Escherichia coli

Brain Res Bull, 1993, 32(2), 123 - 32
Aromatic amino acid decarboxylase (AADC) immunohistochemistry in vertebrate brainstem with an antiserum raised against AADC made in E . coli; Beltramo M et al.; Aromatic L-amino acid decarboxylase (AADC) is involved in the biosynthesis of catecholamines and indolamines . AADC is present in the nervous system, in the chromaffin cells, and in non-neuronal tissues . We tested the capacity of a new polyclonal antibody, obtained by immunization of rabbits with a recombinant protein beta-galactosidase-AADC, to detect monoaminergic neurons in the brainstem as well as monoaminergic paraneurons in the adrenal medulla from goldfish, frog, skink, quail, and mouse . In the adrenal gland we found an immunoreactivity that was consistent with the distributions of the chromaffin cells previously reported . In the brainstem, groups of immunoreactive neurons and several labelled fibers were observed in the five species studied . The raphe region showed cell bodies and processes similar to those previously identified as monoaminergic by other authors . In addition, in medulla oblongata and isthmic tegmentum we found, in goldfish, skink, and quail, neuronal groups similar to mammalian D groups which contain AADC but are devoided of serotonin and catecholamines.

Biochimie, 1993, 75(5), 415 - 22
ColiGene: object-centered representation for the study of E coli gene expressivity by sequence analysis; Perriere G et al.; ColiGene is an object-centered knowledge base for the study of gene expressivity in Escherichia coli by DNA sequence analysis . This system was developed with the knowledge base management system SHIRKA . Objects represented in ColiGene are biological structures such as genes or regulatory signals . They are organized in a hierarchical structure of classes, subclasses and instances . Navigation through the knowledge base and the building of queries are made using a graphical interface . The base is coupled with the data base ACNUC which structures a specialized collection of sequences: EcoSeq . Several tools are also associated to ColiGene, either for sequence analysis or for a more general purpose . Some biological results have been obtained using ColiGene which are summarized here.

Yi Chuan Xue Bao, 1993, 20(4), 374 - 80
{Cloning of beta-amylase gene from B . substitute and its expression in E . coli}; Wang W et al.; The gene coding for beta-amylase with raw starch-digestion ability was isolated from B . substitute R2 which was selected in this lab . The procedure used in this screening was developed in this work by the method named nutrition-restriction . The DNA fragment containing the beta-amylase gene was 5.25 kb in length . There was no difference in enzymological properties between the beta-amylase produced by the gene-donor strain and the expression product of E . coli . The yield of the cloned beta-amylase was over 500 IU/ml in our laboratory culture condition, the RDA value was 57%, and all of this enzyme were found to be secreted into medium.

Arch Virol, 1993, 133(1-2), 223 - 31
Nucleotide sequence and E . coli expression of the coat protein gene of the yellowing strain of soybean dwarf luteovirus; Smith OP et al.; The nucleotide sequence of the coat protein gene of the yellowing (Y) strain of soybean dwarf virus (SbDV) was determined from cloned cDNA . The gene contains 600 nucleotides and encodes a protein of 200 amino acids with a calculated M(r) of 22,200 . The identity of the open reading frame (ORF) encoding the coat protein was confirmed by expression of an Escherichia coli beta-galactosidase fusion protein and detection by a dot blot immunoassay . Sequence comparisons of the deduced coat protein amino acids to several luteoviruses demonstrated that the SbDV-Y coat protein ORF shares greatest similarity with bean leaf roll virus (BLRV) (65%).

Adv Exp Med Biol, 1993, 342, 69 - 74
Identification, expression in E . coli and insect cells of the non-structural protein NS2 encoded by mRNA2 of bovine coronavirus (BCV); Boireau P et al.; The coding part of mRNA 2 (ORF2) of BCV (F15 strain) was cloned and sequenced . The comparison of our sequence data with the sequence of the same ORF of BCV Quebec strain previously published revealed a major difference in the length of the C-terminal part of the NS2 protein . In vitro transcription and translation of ORF2 resulted in the synthesis of a single protein migrating with a Mr of 31 kDa . The ORF2 was fused in frame with the glutathione S transferase gene (GSH) in the pGEX vector . The fusion protein was synthesized as inclusion bodies which were concentrated and used to raise a monospecific antiserum . Alternatively the fusion protein was solubilized, purified by affinity chromatography and cleaved with Factor Xa to yield pure recombinant NS2 . The ORF2 was also expressed in the baculovirus system and the recombinant proteins expressed in pro- and eukaryotic systems were compared on the basis of their size and immunoreactivity . Immunoprecipitation performed with the monospecific antiserum allowed us to identify NS2 in HRT18 infected cells, to follow its kinetic of synthesis, and to ascertain that NS2 was not incorporated in the virion as a minor structural component.

Biochimie, 1993, 75(12), 1083 - 90
Selectivity and specificity in the recognition of tRNA by E coli glutaminyl-tRNA synthetase; Rogers MJ et al.; The specific recognition by Escherichia coli glutaminyl-tRNA synthetase (GlnRS) of tRNA(Gln) is mediated by extensive protein:RNA contacts and changes in the conformation of tRNA(Gln) when complexed with GlnRS . In vivo accuracy of aminoacylation depends on two factors: competition between synthetases, and the context and recognition of identity elements in the tRNA . The structure of the tRNA(Gln):GlnRS complex supports studies from amber and opal suppressor tRNAs, complemented by in vitro aminoacylation of the mutated tRNA transcripts, that the glutamine identity elements are located in the anticodon and acceptor stem of tRNA(Gln) . Recognition of individual functional groups in tRNA, for example the 2-amino group of guanosine, is also evident from the result with inosine-substituted tRNAs . Communication between anticodon and acceptor stem recognition is indicated by mutants in GlnRS isolated by genetic selection with opal suppressor tRNAs which are altered in interactions with the inside of the L-shaped tRNA . We have also used genetic selection to obtain mutants of GlnRS altered in acceptor stem recognition with relaxed specificity for amber suppressor tRNAs, and a more extensive mutational analysis shows the importance of the acceptor binding domain to accurate recognition of tRNA.

Appl Theor Electrophor, 1993, 3(6), 271 - 5
Purification of recombinant adenomatous polyposis coli polypeptide chains from E . coli extracts by continuous-elution electrophoresis; Kraus C et al.; A polypeptide chain encoded by the exons 1-3 of the human adenomatous polyposis coli (APC) tumor suppressor gene was expressed as a maltose binding fusion protein (MBP) in E . coli to be used for immunization purposes . It turned out, that the APC-MBP fusion product of 60 kDa was deposited in bacterial inclusion bodies and, in addition, it was not retarded by an amylose affinity column, most probably due to an altered conformation of the chimeric molecule . For this reason, we established an alternative purification scheme, which took advantage of SDS-extraction followed by a high-resolution two-step continuous-elution electrophoresis (CEE) procedure . This purification method allowed us to obtain high yields of pure human APC exon 1-3-encoded proteins . The final yield of the pure APC polypeptide chains was estimated to represent 5-8% of the amount of SDS-extracted E . coli lysate subjected to the first cycle of CEE . The purified APC molecules were successfully used for the development of specific antibodies . The CEE procedure described here represents a general purification method which is valuable in cases where fusion proteins are deposited as inclusion bodies in bacteria, or if affinity chromatography is precluded due to a conformation-induced lack of ligand binding of the chimeric molecule.

Med Dosw Mikrobiol, 1993, 45(4), 433 - 8
{Identification of enteropathogenic strains of E . coli (EPEC) by application of the latex . II . A trial of applying the test in diagnostic examinations}; Jagielski M et al.; The study was aimed at evaluation of diagnostic usefulness, prepared by own method, of a set of latex reagents for identification of enteropathogenic strains of E . coli (EPEC) . EPEC strains in feces of 334 children with diarrhoea were searched by application at the same time of two methods: used routinely to this time slide agglutination test with commercial OK sera for enteropathogenic serotypes of E . coli and by a latex test with application of warmed culture of investigated strain in peptone water and polystyrene latex coated with immune globulins for O antigens of EPEC serotypes . By application of both methods EPEC strains were found in 50 (15.04%) children, by a latex test in 94 (28.1%) and by agglutination with OK sera in 67 (20.1%) of tested patients . Conformity of results obtained by both tests amounted to 82% . The latex test was found more useful for identification of EPEC strains because of simplicity of performance, higher sensitiveness and better repeatability of results, when compared with agglutination test with live E . coli and OK sera.

Biomater Artif Cells Immobilization Biotechnol, 1993, 21(5), 629 - 36
Genetically engineered E . coli cells containing K . aerogenes gene, microencapsulated in artificial cells for urea and ammonia removal; Prakash S et al.; Microencapsulated genetically engineered E . coli cells can efficiently remove urea without any increase in the ammonia levels in the medium . A 100 mg . alginate encapsulated bacteria rapidly reduces urea in a 100 ml . solution . The original urea concentration 100.00 +/- 1.00 mg./dl . fell to 1.55 +/- 0.13 mg./dl . in 30 minutes . There was no increase in the ammonia in the reaction medium . Extrapolated results shows that urea depletion capacity of encapsulated bacteria is sufficient to remove urea during kidney failure . Using single pool model, 40 gm . of encapsulated genetically engineered E . coli can lower urea (100 mg./dl.) in 40 litres of the body water to 1.60 mg./dl . within 30 minutes . Also, 40.00 gm . bacteria can lower ammonia (758.00 microM/l), in 40 litres of body water, to 90.42 microM/l in 20 minutes . Further studies will be required for multi-compartmental models in the physiological conditions.

Acta Paediatr, 1993 Jan, 82(1), 6 - 11
Inhibition of adhesion of S-fimbriated E . coli to buccal epithelial cells by human skim milk is predominantly mediated by mucins and depends on the period of lactation; Schroten H et al.; Expression of S-fimbriae is frequent in Escherichia coli strains causing sepsis and meningitis in the newborn period . We analysed the ability of human skim milk to inhibit adhesion of S-fimbriated E . coli to human buccal epithelia . Adhesion was inhibited by up to 90% using colostrum (5%) and up to 50% with mature milk (5%), indicating that this anti-infective mechanism depends on the period of lactation . Elimination of up to 99% of immunoglobulins and 91% of lactoferrin by affinity chromatography had no effect on the inhibition of adhesion . After separation of high- (> 10 kD) and low-molecular-weight fractions of skim milk, only the fraction > 10 kD was found to be able to inhibit bacterial adhesion . In order to further characterize receptor molecules for bacteria, we investigated binding of isolated S-fimbriae to glycoprotein bands on Western blot strips . Fimbriae mainly bound to a high-molecular-weight band (> 200 kD) . According to molecular weight and staining behaviour, this band most likely represents mucins . We conclude that carbohydrate residues on secreted mucins of human skim milk are able to inhibit bacterial adhesion to mucosal surfaces . This could provide protection against neonatal sepsis and meningitis caused by E . coli.

