recombinant HIL-2 . The results suggest that the species hierarchy (that MIL-2 derived from native cell culture does not act on human T-cells) is due to the protein moiety, not the sugar moiety, and is to be ascribed to the difference in binding affinity of MIL-2 and HIL-2 to murine and human responder cells respectively, and that recombinant MIL-2 shares identical biological and immunological activities with recombinant HIL-2 . Thus, MIL-2 might be a convenient tool for extensive studies of the pharmacological and physiological activities of IL-2 in murine models.

Biochimie, 1987 Oct, 69(10), 1065 - 70
Open reading frames in the control regions of the phenylalanyl-tRNA synthetase operon of E . coli; Springer M et al.; The pheST operon codes for the two subunits of phenylalanyl-tRNA synthetase and it expression is controlled by attenuation in a way similar to many amino acid biosynthetic operons . The nucleotide sequence of the control regions of the operon indicates the presence of several open reading frames besides that of the leader peptide . One of these open reading frames, called the alternative leader peptide, starts at about the same place as the leader peptide and ends after the terminator of the attenuator . Another open reading frame, called the terminator peptide, starts after the terminator and covers about half the distance to pheS, the first structural gene of the operon . The present report shows that, in fact, the only open reading frame to be translated efficiently is the leader peptide itself . The alternative leader peptide and the terminator peptide are both translated at a negligible rate.

Biochimie, 1987 Oct, 69(10), 1031 - 41
The function, structure and regulation of E . coli peptide chain release factors; Craigen WJ et al.; The termination of protein synthesis in Escherichia coli depends upon the soluble protein factors RF1 or RF2 . RF1 catalyzes UAG and UAA dependent termination, while RF2 catalyzes UGA and UAA dependent termination . The proteins have been purified to homogeneity, their respective genes isolated, and their primary structures deduced from the DNA sequences . The sequences reveal considerable conserved homology, presumably reflecting functional similarities and a common ancestral origin . The RFs are encoded as single copy genes on the bacterial chromosome . RF2 exhibits autogenous regulation in an in vitro translation system . The mechanism of autoregulation appears to be an in-frame UGA stop codon that requires a 1+ frameshift for the continued synthesis of the protein . Frameshifting prior to the inframe stop codon occurs at a remarkably high frequency by an unknown mechanism . Future studies will be directed at understanding how RFs interact with the ribosomal components, and further defining the mechanism of RF2 frameshifting.

Bioorg Khim, 1987 Oct, 13(10), 1425 - 7
{Increased selectivity of directed RNA hydrolysis mediated by RNAse H from E . coli using mixed oligo(deoxyribo-ribo)nucleotides}; Metelev VG et al.; Chimeric oligo(deoxyribo-ribo)nucleotides appeared to be a valuable tool to achieve the high selectivity of RNA cleavage as shown by RNA-ase H-mediated hydrolysis of TMV RNA directed by d(TGTGTATGCC), d(TGTGTAT), d(TGTGTAT)GCCAU and d(TGTGTAT)ppGCCAU.

Biochem Biophys Res Commun, 1987 Sep 30, 147(3), 1129 - 36
The generation of the rifamycin binding site in the beta subunit of E . coli RNA polymerase through subunit interactions; Lowder JF et al.; Spectral studies were performed using rifampicin quinone to investigate the generation of the rifamycin binding site in the beta subunit of E . coli RNA polymerase due to subunit interactions . The spectrum of rifampicin quinone is not significantly altered in the presence of the beta subunit . In the case of the alpha 2 beta subassembly, a negative difference spectral band at 330 nm is observed for rifampicin quinone, whereas in the presence of either the holoenzyme or core polymerase a positive band at 348 and a negative one at 318 are observed . In affinity labeling studies using 3-(2-bromo{1-14C}acetamidoethyl)-thiorifamycin, it was demonstrated that the isolated beta subunit is nonspecifically modified by this reagent . However, in the case of both the alpha 2 beta subassembly and core polymerase, the beta subunit is specifically modified.

Cell, 1987 Sep 25, 50(7), 1071 - 9
Hemimethylation prevents DNA replication in E . coli; Russell DW et al.; The DNA adenine methylase of E . coli methylates adenines at GATC sequences . Strains deficient in this methylase are transformed poorly by methylated plasmids that depend on either the pBR322 or the chromosomal origins for replication . We show here that hemimethylated plasmids also transform dam- bacteria poorly but that unmethylated plasmids transform them at high frequencies . Hemimethylated daughter molecules accumulate after the transformation of dam- strains by fully methylated plasmids, suggesting that hemimethylation prevents DNA replication . We also show that plasmids purified from dam+ bacteria are hemimethylated at certain sites . These results can explain why newly formed daughter molecules are not substrates for an immediate reinitiation of DNA replication in wild-type E . coli.