Vaccine, 1993, 11(2), 201 - 6
Pili in microspheres protect rabbits from diarrhoea induced by E . coli strain RDEC-1; McQueen CE et al.; We tested whether pilus proteins of rabbit diarrhoeagenic Escherichia coli (RDEC-1), incorporated into biodegradable microspheres, could function as safe and effective oral immunogens in the rabbit diarrhoea model . The RDEC-1 adhesin, AF/R1, incorporated into poly(D,L-lactide-co-glycolide) microspheres, was administered intraduodenally . Vaccinated and unvaccinated rabbits were challenged with RDEC-1 and killed 1 week later . Vaccination with AF/R1 in microspheres did not cause diarrhoea or weight loss . After challenge, rabbits given AF/R1 in microspheres, in contrast to unvaccinated animals, remained in good health . RDEC-1 attachment to caecal epithelium of vaccinated rabbits was reduced (p = 0.02), whereas numbers of RDEC-1 in intestinal fluids were little affected . Also, in vaccinated animals, biliary anti-AF/R1 IgA levels were increased, and AF/R1-induced blast-cell transformation was vigorous in spleen cell cultures . We conclude that vaccination with AF/R1 in microspheres was safe and protected rabbits against RDEC-1 disease, probably by interfering with adherence of the bacteria to the intestinal mucosa . The interference might have been due to the presence of specific antibodies secreted in bile.

Vaccine, 1993, 11(2), 155 - 8
Immunization of rabbits with enterotoxigenic E . coli colonization factor antigen (CFA/I) encapsulated in biodegradable microspheres of poly (lactide-co-glycolide); Edelman R et al.; We have searched for an effective oral delivery system for a purified enterotoxigenic Escherichia coli (ETEC) fimbrial adhesin, CFA/I, which elicits anti-colonization immunity . Purified CFA/I antigen encapsulated in biodegradable polymer microspheres of poly (DL-lactide-co-glycolide) (PLG) induced a vigorous, systemic CFA/I IgG antibody response in rabbits immunized once via intragastric tube; oral, unencapsulated CFA/I induced little or no circulating antibody . CFA/I-specific, S-IgA coproantibody was detected in one of three rabbits fed with the CFA/I-PLG microsphere vaccine . We conclude that PLG microspheres protected CFA/I from degradation in the stomach and effectively delivered the antigen for processing by the host's immune system.

J Drug Target, 1993, 1(4), 331 - 40
Intestinal tissue distribution and epithelial transport of the oral immunogen LTB, the B subunit of E . coli heat-labile enterotoxin; Lazorova L et al.; LTB provokes a systemic immune response and exerts adjuvant effects on mucosal immune responses to unrelated antigens . The binding and uptake of fluorescein-labelled LTB in the normal villus epithelium was compared to that in Peyer's patch dome epithelium in mouse intestine . LTB was bound by the GM1-receptor and taken up extensively by both tissues, indicating that not only the Peyer's patches but also the normal villus epithelium play a significant role in the transport of orally administered antigens . These results were supported by transport studies in the human intestinal epithelial cell line Caco-2 using 125I-LTB . After 2 h incubation, 5.1 +/- 0.1% and 5.9 +/- 0.1% of the added radioactivity was transported in the apical to basolateral and basolateral to apical direction, respectively . Less than 1% of the transported radioactivity was immunoprecipitated with anti-LTB antiserum indicating that LTB was extensively degraded during the transport . The results suggest that normal enterocytes play a significant role in the binding, uptake and transport of orally administered LTB.

Cell Mol Biol Res, 1993, 39(4), 393 - 9
Photocrosslinking analysis of protein-RNA interactions in E . coli transcription complexes; Hanna MM; Regulation of transcription involves numerous specific protein-nucleic acid interactions . We have utilized photochemical crosslinking to identify interactions between Escherichia coli transcription proteins and the nascent RNA in several transcription complexes, including initiation, elongation, and antitermination complexes . We have developed new nucleotide analogs, 5-APAS-UTP and 5-APAS-CTP, which are tagged with photocrosslinking groups on base positions that do not interfere with normal Watson-Crick base-pairing . These analogs are incorporated at internal positions in RNA by E . coli RNA polymerase without disrupting RNA secondary structures . We have also used 8-azido-ATP, which can be incorporated uniquely into the 3' end of the RNA, to analyze interactions at the enzyme active site . Interactions between the RNA and the polymerase subunits, and the effect of various transcription factors, including NusA, NusB, NusE, and NusG, have been examined in complexes containing RNAs from 4 to approximately 80 nucleotides . At almost every RNA position examined, both the beta and beta' subunits are contacted, but never the alpha subunit or NusA . An effect of NusA on the core labeling has been observed in some complexes, however . Sigma is contacted by nucleotides within three nucleotides of the +1 position on the DNA.

Nucleic Acids Symp Ser, 1993, (29), 211 - 3
The recognition of E . coli glutamine tRNA by glutaminyl-tRNA synthetase; Rogers MJ et al.; A variety of genetic, biochemical and structural studies have been used to determine factors ensuring the accuracy of recognition by aminoacyl-tRNA synthetases for tRNA . The identity elements of Escherichia coli tRNA(Gln) are located mainly in the anticodon and acceptor stem, and ensure the accurate recognition of the tRNA by glutaminyl-tRNA synthetase . We summarize a number of experimental techniques to define the accuracy of aminoacylation in vivo and in vitro.

Nucleic Acids Symp Ser, 1993, (29), 207 - 8
Discrimination among E . coli tRNAs with a long variable arm; Asahara H et al.; In E . coli, tRNA(Ser), tRNA(Leu) and tRNA(Tyr) have a long variable arm composed of more than ten nucleotides (class II tRNAs) . In order to study how leucyl- and seryl-tRNA synthetase discriminate their cognate tRNA isoacceptors from the other class II tRNAs, kinetic parameters of various mutated class II tRNA transcripts with leucyl- and seryl-tRNA synthetase were determined . Leucyl-tRNA synthetase recognizes A73 and A14 or its vicinity . Seryl-tRNA synthetase recognizes the long variable arm base-nonspecifically . C2-G71 in the acceptor stem functions as a negative identity element against seryl-tRNA synthetase . Difference in the tertiary structure among class II tRNA molecules plays a crucial role in discrimination by these two synthetases.

Nucleic Acids Res, 1992 Dec 11, 20(23), 6143 - 51
Genome-related datasets within the E . coli Genetic Stock Center database; Berlyn MB et al.; The contents of the E . coli Genetic Stock Center database and the availability in electronic form of the subset of information most relevant to sequence databases are described . The database uses the long-standing Stock Center records (developed and curated by Dr B.J.Bachmann) in describing genotypes of mutant derivatives of E.coli K-12 in terms of alleles, structural mutations, mating type, and plasmids as well as the derivation, names and originators of the strain, and references . The database includes descriptions of mutations, mutation properties, genes, gene properties, and gene products, with EC number identifiers for enzymes . Sequence information is not included, but entries refer to sequence database accession numbers for sequenced regions . A gene is described as a subtype of a more general category of chromosome interval called Site . Since sites are used to describe any chromosomal interval, mapping information is associated with sites . Alleles are described as mutations of those sites and they are not primary map objects, but inherit map position information from the corresponding site description . The database design is intended to preserve richness of detail where it is known and uncertainty of measurements or information as it occurs in order to represent the stock center records as accurately as possible.

Nature, 1992 Dec 10, 360(6404), 606 - 10
A whole genome approach to in vivo DNA-protein interactions in E . coli; Wang MX et al.; The increasingly rapid pace at which genomic DNA sequences are being determined has created a need for more efficient techniques to determine which parts of these sequences are bound in vivo by the proteins controlling processes such as gene expression, DNA replication and chromosomal mechanics . Here we describe a whole-genome approach to identify and characterize such DNA sequences . The method uses endogenous or artificially introduced methylases to methylate all genomic targets except those protected in vivo by protein or non-protein factors interfering with methylase action . These protected targets remain unmethylated in purified genomic DNA and are identified using methylation-sensitive restriction endonucleases . When the method was applied to the Escherichia coli genome, 0.1% of the endogenous adenine methyl-transferase (Dam methylase) targets were found to be unmethylated . Five foreign methylases were examined by transfection . Database-matched DNA sequences flanking the in vivo-protected Dam sites all fell in the non-coding regions of seven E . coli operons (mtl, cdd, flh, gut, car, psp and fep) . In the first four operons these DNA sequences closely matched the consensus sequence that binds to the cyclic AMP-receptor protein . The in vivo protection at the Dam site upstream of the car operon was correlated with a downregulation of car expression, as expected of a feedback repressor-binding model.

Int J Biol Macromol, 1992 Dec, 14(6), 338 - 42
A peptide from Tetrahymena disrupts subunit organization of E . coli RNA polymerase; Andersen HA; Incubation of the E . coli RNA polymerase with a polypeptide factor from the protozoan Tetrahymena reduces the affinity of the holoenzyme for DNA . SDS-polyacrylamide gel electrophoresis of the peptide-treated RNA polymerase showed that the band pattern of the polymerase subunits was strongly altered . The three large subunits, beta', beta and sigma, disappear and a high number of rapidly migrating bands appeared . However, a brief heat treatment of the samples almost restored the original RNA polymerase subunit composition, and in addition a high molecular weight protein band approximately 240 kDa appeared . It is suggested that the Tetrahymena peptide specifically binds to the RNA polymerase and changes the structures of the large subunits.

EMBO J, 1992 Dec, 11(13), 5101 - 9
E.coli MukB protein involved in chromosome partition forms a homodimer with a rod-and-hinge structure having DNA binding and ATP/GTP binding activities; Niki H et al.; mukB mutants of Escherichia coli are defective in the correct partitioning of replicated chromosomes . This results in the appearance of normal-sized anucleate (chromosome-less) cells during cell proliferation . Based on the nucleotide sequence of the mukB gene, the MukB protein of 177 kDa was predicted to be a filamentous protein with globular domains at the ends, and also having DNA binding and nucleotide binding abilities . Here we present evidence that the purified MukB protein possesses these characteristics . MukB forms a homodimer with a rod-and-hinge structure having a pair of large, C-terminal globular domains at one end and a pair of small, N-terminal globular domains at the opposite end; it tends to bend at a middle hinge site of the rod section . Chromatography in a DNA-cellulose column and the gel retardation assay revealed that MukB possesses DNA binding activity . Photoaffinity cross-linking experiments showed that MukB binds to ATP and GTP in the presence of Zn2+ . Throughout the purification steps, acyl carrier protein was co-purified with MukB.

J Bacteriol, 1992 Dec, 174(23), 7827 - 30
Escherichia coli B lacks one of the two initiator tRNA species present in E . coli K-12; Mandal N et al.; We show that the metY locus which specifies tRNA(2fMet) in Escherichia coli K-12 specifies tRNA(1fMet) in E . coli B . This conclusion is based on results of Southern blot analysis of E . coli B and K-12 DNAs and on polymerase chain reaction amplification, cloning, and sequencing of an approximately 200-bp region of DNA corresponding to the metY loci of E . coli B and E . coli K-12 . We also show that the metY locus of E . coli B is transcriptionally active . E . coli strains transformed with the multicopy plasmid vector pUC19 carrying the metY locus of E . coli B overproduce tRNA(1fMet) in E . coli B and E . coli K-12 in contrast to strains transformed with pUC19 carrying the corresponding locus from E . coli K-12, which overproduce tRNA(2fMet).

EMBO J, 1992 Dec, 11(12), 4489 - 96
HU, the major histone-like protein of E . coli, modulates the binding of IHF to oriC; Bonnefoy E et al.; HU is one of the most abundant DNA binding proteins of bacteria . Unlike IHF, integration host factor of Escherichia coli, with which HU shares many properties, including a strong sequence homology and similar predicted structure, HU seems to bind non-specifically to DNA whereas IHF binds to specific sites . In this work we compare the binding characteristics of HU and IHF to a DNA fragment containing the minimal origin of replication of E . coli (oriC) and we analyse the effect of HU on the binding capacity of IHF to this oriC fragment . We show that HU interacts randomly and non-specifically with oriC as opposed to the specific binding of IHF to this same DNA sequence . In addition, we show that HU can modulate the binding of IHF to its specific oriC site . Depending on the relative concentrations of HU and IHF, HU is able either to activate or to inhibit the binding of IHF to oriC.

Biokhimiia, 1992 Dec, 57(12), 1902 - 12
{Functionally important residues of glutamic acid in E . coli pyrophosphatase . I . Chemical modification and localization in the primary structure}; Raznikov AV et al.; Inorganic pyrophosphatase of E . coli is rapidly and irreversibly inactivated by 5-ethyl-5-phenylisoxazolium-3'-sulfonate (Woodward's reagent K) . The appearance in the absorption spectrum of a maximum at 340 nm testifies to the formation of an enzyme enol ester with the inhibitor . The non-hydrolyzable substrate analog CaPP1 partly protects the enzyme from inactivation . A peptide has been isolated from a tryptic hydrolysate of inactivated enzyme which contains an amino acid residue whose modification is critical for the enzyme activity . This peptide corresponds to residues 95-104 of pyrophosphatase and contains four dicarboxylic acid residues . A peptide containing a modified glutamic acid residue was isolated from modified pyrophosphatase hydrolyzed by protease v8 . This peptide represents a fragment of a tryptic modified peptide and has a Glu-Ala-Gly-Glu (residues 98-1C1) structure . It is concluded that inactivation of E . coli pyrophosphatase by Woodward's reagent K is a result of selective modification of Glu98, apparently by the most reactive dicarboxylic amino acid within the enzyme active center.

J Protein Chem, 1992 Dec, 11(6), 589 - 94
Sulfhydryl modification of E . coli Cpn60 leads to loss of its ability to support refolding of rhodanese but not to form a binary complex; Mendoza JA et al.; Differential chemical modification of E . coli chaperonin 60 (cpn60) was achieved by using one of several sulfhydryl-directed reagents . For native cpn60, the three cysteines were accessible for reaction with N-ethylmaleimide (NEM), while only two of them are accessible to the larger reagent 4,4'-dipyridyl disulfide (4-PDS) . However, no sulfhydryl groups were modified when the even larger reagents 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) or 2-(4'-(iodoacetamido)anilino) naphthalene-6-sulfonic acid (IAANS), were employed, unless the chaperonin was unfolded . The cpn60 that had been covalently modified with NEM or IAANS, was not able to support the chaperonin-assisted refolding of the mitochondrial enzyme rhodanese, which also requires cpn10 and ATP hydrolysis . However, both modified forms of cpn60 were able to form binary complexes with rhodanese, as demonstrated by their ability to arrest the spontaneous refolding of the enzyme . That is, chemical modification with these sulfhydryl-directed reagents produced a species that was not prevented from interaction with partially folded rhodanese, but that was prevented from supporting a subsequent step(s) during the chaperonin-assisted refolding process.

Brain Res Mol Brain Res, 1992 Dec, 16(3-4), 311 - 5
Authentic and artifactual detection of the E . coli lacZ gene product in the rat brain by histochemical methods; Rosenberg WS et al.; The accurate detection of a marker gene is fundamental to the assessment of any gene delivery protocol . The use of E . coli lacZ as such a marker gene has become common in studies on gene transfer to the central nervous system . The straightforward histochemical assay that is available to detect the gene product, beta-galactosidase; has made it an attractive system . However, using standard protocols, we have found dramatic non-E . coli lacZ staining in cells with neuronal, glial and endothelial morphology in the normal, adult rat brain . This false staining is primarily in the brainstem, but is evident in cortical and subcortical regions as well . This endogenous reactivity is independent of substrate concentration within the range tested, but is exquisitely sensitive to even small fluctuations in pH . In light of these findings, one must carefully examine any findings of E . coli lacZ gene expression in the rat brain based solely on histochemical analysis of tissue sections.

Wei Sheng Wu Xue Bao, 1992 Dec, 32(6), 456 - 8
{The effect of plasmids on the resistance of E . coli to phages}; Hu Y; The introduction of the ColV, I-K94 or R124 plasmid into Escherichia coli K12 resulted in resistance to certain phages . Derivatives of E . coli carrying the plasmid R124 and ColV, I-K94 were resistance to the phages T4, Mel comparing with the plasmid-free parent and the plasmid ColV, I-K94 conferred resistance to the phage Tull* . It suggested that an envelope change caused by the plasmids might be responsible for the resistance because most of the phages fell to absorb to the plasmid-bearing E . coli cells.

Indian J Biochem Biophys, 1992 Dec, 29(6), 512 - 5
Reconstitution of denatured E . coli alkaline phosphatase with E . coli ribosome; Das B et al.; E . coli alkaline phosphatase was denatured by physical/chemical means . In vitro reconstitution of this denatured enzyme was assisted by 70S E . coli ribosome, as shown by the recovery of its catalytic competence . Almost total recovery of activity of the totally inactivated enzyme was obtained in presence of equimolar concentration of 70S ribosome at 50 degrees C.

Zentralbl Hyg Umweltmed, 1992 Dec, 193(4), 379 - 94
{Long-term study of the persistence of E . coli in waters of different composition}; Loof M; The survival of E . coli ATCC 11229 at different initial concentrations was determined in water of standardized hardness, in tap water from Kiel and in groundwater from the Segeberger forest with pH-values of 4.5, 6.9 and 8.5 . In groundwater at pH 4.5 the bacteria died relatively soon, whereas otherwise in relation to the test conditions long persistence times have been determined . The persistence of E . coli in tap water from Kiel and in water of standardized hardness differed from that in groundwater with comparable pH-values of pH 6.9 and pH 8.5 . In groundwater the colony forming units generally decreased slightly, whereas in tap water and in water of standardized hardness high initial bacterial concentrations resulted in growth, low ones on the other hand in a decrease of the colony forming units . In the area of middle initial bacterial concentrations a significant growth of E . coli (frequently not before 50 days) as well as a die-off could be observed in parallel samples . The influence of the water volume of the sample and the conditioning of the bacteria (unwashed - washed) were of secondary importance.

Zentralbl Hyg Umweltmed, 1992 Dec, 193(4), 342 - 9
{Biological safety investigations of the production of human insulin by genetically engineered E . coli K-12 cells . 1 . Survival capacity of the production strain in the digestive tract of Göttinger miniature swine}; Mieschendahl M et al.; The residence time of genetically engineered Escherichia coli K-12 cells in the digestive tract was tested by feeding Gottinger minipigs 10(10)-10(11) cells of strain W3110iqM15 (pSW3) . This strain is a production strain for human insulin and carries the plasmid pSW3 which contains the genes for human insulin . The test strain could be selected as white colonies grown on McConkey ampicillin plates and could be identified by complementation analysis due to its lacZ M15 deletion . The strain could be isolated from the faeces of the animals up to 72 h after feeding, thereafter no cells of the phenotype of the test strain grew on McConkey ampicillin plates . So the production strain for human insulin W3110iqM15 (pSW3) was not able to colonize the digestive tract of the Gottinger minipig . Feeding the genetically engineered E . coli K-12 cells had no influence on the welfare of the animals.

Protein Eng, 1992 Dec, 5(8), 785 - 9
Probing the role of threonine and serine residues of E . coli asparaginase II by site-specific mutagenesis; Derst C et al.; Site-specific mutagenesis has been used to probe amino acid residues proposed to be critical in catalysis by Escherichia coli asparaginase II . Thr12 is conserved in all known asparaginases . The catalytic constant of a T12A mutant towards L-aspartic acid beta-hydroxamate was reduced to 0.04% of wild type activity, while its Km and stability against urea denaturation were unchanged . The mutant enzyme T12S exhibited almost normal activity but altered substrate specificity . Replacement of Thr119 with Ala led to a 90% decrease of activity without markedly affecting substrate binding . The mutant enzyme S122A showed normal catalytic function but impaired stability in urea solutions . These data indicate that the hydroxyl group of Thr12 is directly involved in catalysis, probably by favorably interacting with a transition state or intermediate . By contrast, Thr119 and Ser122, both putative target sites of the inactivator DONV, are functionally less important.

Biochem Biophys Res Commun, 1992 Nov 16, 188(3), 1131 - 8
Expression of bovine adrenodoxin in E . coli and site-directed mutagenesis of /2 Fe-2S/ cluster ligands; Uhlmann H et al.; Expression systems for adrenodoxin into the periplasm and the cytoplasm of E . coli have been developed as a prerequisite for site-directed mutagenesis studies . In both systems the /2Fe-2S/ cluster of the protein was correctly assembled, the cytoplasmic one gives, however, a tenfold higher expression level . To determine which of the five cysteines at positions 46, 52, 55, 92, and 95 coordinate the /2Fe-2S/ center, they have been individually mutated into serines . From these mutants, only C95S forms a functionally active holoprotein . Thus, residues 46, 52, 55, and 92 are the cysteines that coordinate the /2Fe-2S/ cluster in adrenodoxin.

Nucleic Acids Res, 1992 Nov 11, 20(21), 5633 - 40
Molecular mimicry in translational control of E . coli threonyl-tRNA synthetase gene . Competitive inhibition in tRNA aminoacylation and operator-repressor recognition switch using tRNA identity rules; Romby P et al.; We previously showed that: (i) E.coli threonyl-tRNA synthetase (ThrRS) binds to the leader of its mRNA and represses translation by preventing ribosome binding to its loading site; (ii) the translational operator shares sequence and structure similarities with tRNA(Thr); (iii) it is possible to switch the specificity of the translational control from ThrRS to methionyl-tRNA synthetase (MetRS) by changing the CGU anticodon-like sequence to CAU, the tRNA(Met) anticodon . Here, we show that the wild type (CGU) and the mutated (CAU) operators act as competitive inhibitors of tRNA(Thr) and tRNA(fMet) for aminoacylation catalyzed by E.coli ThrRS and MetRS, respectively . The apparent Kd of the MetRS/CAU operator complex is one order magnitude higher than that of the ThrRS/CGU operator complex . Although ThrRS and MetRS shield the anticodon- and acceptor-like domains of their respective operators, the relative contribution of these two domains differs significantly . As in the threonine system, the interaction of MetRS with the CAU operator occludes ribosome binding to its loading site . The present data demonstrate that the anticodon-like sequence is one major determinant for the identity of the operator and the regulation specificity . It further shows that the tRNA-like operator obeys to tRNA identity rules.

J Mol Evol, 1992 Nov, 35(5), 436 - 43
The role of anticodon bases and the discriminator nucleotide in the recognition of some E . coli tRNAs by their aminoacyl-tRNA synthetases; Shimizu M et al.; The T7 polymerase transcription system was used for in vitro synthesis of unmodified versions of the E . coli tRNA mutants that insert asparagine, cysteine, glycine, histidine, and serine . These tRNAs were used to qualitatively explore the role of some anticodon bases and the discriminator nucleotide in the recognition of tRNA by aminoacyl-tRNA synthetases . Coupled with data from earlier studies, these new results essentially complete a survey of all E . coli tRNAs with respect to the involvement of anticodon bases and the discriminator nucleotide in tRNA recognition . It is found that in the vast majority of tRNAs both of these elements are significant components of tRNA identity . This is not universally true, however . Anticodon sequences are unimportant in tRNA(Ser), tRNA(Leu), and tRNA(Ala) while the discriminator base is inconsequential in tRNA(Ser) and tRNA(Thr) . The significance of these results for origin-of-life studies is discussed.