Cell, 1987 Sep 25, 50(7), 1039 - 46
Initiation of transcription at the bacterial glnAp2 promoter by purified E . coli components is facilitated by enhancers; Ninfa AJ et al.; The initiation of transcription from the nitrogen-regulated promoter glnAp2 requires RNA polymerase containing sigma 54, the transcriptional activator NRI, and the protein kinase NRII, responsible for the conversion of NRI to the active NRI-phosphate . NRI-phosphate does not increase the ability of sigma 54-containing RNA polymerase to bind to the promoter, but rather stimulates the conversion of an initial promoter:polymerase complex to the transcriptionally active open complex . The presence on the DNA template of high-affinity binding sites for NRI/NRI-phosphate, normally located 130 and 100 bp upstream of the site of transcription initiation, results in a 4- to 5-fold lowering of the concentration of NRI required for the formation of the open complex . These high-affinity NRI binding sites facilitate open complex formation when they are moved to positions 700 bp further upstream or 950 bp downstream of glnAp2 on linear DNA templates.

Nature, 1987 Sep 24-30, 329(6137), 348 - 51
The heat shock response of E . coli is regulated by changes in the concentration of sigma 32; Straus DB et al.; Cells subjected to a heat shock, or a variety of other stresses increase the synthesis of a set of proteins, known as heat shock proteins . This response is apparently universal, occurring in the entire range from bacterial to mammalian cells . In Escherichia coli heat shock protein synthesis transiently increases following a shift from 30 degrees C to 42 degrees C as a result of changes in transcription initiation at heat shock promoters . Heat shock promoters are recognized by RNA polymerase containing a sigma factor of relative molecular mass (Mr) 32,000 (32K) E sigma 32 and not E sigma 70, the major form of RNA polymerase holoenzyme . To determine whether changes in the concentration of sigma 32 regulate this response, we measured the amount of sigma 32 before and after shift to high temperature and found that it increased transiently during heat shock as a result of changes in sigma 32 synthesis and stability . Our results indicate that sigma 32 is directly responsible for regulation of the heat shock response.

FEBS Lett, 1987 Sep 14, 221(2), 226 - 30
Subcellular localization of a PhoE-LacZ fusion protein in E . coli by protease accessibility experiments reveals an inner-membrane-spanning form of the protein; Tommassen J et al.; Protease accessibility experiments were employed to localize a PhoE-LacZ hybrid protein, encompassing a large N-terminal fragment of the outer membrane PhoE protein of E . coli, fused to beta-galactosidase, at the subcellular level . In previous studies, this protein was shown to co-fractionate with the outer membrane, whereas immunocytochemical methods suggested a cytoplasmic location . The present results confirm the latter localization . Moreover, it appears that a minor amount of hybrid protein spans the inner membrane, with the PhoE moiety in the periplasm and the beta-galactosidase moiety in the cytoplasm . These membrane-spanning proteins might be responsible for the lethal jamming of the export machinery, observed upon induction of synthesis of the protein.

Nucleic Acids Res, 1987 Sep 11, 15(17), 6843 - 54
The effects of covalent additions of a psoralen on transcription by E . coli RNA polymerase; Shi YB et al.; Synthetic DNA substrates containing a site-specifically engineered psoralen monoadduct or diadduct were used to characterize the response of the E . coli RNA polymerase elongation complex to these lesions . The psoralen derivative HMT (4'-hydroxymethyl-4,5', 8-trimethylpsoralen) was site specifically placed into two synthetic double-stranded DNA fragments each of which contained an E . coli RNA polymerase promoter at one end . The HMT molecule was attached to the middle of the DNA fragments as either a furan-side monoadduct or an interstrand diadduct . Transcription off the HMT crosslinked DNA templates showed that E . coli RNA polymerase terminated at the HMT diadduct site, i . e., one nucleotide before the modified thymidine residue on the template strand . The furan-side monoadduct when on the template strand also blocked transcription by the polymerase . However, no effect on transcription was observed when the monoadduct was located on the non-template strand.