Mol Gen Genet, 1992 Nov, 235(2-3), 173 - 8
G:C-->T:A and G:C-->C:G transversions are the predominant spontaneous mutations in the Escherichia coli supF gene: an improved lacZ(am) E . coli host designed for assaying pZ189 supF mutational specificity; Akasaka S et al.; Escherichia coli K12 strain KS40 and plasmid pKY241 were designed for easy screening of supF mutations in plasmid pZ189 . KS40 is a nalidixic acid-resistant (gyrA) derivative of MBM7070 (lacZ(am)CA7020) . Using in vitro mutagenesis, an amber mutation was introduced into the cloned gyrA structural gene, of E . coli, to give pKY241, a derivative of pACYC184 . When KS40 containing pKY241 (designated KS40/pKY241) is transformed with pZ189, nalidixic acid-resistant GyrA protein is produced from the chromosomal gyrA gene and wild-type GyrA protein from pKY241 because of the suppression of the gyrA amber mutation by supF . It is known that the wild-type, otherwise nalidixic acid-sensitive, phenotype is dominant over the nalidixic acid-resistant phenotype . Thus, KS40/pKY241 gives rise to nalidixic acid-sensitive colonies when it carries a pZ189 plasmid with an active supF suppressor tRNA . If the supF gene on the plasmid carries an inactivating mutation then KS40/pKY241 will form nalidixic acid-resistant colonies . By using this system, the spontaneous mutational frequency of the supF gene on pZ189 was calculated to be 3.06 x 10(-7) per replication . Among 51 independent supF mutations analyzed by DNA sequencing, 63% were base substitutions, 25% IS element insertions, 9.6% deletions and 1.9% single-base frameshifts . The base substitutions included both transversions (84.8%) and transitions (15.2%), the largest single group being G:C to T:A transversions (45.4% of the base substitutions) . These results demonstrate that the KS40/pKY241 system we have developed can be used to characterize the DNA sequence changes induced by mutagens that give very low mutational frequencies.

Acta Chem Scand, 1992 Nov, 46(11), 1114 - 21
The function of the 5-hydroxymethyl group of lactose in enzymatic hydrolysis with beta-galactosidase from E . coli; Adelhorst K et al.; A series of 6-substituted methyl lactoside derivatives together with methyl allolactoside and (6S)-methyl {6-2H}lactoside have been synthesized and characterized by NMR spectroscopy . All compounds were tested as substrates for the enzyme beta-galactosidase from E . coli using progress curve kinetic methology both in single-substrate and competition experiments . The results show that the hydrolysis of methyl lactoside to a large extent takes place through an intramolecular transglycosidation reaction via allolactoside . Furthermore, methyl 6-amino-6-deoxy-D-glucopyranoside proved to be an ihibitor for the enzymatic hydrolysis.

Biotechniques, 1992 Nov, 13(5), 756 - 62
Production of recombinant porcine tumor necrosis factor alpha in a novel E . coli expression system; Su X et al.; DNA sequences encoding porcine tumor necrosis factor alpha (TNF alpha) were reconstructed from a genomic-derived PCR product for expression in Escherichia coli . A synthetic DNA primer containing most of exon III was fused to exon IV sequences by means of PCR . The fused product was then inserted into the novel FLAG vector by restriction and ligation . This initial recombinant construct was propagated in single-strand form through use of a helper phage and subjected to oligonucleotide-directed mutagenesis for the purpose of introducing additional coding sequences from exons II and III . The final construct encoded a fusion protein consisting of the Omp-A signal peptide, a seven-amino acid FLAG peptide and the soluble form of porcine TNF alpha . Bacteria containing this construct produced a protein which was recognized by anti-FLAG monoclonal antibody in Western blots and which was purified by anti-FLAG immunoaffinity chromatography . The purified material was cleaved with enterokinase to remove the FLAG peptide . Both the enterokinase-cleaved form and the uncleaved form were shown to have TNF activity in a WEHI cell cytotoxicity assay.

J Biotechnol, 1992 Nov, 26(2-3), 275 - 88
Expression in E . coli and purification of intracellular proteins by fusion to cyclomaltodextrin glucanotransferase; Hellman J et al.; A plasmid expression vector was constructed to direct the synthesis of foreign proteins in Escherichia coli as fusions with cyclomaltodextrin glucanotransferase (CGT) with cytoplasmic location (delta ssCGT) . The ability of CGT to bind to covalently immobilized cyclodextrins was utilized in purifying fused target proteins . A large proportion of the cytoplasmically synthesized delta ssCGT formed inclusion bodies which adopted the active conformation at considerably high refolding concentration (67 microM delta ssCGT solution) . By lowering the cultivation temperature the proportion of the soluble delta ssCGT was slightly increased . Intracellularly expressed delta ssCGT provides a potential affinity handle which forms easily refoldable inclusion bodies increasing the yield and stability, and possibly allows the expression of lethal target proteins . Interestingly, the interaction between one model fusion protein delta ssCGT-CAT (CAT, chloramphenicol acetyltransferase) and the E . coli heat shock protein GroEL was observed.

Mol Biol Rep, 1992 Nov, 17(1), 61 - 70
Shuttle vectors conferring hygromycin B resistance to E . coli and to mammalian cells . Differential expression of carboxyterminal fusion proteins; Asselbergs FA et al.; Shuttle vectors expressing resistance to hygromycin B in both E . coli and in mammalian cells were constructed . A combination of the simian virus 40 early promoter upstream of the native bacterial promoter of the neo gene from transposon Tn5 was found to express hygromycin B resistance better in both types of host cells than a combination of the Tn5 promoter followed by the promoter of the Herpes simplex virus thymidine kinase gene . Hygromycin phosphotransferase fusion proteins with extensions at the carboxyterminus were also tested and found to be marginally less effective as selection markers in eukaryotic cells but virtually inactive in E . coli.

Virology, 1992 Nov, 191(1), 443 - 7
Expression of Epstein-Barr virus membrane antigen gp350/220 in E . coli and in insect cells; Nuebling CM et al.; The Epstein-Barr virus open reading frame BLLF1 encodes the major envelope glycoproteins gp350 and gp220 . Fragments of the gp350/220 gene were expressed in Escherichia coli in order to define regions of the polypeptide chain reacting with human sera . The C-terminal half of the protein was sufficient for recognition by all VCA-positive sera tested . A membrane anchor truncated version of gp350/220 was expressed in insect cells using the baculovirus system . Proteins of different sizes were specifically detected in the cells while a glycosylated 220-kDa protein was secreted . The insect cells were tested for their suitability as tools for performing monospecific immunofluorescence.

FEBS Lett, 1992 Oct 19, 311(2), 139 - 42
Crucial role of pyrophosphate in the aminoacylation of E . coli tRNA(Phe) by yeast phenylalanyl-tRNA synthetase; Khvorova AM et al.; Rapid inactivation of the yeast phenylalanyl-tRNA synthetase in the course of aminoacylation of the heterologous E . coli tRNA(Phe) is observed . This inactivation occurs due to the formation of the tight complex of the enzyme with the pyrophosphate formed during the aminoacylation reaction . This complex is shown to be the normal intermediate of the reaction . Possible inactivation mechanism and correlation between structural differences of yeast and E . coli tRNAs(Phe) with the changes in the enzymatic mechanism of aminoacylation are discussed.

Zentralbl Bakteriol, 1992 Oct, 277(3), 389 - 402
Escherichia coli types isolated from porcine E . coli infections in Saxony from 1963 to 1990; Wittig W et al.; Between 1963 and 1990, 4221 Escherichia coli strains were isolated from 4221 cases of porcine neonatal colibacillosis and 16826 E . coli strains from 16064 cases of E . coli enterotoxicosis of weaned pigs . They belonged to serotypes which, due to their enterotoxigenicity or verotoxigenicity, are considered to be pathogenic for pigs . Nonhaemolytic enterotoxigenic strains characterized by one of the fimbrial antigens K99 (F5), 987p (F6), and F41 and one of at least 8 different A antigens were only found in suckling piglets . Haemolytic serotypes lacking the K88 (F4), K99, 987p, and F41 fimbrial antigens as well as A antigens were only isolated from pigs with E . coli enterotoxicosis of weaned pigs, which can occur already some days before weaning, while enterotoxigenic types carrying the fimbrial antigen K88 were found in both neonatal colibacillosis and E . coli enterotoxicosis of weaned pigs . In the 28 years considered, the type O149: (K91), K88 dominated in neonatal colibacillosis and, since 1972, in E . coli enterotoxicosis of weaned pigs, too . It was mainly nonhaemolytic during the first years and always haemolytic later . The emergence of type O147: (K89) coincided with a gradual disappearance of type O147: (K89), K88, both types differing in their biochemical behaviour.

Protein Eng, 1992 Oct, 5(7), 605 - 10
3-D structure of a mutant (Asp101-->Ser) of E.coli alkaline phosphatase with higher catalytic activity; Chen L et al.; Mutagenesis of the absolutely conserved residue Asp101 of the non-specific monoesterase alkaline phosphatase (E.C . 3.1.3.1) from E . coli has produced an enzyme with increased kcat . The carboxyl group of the Asp101 residue has been proposed to be involved in the positioning of Arg166 and the formation of the helix that contains the active site Ser102 . The crystal structure of the Asp101-->Ser mutant has been refined at 2.5 A to a final crystallographic R-factor of 0.173 . The altered active site structure of the mutant is compared with that of the wild-type as well as with the structures of the mutant enzyme soaked in two known alkaline phosphatase inhibitors (inorganic phosphate and arsenate) . The changes affect primarily the side chain of Arg166 which, by losing the hydrogen bond interaction with the carboxyl side chain of Asp101, becomes more flexible . This analysis, in conjunction with product inhibition studies of the mutant enzyme, suggests that at high pH (> 7) the enzyme achieves a quicker catalytic turnover by allowing a faster release of the product.

Biochem Int, 1992 Oct, 28(2), 353 - 62
One-step purification of E . coli elongation factor Tu; Knudsen CR et al.; The tuf A gene, encoding the E . coli elongation factor Tu, was cloned in the pGEX gene fusion system . Upon expression EF-Tu is fused to glutathione-S-transferase serving as a purification handle with affinity for glutathione immobilised on agarose . This allows purification of EF-Tu in a one-step procedure . The construct was designed in order to make possible the release of authentic EF-Tu by cleaving the fusion protein with the protease factor Xa.

Ryumachi, 1992 Oct, 32(5), 437 - 45
{Reassessment of measurement of anti double-stranded DNA antibodies by Farr's assay using double-stranded DNA from E . coli and application of DNA binding Activities in high salt solution}; Miyawaki S et al.; Farr's assay using double-stranded (ds) DNA from E . coli is a most sensitive and specific method for the detection of anti ds-DNA antibodies in patients with systemic lupus erythematosus (SLE) . Because of the lack of sufficient DNA antigens, however, final antibody titers were hardly determined when the sera contained antibodies titered more than 100U/ml (or more than 60 per cent by DNA binding activities) . In such sera DNA binding activities were measured by using ds-DNA tracer adjusted final concentration of NaCl to 125 mM . Higher binding activities measured by high-salt tracer are obtained significantly in SLE patients groups with nephrotic syndrome, proteinuria, cast, renal failure, diffuse proliferative nephritis, low serum complement levels, anemia and/or low IgG/IgA levels compared with the patients who lacked these clinical findings . In contrast the patients with digital rash or cramp showed significantly lower high-salt binding activities . The patients with pleuropericarditis tended to have lower bindings . The non-lupus patients including MCTD also had lower levels . These clinical characteristics could not be evaluated by standard Farr's assay . High-salt bindings suggest the presence of high avidity antibodies and also partly may mean the high levels of low avidity antibodies . The application of high-salt binding activities, thus, is a useful tool for the evaluation of clinical characteristics of SLE patients who had high levels of anti ds-DNA antibodies by standard Farr's assay.

Microbiologica, 1992 Oct, 15(4), 397 - 8
Transfection of E . coli with lambda DNA by electroporation; Magistrelli C et al.; In the ambit of the B . subtilis genoma sequencing and mapping project, we have set up an electroporation method to transfect E . coli cells with lambda DNA . This methodology presents features that make it preferable to traditional in vitro packaging for some purposes . Here we will illustrate the experimental procedure and the possible applications.

Plant Mol Biol, 1992 Oct, 20(2), 301 - 6
Biosynthesis of active spinach-chloroplast thioredoxin f in transformed E . coli; Aguilar F et al.; The recently cloned gene for spinach chloroplast thioredoxin f was subcloned in a modified pKK233-2 expression vector and used for transformation of Escherichia coli cells containing the Iq plasmid . After induction with IPTG (isopropyl-beta-D-thiogalactoside) the transformed cells produce the chloroplast protein in large amounts as insoluble deposit within the cell . The protein has been solubilized, purified and analysed for activity . It shows no difference in catalytic activity from native spinach chloroplast thioredoxin f . Its electrophoretic behaviour suggests that the native thioredoxin f may have a different N-terminus than was assumed on the basis of the protein sequencing results.

Mutat Res, 1992 Oct, 272(2), 161 - 73
An evaluation of the E . coli K-12 uvrB/recA DNA repair host-mediated assay . II . In vivo results for 36 compounds tested in the mouse; Hellmer L et al.; The aim of this study was to further evaluate the E . coli K-12 DNA repair host-mediated assay, as a short-term in vivo genotoxicity test, to be used as a complement to the micronucleus test in the routine testing of chemicals and drugs . The assay involves the administration of the test substance to mice by the route of choice, followed by the intravenous administration of a mixture of DNA repair deficient and proficient derivatives of E . coli K-12 . After an incubation period the relative survival of the two strains was determined in blood, liver, lungs, kidneys and testes of the host . A significant preferential reduction of the DNA repair deficient strain in any organ indicates that the test substance possesses genotoxic properties . A total of 36 substances, 26 carcinogens, 4 weak or non-carcinogens and 6 unclassified substances, were tested in this assay . Positive results were obtained for 23 compounds . Of the carcinogens 18 were positive and of the non-carcinogens 3 were negative . The overall concordance between the assay and carcinogenicity was 72% . In general, alkylating agents and direct-acting nitroso compounds showed genotoxic activity in all organs tested, while the other substances were positive in a limited number of organs . With oral administration, which was the most commonly used administration route in the study, the organ showing a positive response most often was the blood . The results from the present study were compared with results from the micronucleus test, which were available for 26 of the substances . Results were in agreement for 15 of the substances, while 8 substances were positive in the present assay and negative in the micronucleus test: 4-aminobiphenyl, 2-anisidine, epichlorohydrin, formaldehyde, 1- and 2-naphthylamine, 2-nitrophenylenediamine and 4-nitroquinoline-N-oxide . The substances negative in the E . coli DNA repair host-mediated assay, but positive in the micronucleus test were: benzene, catechol and cyclophosphamide . It is concluded from this evaluation that the E . coli K-12 DNA repair host-mediated assay detects a number of carcinogens that are negative in the micronucleus test, while detecting most of the compounds that are positive in the latter . The advantages of this test are that differential DNA repair measures a broad spectrum of genetic damage, an in vitro/in vivo comparison is possible with the same test organisms, results can be obtained from various organs and the test is rapid.(ABSTRACT TRUNCATED AT 400 WORDS)

Avian Dis, 1992 Oct-Dec, 36(4), 881 - 90
Escherichia coli multiplication and lesions in the respiratory tract of chickens inoculated with infectious bronchitis virus and/or E . coli; Nakamura K et al.; Escherichia coli numbers and histopathological changes were studied in the respiratory tract of line 151 chickens intranasally inoculated with infectious bronchitis virus (IBV) and/or virulent E . coli; this line is highly susceptible to IBV . Chickens inoculated with IBV alone showed increased numbers of E . coli in the trachea and had tracheitis, airsacculitis, and bronchiolitis . One of 17 chickens inoculated with IBV alone died with fibrinopurulent serositis . Chickens inoculated with IBV and E . coli had more severe and persistent respiratory lesions than those inoculated with IBV alone . E . coli was isolated from tracheas of chickens inoculated with IBV and E . coli more frequently than from chickens inoculated with IBV alone . In this group, 14 of 27 chickens died with tracheal plugs or with fibrinopurulent serositis . There was neither increased numbers of E . coli nor significant lesions in the respiratory tract of the group inoculated with E . coli alone . These results suggest that IBV may facilitate E . coli invasion into the lower respiratory tract of the chicken.

Rinsho Byori, 1992 Oct, 40(10), 1073 - 9
{Clinical application of anti double-stranded and single-stranded DNA antibodies measurements by enzyme-linked immunosorbent assay (ELISA) using E . coli plasmid DNA}; Miyawaki S et al.; Anti double-stranded (ds) and single-stranded (ss) DNA IgG antibodies were detected in patients with various connective tissue diseases by RECOMBIGEN ELISA Anti ds-DNA Kit and ELISA Anti ss-DNA Kit (Nippon DPC Co) using E . coli plasmid DNA . High levels of anti ds-DNA and anti ss-DNA antibodies were mostly found and distributed in patients with systemic lupus erythematosus(SLE) and allied disorders . The sensitivity of Anti ds-DNA Kit was higher than that of current ELISA kit using calf thymus DNA antigen . In SLE patients with anti DNA antibodies sixty per cent of sera were reacted with both ds- and ss-DNA antigens, and forty per cent of sera were reacted only with ss-DNA antigen; there was no sample reacted positively with ds-DNA antigen alone . RECOMBIGEN ELISA Anti ds-DNA Kit and ELISA Anti ss-DNA Kit are highly sensitive and useful methods to detect anti ds- and/or ss-DNA IgG antibodies in patients with SLE.

Biull Eksp Biol Med, 1992 Oct, 114(10), 439 - 42
{The morphological changes in the erythrocytes and in the blood iron level of rats under the action of E . coli endotoxin}; Bardakhch'ian EA et al.; Development of endotoxin shock in rats is accompanied by decreasing dry mass and content of dense substances in red blood cells . Endotoxin reduces circulating iron concentration as well . During endotoxin administration morphological changes of erythrocytes become stable, appearance of echinocytes promotes elevation of hemostatic potential and enhancement of blood cells aggregability.

FEBS Lett, 1992 Sep 21, 310(1), 63 - 5
Human interleukin-1 receptor antagonist . High yield expression in E . coli and examination of cysteine residues; Steinkasserer A et al.; The human IL-1 receptor antagonist (IL-1ra) was produced in a high yield E . coli expression system, and was purified in a rapid two-step purification . This recombinant IL-1ra molecule possessed full binding activity to the IL-1 receptor (type I) and totally inhibited IL-1-induced PGE2 production by human dermal fibroblasts . Radioalkylation and analysis of V8-derived IL-1ra peptides indicate that the four cysteines present in the IL-1ra are not disulphide-linked.

Pol Tyg Lek, 1992 Sep 7-14, 47(36-37), 808 - 9
{Late episodes of apnea in a premature infant infected with E . coli O111K58}; Maszkiewicz W et al.; Typical episodes of apnoea with paleness and cyanosis have been noted noted in premature baby born on the 28th week of pregnancy with body weight 1,010 g as a result of infection with enteropathogenic strain of E . coli O111K58 on the 21st day of life (3rd day of the infection) . Effective treatment with antibiotics produced recovery.

Carbohydr Res, 1992 Sep 2, 233, 195 - 204
Structural elucidation of the capsular polysaccharide of E . coli serotype K47; de Bruin AH et al.; The capsular polysaccharide from Escherichia coli K47 was investigated using mainly methylation analysis and 1H and 13C NMR spectroscopy and shown to have the following repeating unit: {Formula: see text}

Circ Shock, 1992 Sep, 38(1), 9 - 13
In vivo assessment of regional microvascular albumin leakage during E . coli septic shock in the baboon model; Dormehl IC et al.; Changes in regional microvascular albumin flux during septic shock were studied noninvasively by scintigraphy in the baboon model . Use was made of an i.v . injection of 99mTc-labeled baboon serum albumin . Count ratios of lung to cardiac, liver to cardiac, and abdominal to cardiac regions were measured two-hourly for 6 hr in six control and six septic shock baboons (live E . coli) and compared . Increased ratios obtained during shock pointed to an increase in extravascular albumin . Linear regression lines fitted to these count ratios provided regional albumin leak indices . These indices demonstrated statistically significant increases (P less than 0.05) during septic shock for the abdominal region during the 6-hr study, and for all regions, but especially the abdomen, when data were calculated over 4 hr . Increasing ratios and leak indices correlated with postmortem data and with changes in neutrophil and platelet behaviour previously established during shock . Possible accompanying mediator releases could be responsible for the endothelial damage leading to the increased permeability.

Br Poult Sci, 1992 Sep, 33(4), 769 - 74
Influence of age on the febrile response to E . coli and S . typhimurium endotoxins in growing pullets; Gregorut FP et al.; 1 . The effect of bacterial endotoxin injection was studied in growing pullets of different ages . Commercial chicks were divided into 5 groups according to age . Bacterial endotoxins (E . coli and S . typhimurium) were injected intravenously and rectal temperature was measured over a period of 300 min . 2 . The results showed no significant effect of age on the febrile response induced by bacterial endotoxins, but a slight tendency towards a reduced fever peak was observed with increasing age . The response latency also increased with age.

Appl Microbiol Biotechnol, 1992 Sep, 37(6), 765 - 71
Secretion of genetically-engineered dihydrofolate reductase from Escherichia coli using an E . coli alpha-hemolysin membrane translocation system; Nakano H et al.; Secretion of fusion proteins composed of cytoplasmic protein dihydrofolate reductase (DHFR) and the Escherichia coli alpha-haemolysin (HlyA) C-terminal sequence was examined through the haemolysin secretion machinery of E . coli . DHFR of various lengths was combined with the HlyA C-terminal region, and both secretion and DHFR activity of the fusions were measured . The secretion was found to be inversely correlated with the intracellular DHFR activity . Moreover, when one amino acid (Ile155) in a beta-sheet of the DHFR C-terminal region was replaced with Lys, the enzymatically active DHFR fusion protein was secreted into the medium . We discuss the possibility of a relationship between folding and secretion of HlyA-fused protein in the HlyA secretion system.

EMBO J, 1992 Sep, 11(9), 3209 - 17
Restoration of a lost metal-binding site: construction of two different copper sites into a subunit of the E . coli cytochrome o quinol oxidase complex; van der Oost J et al.; The cupredoxin fold, a Greek key beta-barrel, is a common structural motif in a family of small blue copper proteins and a subdomain in many multicopper oxidases . Here we show that a cupredoxin domain is present in subunit II of cytochrome c and quinol oxidase complexes . In the former complex this subunit is thought to bind a copper centre called CuA which is missing from the latter complex . We have expressed the C-terminal fragment of the membrane-bound CyoA subunit of the Escherichia coli cytochrome o quinol oxidase as a water-soluble protein . Two mutants have been designed into the CyoA fragment . The optical spectrum shows that one mutant is similar to blue copper proteins . The second mutant has an optical spectrum and redox potential like the purple copper site in nitrous oxide reductase (N2OR) . This site is closely related to CuA, which is the copper centre typical of cytochrome c oxidase . The electron paramagnetic resonance (EPR) spectra of both this mutant and the entire cytochrome o complex, into which the CuA site has been introduced, are similar to the EPR spectra of the native CuA site in cytochrome oxidase . These results give the first experimental evidence that CuA is bound to the subunit II of cytochrome c oxidase and open a new way to study this peculiar copper site.