Eur J Pharmacol, 1987 Sep 11, 141(2), 283 - 90
Antagonism by E . coli endotoxin of some cardiovascular effects induced in the rat by two alpha 2-adrenoceptor agonists; Auclair MC et al.; E . coli endotoxin (0.01, 0.1 and 1 microgram/kg i.v.) 1 h before alpha 2-adrenoceptor agonists B-HT 933 and clonidine (i.v.) antagonized their bradycardiac and hypotensive effects in intact rats . This antagonism seems not to depend on the presence of the adrenal glands since similar results were obtained in adrenalectomized rats . Endotoxin at higher doses (1 and 10 micrograms/kg) suppressed the hypotensive and reduced the bradycardiac effect of clonidine injected i.c.v . (5 micrograms/kg) . In contrast, endotoxin (up to 100 micrograms/kg) did neither increase arterial pressure nor heart rate in the pithed rat . This suggests the participation of a central site of action for endotoxin . However, E . coli endotoxin (1, 10 or 100 micrograms/kg) did not decrease the inhibition by clonidine of the tachycardia induced by stimulation of the cardioacceleratory nerve . This excludes peripheral presynaptic alpha 2-adrenoceptor blockade by endotoxin . Only 100 micrograms/kg decreased the pressor response to clonidine in the pithed rat . These results show that E . coli endotoxin is a potent modificator of the cardiovascular regulation in the rat . It antagonized central alpha 2-adrenoceptor mediated cardiovascular effects at doses lower than those acting on postsynaptic peripheral alpha-adrenoceptors.

Mutat Res, 1987 Sep, 184(2), 121 - 8
Expression of the truncated E . coli O6-methylguanine methyltransferase gene in repair-deficient human cells and restoration of cellular resistance to alkylating agents; Ishizaki K et al.; We have constructed a truncated E . coli O6-methylguanine methyltransferase (MT) gene (ada gene) to express the MT activity for O6-methylguanine and O4-methylthymine but not for methylphosphotriester in human cells and transferred it into Mer- HeLa MR cells . The transfectant cells expressed the truncated E . coli MT were resistant to alkylating agents as same as the transfectant cells with the intact ada gene in cell killing, sister-chromatid exchange induction and host-cell reactivation of adenovirus 5 . These results strongly suggest that methylphosphotriester may not contribute to the biological effect of alkylating agents in human cells.

Mutagenesis, 1987 Sep, 2(5), 375 - 81
The toxicity and mutagenicity of the anti-tumour drug 5-aziridino-2,4-dinitrobenzamide (CB1954) is greatly reduced in a nitroreductase-deficient strain of E . coli; Venitt S et al.; In the 1970s it was shown that the monofunctional alkylating agent CB1954 (5-aziridino-2,4-dinitrobenzamide) kills the Walker rat carcinoma 256 in vivo and in vitro, but is inactive against several other tumours . In studies with bacteria, the large differences in survival in DNA-repair-proficient and deficient strains of Escherichia coli B treated with CB1954 were characteristic of a difunctional cross-linking agent . It was concluded that DNA was the only target large enough to receive significantly more than one lethal hit per molecule and that mono-alkylation alone could not account for the lethal effects of CB1954 . In 1986 it was shown that CB1954 induced DNA interstrand cross-links in CB1954-sensitive cultured Walker 256 cells, but not in resistant Chinese hamster V79 cells, suggesting that the sensitivity of Walker cells results mainly from their activation of the drug to a difunctional agent by nitroreduction . To test this, we assayed the toxicity and mutagenicity of CB1954 in nitroreductase-plus and -minus strains of E . coli WP2uvrA . Agar-overlay assays showed that CB1954 was mutagenic to several strains of E . coli WP2, in a dose range 1-100 micrograms per plate, with slopes (mutants/nmol) of 7.1, 1.05 and 0.16 for WP2uvrA pKM101, WP2uvrA and WP2 . Assays with nitrofurazone showed that these strains possessed nitroreductase activity . However, E . coli NFR-343, a nitrofurazone-resistant mutant of WP2uvrA which lacks nitroreductase activity was markedly less sensitive to the mutagenicity of CB1954, giving a mean slope of 0.12 compared with 1.15 for WP2uvrA . Aroclor-induced rat-liver S9 did not change these responses.(ABSTRACT TRUNCATED AT 250 WORDS)

Bioorg Khim, 1987 Sep, 13(9), 1176 - 85
{Formation and properties of artificial polycistrons containing truncated genes for E . coli tryptophan operon and phage M13 envelope protein}; Kravchenko VV et al.; Using gene fragments encoding the leader peptide of E . coli tryptophane operon (as duplicated fragment HhaI-140) or M13 phage coat protein (as TaqI-381 or HaeIII-1623 fragments) and basing on pDS1 family of plasmids, expression vectors have been constructed which contained transcription promoters Ptrp, PVIII, and Pv + PVIII, respectively . An artificial gene for human leukocyte interferon alpha 2 (ifn-alpha 2) has been cloned into these plasmids, so that its transcription was a part of polycistronic mRNA and preceding translation was terminated upstream to the ribosome binding site and starting codon of the interferon gene . E . coli cells harbouring these recombinant plasmids provided high level of the interferon biosynthesis . The effect of the mRNA length on the amount of protein synthesised under control of the M13 coat protein transcription-translation signals has been found.