Biochem Biophys Res Commun, 1992 Aug 31, 187(1), 425 - 31
Site directed mutagenesis of two cysteine residues in the E . coli ogt O6-alkylguanine DNA alkyltransferase protein; Harris LC et al.; The E . coli ogt O6-alkylguanine-DNA alkyltransferase has two cysteine residues positioned identically with respect to cysteines in the E . coli ada O6-alkylguanine-DNA alkyltransferase . In order to assess their function, these residues were each substituted by a glycine to generate altered forms of the ogt protein . Mutagenesis of cysteine-139, located within a 'PCHRV' region of homology, eliminated functional activity confirming that this residue is the methyl-accepting cysteine in the active site of the protein . Substitution of cysteine 102 within the sequence 'LRTIPCG' had little effect on the ogt protein activity demonstrating that this cysteine is not directly involved with the transfer of O6-methylguanine adducts.

Cancer Lett, 1992 Aug 31, 65(3), 201 - 7
Polyclonal and monoclonal antibodies monospecific to MMTV LTR orf protein produced in E . coli; Haga S et al.; Monoclonal and polyclonal antibodies specific to an open reading frame of the mouse mammary tumor virus long terminal repeat were generated using an open reading frame-beta-galactosidase fusion protein produced in E . coli . Both antibodies reacted with the open reading frame-beta-galactosidase fusion protein but not with beta-galactosidase alone using an immunoblotting technique . It is concluded that these antibodies were specific for the protein encoded by the open reading frame of the mouse mammary tumor virus long terminal repeat.

Nucleic Acids Res, 1992 Aug 25, 20(16), 4331 - 8
An assessment of neural network and statistical approaches for prediction of E . coli promoter sites; Horton PB et al.; We have constructed a perceptron type neural network for E . coli promoter prediction and improved its ability to generalize with a new technique for selecting the sequence features shown during training . We have also reconstructed five previous prediction methods and compared the effectiveness of those methods and our neural network . Surprisingly, the simple statistical method of Mulligan et al . performed the best amongst the previous methods . Our neural network was comparable to Mulligan's method when false positives were kept low and better than Mulligan's method when false negatives were kept low . We also showed the correlation between the prediction rates of neural networks achieved by previous researchers and the information content of their data sets.

Nucleic Acids Res, 1992 Aug 25, 20(16), 4339 - 46
RNA-DNA hybridization promoted by E . coli RecA protein; Kirkpatrick DP et al.; RecA protein of E . coli plays a central regulatory role that is induced by damage to DNA and results in the inactivation of LexA repressor . In vitro, RecA protein binds preferentially to single-stranded DNA to form a nucleoprotein filament that can recognize homology in naked duplex DNA and promote extensive strand exchange . Although RecA protein shows little tendency at neutral pH to bind to RNA, we found that it nonetheless catalyzed at 37 degrees C the hybridization of complementary RNA and single-stranded DNA sequences . Hybrids made by RecA protein at 37 degrees C appeared indistinguishable from ones prepared by thermal annealing . RNA-DNA hybridization by RecA protein at neutral pH required, as does RecA-promoted homologous pairing, optimal conditions for the formation of RecA nucleoprotein filaments . The cosedimentation of RNA with those filaments further paralleled observations made on the formation of networks of nucleoprotein filaments with double-stranded DNA, an instrumental intermediate in homologous pairing in vitro . These similarities with the pairing reaction support the view that RecA protein acts specifically in the hybridization reaction.

Nucleic Acids Res, 1992 Aug 25, 20(16), 4221 - 7
A ribosomal ambiguity mutation in the 530 loop of E . coli 16S rRNA; O'Connor M et al.; A series of base substitution and deletion mutations were constructed in the highly conserved 530 stem and loop region of E . coli 16S rRNA involved in binding of tRNA to the ribosomal A site . Base substitution and deletion of G517 produced significant effects on cell growth rate and translational fidelity, permitting readthrough of UGA, UAG and UAA stop codons as well as stimulating +1 and -1 frameshifting in vivo . By contrast, mutations at position 534 had little or no effect on growth rate or translational fidelity . The results demonstrate the importance of G517 in maintaining translational fidelity but do not support a base pairing interaction between G517 and U534.

FEBS Lett, 1992 Aug 24, 308(3), 258 - 60
Tobacco chloroplast ribosomes contain a homologue of E . coli ribosomal protein L28; Yokoi F et al.; The genes for ribosomal proteins L28 and L33 constitute an operon (rpmBG) in E . coli, but in plant chloroplasts L33 is encoded by the chloroplast DNA and L28 seems to be encoded by the nuclear genome . A 15 kDa protein was isolated from the 50 S subunit of tobacco chloroplast ribosomes and its N-terminal amino acid sequence was determined . A cDNA for this protein was cloned and analyzed . The cDNA encodes a 151 amino acid protein consisting of a predicted transit-peptide of 74 amino acids and a mature protein of 77 amino acids . The mature protein is homologous to E . coli L28, hence we named it chloroplast L28 (CL28) . This is the first report on the presence of an E . coli L28-like protein in another organism.

FEBS Lett, 1992 Aug 3, 307(3), 375 - 8
Expression and purification of mouse TIMP-1 from E . coli; Cocuzzi ET et al.; Tissue inhibitors of metalloproteinases (TIMPs) constitute a family of secreted glycoproteins involved in regulating extracellular matrix degradation in both normal and malignant tissues . We have expressed a cDNA clone of mouse TIMP-1 as a 22-kDa protein with 12 cysteine residues in E . coli and purified protein that shows inhibitory activity against collagenase following renaturation by chemical means . The low specific activity and circular dichroism measurements suggest, however, that the renaturation of the mouse recombinant (non-glycosylated) protein is not efficient under the conditions we have used, indicative of either thermodynamic instability or the transition to kinetic intermediates which have very low in vitro refolding rates.

Proteins, 1992 Aug, 13(4), 352 - 63
Amino acid substitution analysis of E . coli thymidylate synthase: the study of a highly conserved region at the N-terminus; Kim CW et al.; Amino acid substitution analysis within a highly conserved region of Escherichia coli thymidylate synthase (TS), using suppression of amber mutations by tRNA suppressors, has yielded a bank of 124 new mutationally altered TS proteins . These mutant proteins have been used to study the structure-function relationship of the Escherichia coli TS protein at the N-terminus corresponding to residues 20 through 35 . This region contains a block of amino acids whose sequence has been well conserved among other known TS proteins from various organisms . Positions 20 through 25 contain a surface loop structure and positions 26 through 35 encompass a beta-strand . We find that residues surrounding a beta-bulge structure within the beta-strand are particularly sensitive to amino acid substitution, suggesting that this structure is maintained by a highly ordered packing arrangement . Three residues in the surface loop that are present at the base of the substrate binding pocket are also sensitive to amino acid substitution . The remainder of the conserved sites, including those at the dimer interface, are tolerant to most, if not all, of the substitutions tested.

Appl Biochem Biotechnol, 1992 Aug, 36(2), 137 - 52
Quantitation of E . coli protein impurities in recombinant human interferon-gamma; Chen AB et al.; A multiple antigen ELISA for E . coli proteins (ECPs) that may be present in purified recombinant human interferon-gamma (rIFN-gamma) was developed . SDS-PAGE and Western blotting analyses showed that the assay antibodies reacted with a wide spectrum of ECPs in the standard and with ECPs in a production run . In spike recovery studies, rIFN-gamma at concentrations of 0.05 mg/mL and higher augmented the immunoreactivity of the ECPs in the standard curve (1.3-40.0 ng ECPs/mL) by approx 50% . To determine ECP content in purified rIFN-gamma, 0.2 mg/mL of rIFN-gamma was added to the standard curve diluent to compensate for enhanced immunoreactivity . The assay was precise (interassay precision of ECP controls < or = 4.1 %CV) and accurate with recoveries of 111-115% of expected for ECPs (15-40 ng/mL) spiked into purified rIFN-gamma (1 mg/mL) . Linearity of dilution for ECPs spiked into rIFN-gamma was obtained (r = 0.999) . Moreover, linearity of dilution was obtained for ECPs in "in-process" samples, demonstrating the required condition of antibody excess for this type of multiple antigen ELISA . ECPs were not detectable in several purified lots of rIFN-gamma . Therefore, these lots contained < 1.3 ppm ECPs.

Biochem Int, 1992 Aug, 27(4), 745 - 53
Effect of E . coli ribosomal protein S1 on the fidelity of the translational elongation step: reading and misreading of poly(U) and poly(dT); Potapov AP et al.; Ribosomal protein S1 was selectively removed from E . coli ribosomes by affinity chromatography and the effect of added S1 on the translation of poly(dT) {which is read as poly(U) in the presence of neomycin} and on the misreading of poly(U) and poly(dT) were examined . S1 enhances the translation of poly(dT) at low template concentration, which is similar to the effect of S1 on poly(U) translation . The misreading of poly(dT) by E . coli ribosomes is at a lower level than is the case with poly(U) . This low misreading is the same for "S1-dependent" and "S1-independent" modes of translation . On the other hand, the misreading of poly(U) is significantly reduced when S1 is present . These results thus indicate that S1 not only facilitates the binding of mRNA to the ribosome as already known, but also plays a role in the correct codon-dependent selection of aminoacyl-tRNA.

Biochem Int, 1992 Aug, 27(4), 601 - 11
OmpT proteolysis of E . coli initiation factor IF2 . Elimination of a cleavage site by site-directed mutagenesis; Lassen SF et al.; A serious problem during purification of the E . coli initiation factor IF2 is a significant loss of native IF2 due to partial degradation . We have previously shown that the major fragment, IF2 gamma (65 kDa), is the result of cleavage of IF2 alpha at the peptide bond between lysine 289 and arginine 290 . In this paper we demonstrate that the cleavage is a result of proteolysis by outer membrane protease OmpT occurring immediately after cell lysis and in the S30 supernatant . By protein engineering we have constructed an IF2 mutant Lys289-greater than Met and shown that the IF2 gamma cleavage site in this mutant protein is insensitive to cleavage by OmpT . However the mutant protein is cleaved by OmpT between arginine 279 and alanine 280, which is a novel sequence specificity for this protease.

Mutat Res, 1992 Aug, 268(2), 287 - 95
Enhancing effect of heterocyclic amines and beta-carbolines on UV or chemically induced mutagenesis in E . coli; Shimoi K et al.; Most heterocyclic amines and beta-carbolines--harman, norharman, harmine, harmaline--enhanced UVC (254 nm) induced mutagenesis without microsomal activation in E . coli B/r WP2 . 3-Amino-1,4-dimethyl-5H-pyrido{4,3-b}indole (Trp-P-1) was most effective and increased UVAB (295-400 nm) induced mutations as well as UVC induced ones . Trp-P-1 enhanced the frequencies of mutations induced by not only UV but also 4-nitroquinoline-1-oxide (4NQO) or 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide (AF2), while it showed little effect on N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) or gamma-ray induced mutagenesis . Trp-P-1 decreased the survival of UVC irradiated cells of CM571recA . However, these effects of Trp-P-1 on UVC induced mutagenesis and lethality were not observed in WP2suvrA which is excision repair deficient . The alkaline sucrose gradient sedimentation analysis demonstrated that Trp-P-1 blocked the incision step in DNA excision repair . Further, pretreatment with Trp-P-1 before UVC irradiation showed no effect on UVC induced mutagenesis . Similar effects were also seen in the case of harman or norharman . These results suggest that heterocyclic amines and beta-carbolines inhibit DNA excision repair directly or indirectly, thus enhancing UV or chemically induced mutagenesis.

J Pediatr Gastroenterol Nutr, 1992 Aug, 15(2), 105 - 11
Comparative effects of atrial natriuretic peptide and E . coli heat-stable toxin on rat intestinal transport; Guarino A et al.; Conflicting data have been published in favor of or against a secretory effect of atrial natriuretic peptide (ANP) in the intestine . The reported effects resemble that of Escherichia coli heat-stable enterotoxin (ST) . In this work the effects of ANP were studied in well established experimental systems and compared with that of ST . Both peptides induced a prompt secretion of water, Na, and Cl with no effects on K net transport in the in vivo rat perfused jejunum . The addition of ST, but not of ANP, evoked an increase of short circuit current in rat intestinal mucosa mounted in Ussing chambers . ST induced a significant increase in guanylate cyclase activity in intestinal homogenates, whereas ANP showed no effect . No binding sites for ANP were detected in basolateral or brush border membranes, nor in isolated enterocytes by a suction filtration technique . In conclusion, ANP acts as a short-lived intestinal secretagogue in the rat . Its mechanism of action is different from that of E . coli ST and appears to be indirect, since is not mediated by specific intestinal receptors and is not evident in vitro.

Cell Biophys, 1992 Aug-Dec, 21(1-3), 81 - 91
Recombinant human monoclonal antibodies . Basic principles of the immune system transferred to E . coli; Fuchs P et al.; To produce human monoclonal antibodies in bacteria, a gene repertoire of IgM variable regions was isolated from human peripheral B lymphocytes by the polymerase chain reaction . Alternatively, synthetic antibody genes with random hypervariable regions are being generated that may provide libraries of even higher complexity . For the selection of specific monoclonal antibodies from these libraries, we have developed two E . coli vector systems that facilitate the surface display of an antibody physically linked to its own gene . The phagemid pSEX encodes a fusion protein of an antigen binding domain (Fv-antibody) with the docking protein (pIII) of filamentous phages . Specific antibody genes can therefore be enriched by antigen affinity chromatography . The plasmid pAP1 encodes a fusion protein of an Fv-antibody with a bacterial cell-wall protein . Bacteria carrying this plasmid express functional Fv-antibodies tightly bound to their surface . This should enable the selection of single cells with a fluorescence-assisted cell sorter (FACS) using labeled antigen or by adsorption to immobilized antigen . These vectors permit three major principles of the antibody response to be mimicked in E . coli: 1 . Generation of a highly complex antibody repertoire; 2 . Clonal selection procedures for library screening; and 3 . The possibility of increasing a given affinity by repeated rounds of mutation and selection.

Cell Biophys, 1992 Aug-Dec, 21(1-3), 69 - 79
Regulated secretion and purification of recombinant antibodies in E . coli; Dubel S et al.; A plasmid for optimized protein expression of recombinant Fv antibodies (pOPE) in E . coli was used to express the variable domains of the murine monoclonal antibody HD39 specific for the human B-cell surface antigen CD22 . The production of Fv antibodies by pOPE can be regulated over a wide range by varying the IPTG concentration . Antibodies that can discriminate between secreted and nonsecreted Fv antibody fragments were used to show that secretion is the limiting step for the production of functional Fv antibodies . IPTG concentrations above 20 microM increased the total antibody production, but did not yield larger amounts of secreted Fv antibodies . The addition of five histidines to the C terminus facilitates an easy single-step enrichment procedure based on immobilized metal affinity chromatography.

FEBS Lett, 1992 Jul 28, 307(2), 173 - 6
Effect of tandemly repeated AGG triplets on the translation of CAT-mRNA in E . coli; Ivanov I et al.; It has been shown that tandems of rare arginine codons AGG have a strong inhibitory effect on translation of mRNA in E . coli {5} . This has been explained by the rate-limiting interaction of these codons with the less abundant tRNA(AGG) {6} . In this study tandemly repeated AGG triplets were introduced into the chloramphenicol acetyltransferase (CAT) gene either upstream of the initiation ATG codon or downstream of it (both in frame and out of frame) and the expression of the modified genes was investigated . We report that the addition of AGG clusters resulted in a substantial inhibitory effect on CAT gene expression independently of their localization in mRNA . This inhibitory effect is explained by a competition of the tandem AGGAGG with the natural Shine-Dalgarno (SD) sequence (consensus AAGGAGGU) for the 3'-end of the 16S small ribosomal RNA (rRNA).

FEBS Lett, 1992 Jul 20, 306(2-3), 234 - 8
Computational approach towards the three-dimensional structure of E . coli tyrosine aminotransferase; Jager J et al.; We present a new model for E . coli tyrosine aminotransferase based on the X-ray structures of the wild type and Val39Leu mutant of E . coli aspartate aminotransferase and computer simulation studies . Active site characteristics of the model are correlated with experimental observations on the specificity of these enzymes towards aromatic/dicarboxylic acid substrates.

Biochem Biophys Res Commun, 1992 Jul 15, 186(1), 143 - 9
Gene synthesis and expression in E . coli for pump, a human matrix metalloproteinase; Ye QZ et al.; The gene for PUMP (putative metalloproteinase), a human matrix metalloproteinase, was synthesized by a PCR-based method . The DNA fragment of 546 bases containing the PUMP gene was generated by overlap extension of six long oligonucleotides (length ranging from 101 to 116 bases) and subsequent amplification by two short terminal oligonucleotide primers (length from 20 to 48 bases) in one pot without using restriction and ligation enzymes . The synthetic gene was cloned into a T7 expression vector in two ways to express PUMP as a non-fusion protein . Both constructs showed high level expression in E . coli.

Nucleic Acids Res, 1992 Jul 11, 20(13), 3357 - 60
Site-specific dissection of E . coli chromosome by lambda terminase; Kotani H et al.; We have succeeded the targeted cleavage of chromosomes by lambda terminase that introduces double-strand cleavages in DNA recognizing the lambda cos sequence . When chromosomal DNAs of various Escherichia coli K-12 strains were subjected to terminase digestion, all were found to contain two common cleavage sites . Therefore, DNAs from lambda lysogens in which lambda DNA was inserted at different chromosomal sites were specifically cleaved at one more additional site . The two sites, termed ecos1 and ecos2, were mapped at approximately 35.1' and 12.7' of E . coli genetic map . The ecos1 and ecos2 sites were included in qin and qsr' regions, respectively . Therefore, the cleavage sites were associated with cryptic prophages . Sequences at the ecos1 and ecos2 sites showed 98% homology to the lambda cos sequence, indicating high fidelity of sequence recognition by the terminase . Since the strategy for integration of a DNA segment into chromosomal DNA through homologous recombination has been established, the dissection method that uses lambda terminase should be applicable for gene mapping as well as construction of macrophysical maps of larger genomes.

EMBO J, 1992 Jul, 11(7), 2633 - 41
E.coli polynucleotide phosphorylase expression is autoregulated through an RNase III-dependent mechanism; Robert-Le Meur M et al.; It has been previously shown that the pnp messenger RNAs are cleaved by RNase III at the 5' end and that these cleavages induce a rapid decay of these messengers . A translational fusion between pnp and lacZ was introduced into the chromosome of a delta lac strain to study the expression of pnp . In the presence of increased cellular concentrations of polynucleotide phosphorylase, the level of the hybrid beta-galactosidase is repressed, whereas the synthesis rate of the corresponding message is not significantly affected . In the absence of pnp, the level of the hybrid protein increases strongly . Thus, polynucleotide phosphorylase is post-transcriptionally autocontrolled . However, autocontrol is totally abolished in strains where the RNase III site on the pnp message has been deleted or in strains devoid of RNase III . These results suggest that polynucleotide phosphorylase requires RNase III cleavages to autoregulate the translation of its message . Other mutations in the ribosome binding site region support the hypothesis that this 3' to 5' processive enzyme could recognize a specific repressor binding site at the 5' end of pnp mRNA . Implications of these results on the mechanism of regulation and on messenger degradation are discussed.

J Gerontol, 1992 Jul, 47(4), B142 - 5
The association of E . coli peritonitis with an impaired and delayed fever response in senescent rats; Scarpace PJ et al.; Infection is one of the leading causes of death in elderly humans, and the importance of the early diagnosis of severe infection is undisputed . In the elderly a delay in diagnosis is often due to a reduced or absent fever . To understand more fully the pathogenesis of fever in senescence, we assessed the febrile response to E . coli peritonitis in 3-, 12-, and 24-month-old rats . Baseline temperatures were unchanged with age . Following infection with 1 x 10(8) CFU E . coli, a fever was evident in 2.8 h in the young, 3.9 h in the 12-month-old rats, and delayed until 5.8 h in the senescent rats . The magnitude of the fever was quantitatively less in the older rats compared with the two younger age groups throughout the time course of the fever . Because beta-adrenergic-mediated thermogenesis in brown adipose tissue has been implicated in the genesis of fever, we also assessed adenylate cyclase activity in this tissue . There was a progressive age-related decrease in both receptor- and postreceptor-stimulated adenylate cyclase activity . Our findings indicate there is both a delay in the onset of the fever and a reduced febrile response in the senescent rats following E . coli infection.

Klin Padiatr, 1992 Jul-Aug, 204(4), 274 - 6
{Clinical experiences with adrenaline as therapy and prevention of E . coli-L-asparaginase-induced anaphylaxis}; Schwinger W et al.; Management of E.-coli L-asparaginase induced anaphylaxis mainly consists of application of i.v . epinephrine but also in the withdrawal of this drug . Despite of this firm recommendation we have shown in 7 patients with E . coli L-asparaginase induced anaphylaxis, that further application of the drug is possible in the presence of a continuous epinephrine infusion (0.01-0.02 mg/kg) started one hour before and finished one hour after the concomittant infusion of the E.-coli L-asparaginase . In none of the patients there was a second event of anaphylaxis even though most of the patients still had to continue on E.-coli L-asparaginase with an average of more than 6 infusion.

Int J Food Microbiol, 1992 Jul, 16(3), 197 - 208
Evaluation of the MPN, Anderson-Baird-Parker, Petrifilm E . coli and Fluorocult ECD method for enumeration of Escherichia coli in foods of animal origin; Bredie WL et al.; Commercially available beta-D-glucuronidase (GUR) based methods, Petrifilm E . coli (PEC) and Fluorocult ECD (FECD), and ISO standard MPN and Anderson-Baird-Parker (ABP) procedures were evaluated for routine enumeration of E . coli in naturally contaminated foods of animal origin . The methods concerned were classifiable in a sequence of best qualities for: production, MPN > ABP = PEC = FECD; costs, FECD > ABP = PEC > MPN; time per measurement, ABP = PEC = FECD > MPN; practical use, PEC > FECD > ABP > MPN; detection at low contamination, MPN > ABP = PEC > FECD . The ABP and PEC method appeared useful for routine counting of E . coli in raw meat, poultry and meat products, whereas the MPN procedure turned out to be more sensitive, however, impractical and considerably more expensive . The FECD method was inexpensive although suitable for the enumeration of E . coli at higher contamination level (> 50 cfu/g) . The indole and MUG indicators both applied to demonstrate E . coli with the ABP or FECD method proved to be equal in specificity.