Bioorg Khim, 1987 Sep, 13(9), 1164 - 9
{Effect of diadenosine oligophosphates (Ap4A and Ap3A) and their phosphonate analogs on catalytic properties of phenylalanyl-tRNA synthetase from E . coli}; Biriukov AP et al.; The influence of P1,P3-bis(5'-adenosyl)triphosphate (Ap3A), P1,P4-bis(5'-adenosyl)tetraphosphate (Ap4A) and its analogues, containing a residue of methylenediphosphonic acid in various positions of the oligophosphate chain, on the reactions catalysed by phenylalanyl-tRNA synthetase from E . coli MRE-600 has been studied . The compounds do not affect significantly the rate of ATP-{32P}PPi-exchange nor maintain this reaction in the absence of ATP . The diadenosineoligophosphates are shown to be noncompetitive inhibitors of ATP in the tRNA aminoacylation by phenylalanine (for Ap4A Ki = 1,45.10(-3) M) . The phosphonate analogues of Ap4A inhibit the synthesis of Ap3A depending on their structure . The conclusion is thus drawn that the E . coli MRE-600 phenylalanyl-tRNA synthetase does not interact property with Ap4A and its phosphonate analogues.

Dig Dis Sci, 1987 Sep, 32(9), 1017 - 26
Binding of E . coli heat-stable enterotoxin to rat intestinal brush borders and to basolateral membranes; Guarino A et al.; We studied the binding of E . coli heat-stable enterotoxin (STa) to rat brush borders (BB) and to basolateral membranes (BLM) using a biologically active monoiodinated radioligand {( 125I}STa) and highly enriched BB and BLM preparations free of other significant organelle contamination . Binding of {125I}STa to BB was specific; time-, temperature-, and pH-dependent; saturable; and partially reversible . Nonlabeled toxin competitively inhibited the binding of radioligand to BB in a dose-related manner . Scatchard analysis revealed a single class of receptors with an apparent affinity constant of 8.7 +/- 1.5 X 10(8) l/mol . Binding was not affected by amino acids, sugars, and lectins . Proteolytic enzymes significantly decreased binding, although several did so by modifying the radioligand . Trypsin inhibited binding without modifying the radioligand thus supporting the proteinaceous nature of the receptor . Since the enrichment in binding activity in the BB over the homogenate was significantly lower than the enrichment in sucrase activity, we concluded that binding activity is probably associated with other membranous domains, but direct examination revealed no binding activity on basolateral membranes.

Appl Biochem Biotechnol, 1987 Sep-Dec, 16, 1 - 13
A crosslinked preparation of E . coli beta-D-galactosidase; Khare SK et al.; beta-D-Galactosidase from E . Coli was crosslinked using glutaraldehyde and two bisimidoesters . With glutaraldehyde and dimethyl adipimidate (DMA), it is possible to obtain preparations having higher activity than the native enzyme . Glutaraldehyde and DMA gave preparations showing enhanced thermal stability . The preparation crosslinked with DMA, when used for continuous hydrolysis of lactose in milk, was found to be significantly better than the native enzyme.

Biochimie, 1987 Sep, 69(9), 965 - 74
Comparison of active and inactive forms of the E . coli 30S ribosomal subunits; Guerin MF et al.; Dissociation of E . Coli 70S ribosomes in the presence of 0.1 mM Mg++ yields partially inactivated 30S and 50S subunits . This inactivation can be avoided by dissociating the 70S ribosome in a medium containing 10 mM Mg++ . 400 mM Na+ . Comparison of the active and inactive forms of the 30S and 50S subunits has led to the following conclusions: 1) The two forms possess identical (50S subunits) or very similar (30S subunits) hydrodynamic properties . No differences in their morphologies is detectable by electron microscopy . 2) They possess the same protein compositions except for the presence of a larger amount of protein S1 in the inactive than in the active form of the 30S subunit . 3) They differ significantly in functional properties: more efficient association of the active than of the inactive forms with the complementary subunit; extensive dimerization of inactive 30S subunits in the presence of 10 mM Mg++; no dimerization of active 30S subunits under the same conditions; six-fold higher peptidyl transferase activity of active as compared to inactive 50S subunits.

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