Biofizika, 1992 Jul-Aug, 37(4), 733 - 7
{Deviation from a Poisson distribution in a series of identical tests of E . coli cultures as a result of the effect of correlating factors of exo- and endogenous natures}; Gusev VA et al.; Vital cells number (VCN) in the sampling of E . coli populations was experimentally measured and distribution histograms were obtained . In most cases distributions show considerable deviation from Poisson model . VCN distribution histograms are polymodal, dispersion/arithmetical mean ratio may essentially differ from 1 . The more essential differences from Poisson distribution were observed for populations with the higher cell concentration . Computer simulation of the VCN histograms indicated that additional parameters (such as those describing cellular interaction of different nature and/or other factors that influence random behaviour of cells) should be introduced into Poisson model to explain observed variations in VCN distribution histograms.

Ultramicroscopy, 1992 Jul, 42-44 ( Pt B), 1173 - 80
Electrodeposition procedure of E . coli RNA polymerase onto gold and deposition of E . coli RNA polymerase onto mica for observation with scanning force microscopy; Keller RW et al.; Molecules of the transcriptional enzyme E . coli RNA polymerase (RNAP) have been deposited using three different deposition methods: (1) passive adsorption onto gold, (2) electrochemical adsorption onto gold and (3) adsorption onto mica . In all cases SFM imaging was straightforward and reliable, and surface coverage by the protein varied with deposition conditions as expected . To determine the nature of the electrochemical treatment on the gold substrate, cyclic voltammetry was performed with various chemical solutions . Finally, a comparison is made between the SFM images of RNAP obtained with these methods and STM images obtained earlier . Both STM and SFM show strikingly similar results; however, heights and widths of individual molecules differ.

Z Naturforsch {C}, 1992 Jul-Aug, 47(7-8), 621 - 7
Resonance effect of microwaves on the genome conformational state of E . coli cells; Belyaev IYa et al.; The effect of low intensity microwaves on the conformational state of the genome of X-irradiated E . coli cells was studied by the method of viscosity anomalous time dependencies . It has been established that within the ranges of 51.62-51.84 GHz and 41.25-41.50 GHz the frequency dependence of the observed effect has a resonance nature with a resonance half-width of the order of 100 MHz . The power dependence of the microwave effect within the range of 0.1-200 microW/cm2 has shown that a power density of 1 microW/cm2 is sufficient to suppress radiation-induced repair of the genome conformational state . The effect of microwave suppression of repair is well reproduced and does not depend on the sequence of cell exposure to X-rays and microwave radiation in the millimeter band . The results obtained indicate the role of the cell genome in the resonant interaction of cells with low intensity millimeter waves.

Indian J Pathol Microbiol, 1992 Jul, 35(3), 247 - 50
Photoprotection of ultraviolet light irradiated E . Coli . B/r cells; Bhatnagar D; Ultraviolet irradiated E . Coli . B/r cells recover from UV damage when the cells are kept in dark due to dark repair mechanism . Photoprotection by illumination of the cells in near UV light prior to the exposure to UV light increases the capacity of the cells to induce L-arabinose isomerase synthesis in response to inducer, L-arabinose . The survival of the cells is dependent on the UV dose . The increased synthesis of L-arabinose isomerase after photoprotection is due to the amount of cyclic AMP in the cells.

Cell, 1992 Jun 26, 69(7), 1171 - 80
ATP-dependent branch migration of Holliday junctions promoted by the RuvA and RuvB proteins of E . coli; Tsaneva IR et al.; The RuvA and RuvB proteins of E . coli, which are induced as part of the cellular response to DNA damage, act together to promote the branch migration of Holliday junctions . Addition of purified RuvA and RuvB to a RecA-mediated recombination reaction stimulates the rate of strand exchange and the formation of hetero-duplex DNA . Stimulation does not occur via interaction with RecA; instead, RuvA and RuvB act directly upon recombination intermediates (Holliday junctions) made by RecA . We show that RuvAB-mediated branch migration requires ATP and can bypass UV-induced DNA lesions . At high RuvB concentrations, the requirement for RuvA is overcome, indicating that the RuvB ATPase provides the motor force for branch migration . RuvA protein provides specificity by binding to the Holliday junction, thereby reducing the requirement for RuvB by 50-fold . The newly discovered biochemical properties of RuvA, RuvB, and RuvC are incorporated into a model for the postreplicational repair of DNA following UV irradiation.

Cell, 1992 Jun 26, 69(7), 1063 - 5
Movement and resolution of Holliday junctions by enzymes from E . coli; Taylor AF; RuvA and RuvB act together to move Holliday junctions . RuvC cleaves Holliday junctions and apparently acts in concert with RuvA and RuvB . RecG can substitute for RuvABC in the RecBCD pathway of recombination but not in the RecF pathway.

Eur J Biochem, 1992 Jun 15, 206(3), 767 - 73
Selenoprotein synthesis in E . coli . Purification and characterisation of the enzyme catalysing selenium activation; Ehrenreich A et al.; The product of the selD gene from Escherichia coli catalyses the formation of an activated selenium compound which is required for the synthesis of Sec-tRNA (Sec, selenocysteine) from Ser-tRNA and for the formation of the unusual nucleoside 5-methylaminomethyl-2-selenouridine in several tRNA species . selD was overexpressed in a T7 promoter/polymerase system and purified to apparent homogeneity . Purified SELD protein is a monomer of 37 kDa in its native state and catalyses a selenium-dependent ATP-cleavage reaction delivering AMP and releasing the beta-phosphate as orthophosphate . The gamma-phosphate group of ATP was not liberated in a form able to form a complex with molybdate . It was precluded that any putative covalent or non-covalent ligand of SELD not removed during purification participated in the reaction . In a double-labelling experiment employing {75Se}selenite plus dithiothreitol and {gamma-32P}ATP the 75Se and 32P radioactivities co-chromatographed on a poly(ethyleneimine)-cellulose column . No radioactivity originating from ATP eluted in this position when {alpha-32P}ATP or {beta-32P}ATP or {14C}ATP were offered as substrates . The results support the speculation that the product of SELD is a phosphoselenoate with the phosphate moiety derived phosphoselenoate from the gamma-phosphate group of ATP . The alpha,beta cleavage of ATP is also supported by the finding that neither adenosine 5'-{alpha,beta-methylene}triphosphate nor adenosine 5'-{beta,gamma-methylene}triphosphate served as substrates in the reaction.

FEBS Lett, 1992 Jun 15, 304(2-3), 216 - 20
Membrane-derived oligosaccharides (MDO's) promote closing of an E . coli porin channel; Delcour AH et al.; The outer membrane of Escherichia coli is a diffusion barrier for macromolecules, but allows the passage of small hydrophilic solutes through non-specific channels, the porins . Some electrophysiological studies find reconstituted porins in a mostly open state, while those done with the patch-clamp technique performed on live cells suggest that the vast majority of the native channels are closed . We present here current measurements through porins from reconstituted outer membrane, which demonstrate that bacterial metabolites, the MDO's, which bathe the periplasmic side of the outer membrane, induce the channels to close . These findings illustrate that the degree of openness of porins can be regulated by compounds naturally found in bacteria.

Zentralbl Bakteriol, 1992 Jun, 277(1), 22 - 7
Identification of aerobactin genes in clinical isolates of E . coli using a non-radioactive DNA probe; Graser Y et al.; A digoxigenin-labelled gene probe was used for the identification of aerobactin genes in 21 E . coli strains from clinical isolates by means of a colony hybridization test . The results were compared with those obtained by previous hybridization experiments with radiolabelled DNA probes as well as with a crossfeeding-assay . It could be demonstrated that only after an additional proteinase K treatment of the filters before hybridization and after a repeated blocking during the immunological detection procedure, specific hybridization signals could be observed.

Biochimie, 1992 Jun, 74(6), 581 - 4
Overproduction and purification of lysyl-tRNA synthetase encoded by the herC gene of E coli; Nakamura Y et al.; Lysyl-tRNA synthetases are synthesized from two distinct genes in E coli, lysS and lysU, but neither gene product has been purified distinctively by using overproducing systems . The lysS gene has been identified by a herC mutation which restores maintenance of the mutant ColE1 replicon . The herC gene product was overproduced by using a tac promoter fusion and purified to homogeneity . The purified HerC protein possesses a lysyl-tRNA synthetase activity as predicted by the sequence identity of herC to lysS . The procedure is useful for rapid mass-scale purification of lysyl-tRNA synthetase.

Biochem Biophys Res Commun, 1992 May 29, 185(1), 41 - 6
Expression of E . coli tag gene encoding 3-methyladenine glycosylase I in NIH-3T3 murine fibroblasts; Taverna P et al.; NIH-3T3 fibroblasts were co-transfected with pSV2neo and pSG5tag by the calcium-phosphate precipitation method . The stable integration of the tag gene sequence and its transcription was verified by Southern and Northern blot analysis . 3-Methyladenine glycosylase activity in pSG5tag transfected 3T3 cells was approximately 400 times higher than in cells transfected with the control plasmid pSG5 or in the parental cells and was inhibited by 3-methyladenine . Bacterial tag gene can thus be expressed in mammalian cells and the encoded enzyme is functionally active . These transfected cells could serve as an important tool to investigate the importance of the repair of N3-adenine as a mechanism of protection against the mutagenicity and cytotoxicity of alkylating agents.

Biochem Biophys Res Commun, 1992 May 15, 184(3), 1496 - 503
Similar-sized daughter-strand gaps are produced in the leading and lagging strands of DNA in UV-irradiated E . coli uvrA cells; Wang TC et al.; The nascent DNA synthesized after UV irradiation contained discontinuity, i.e., daughter-strand gaps . The sizes of these gaps produced in the leading and lagging strands of UV-irradiated Escherichia coli cells were determined by using strand-specific DNA probes . The DNA isolated from irradiated uvrA delta(lac-pro) cells was hybridized with the 32P-labeled single-stranded DNA probes . After digestion with S1 nuclease, the sizes of the bound radioactive DNA fragments were determined by electrophoresis in an alkaline agarose gel . It was found that the average size of gaps produced in the leading strand was about 0.12 kb, whereas those produced in the lagging strand was slightly smaller than 0.12 kb . No gaps larger than 0.5 kb were detected.

Nucleic Acids Res, 1992 May 11, 20(9), 2335 - 9
In vitro study of E.coli tRNA(Arg) and tRNA(Lys) identity elements; Tamura K et al.; Various tRNA transcripts were constructed to study the identity elements of E.coli tRNA(Arg) and tRNA(Lys) . Exchange of the anticodon of the major tRNA(Arg) from ACG to either CCG or CCU did not result in a significant loss of arginine acceptor activity, whereas not only that to UUU but also that to ACA or ACC decreased the activity . Base substitutions and deletion at A20 also impaired the arginine charging activity by over 50-fold . Arginine charging activity was introduced by either substitution of the anticodon from UAC to ACG in tRNA(Val) or from UUU to UCU in tRNA(Lys) . Only a single base substitution at the third position of tRNA(Trp) anticodon (CCA) from A to G also gave rise to arginine charging activity, which was elevated to a comparable level to that of the tRNA(Arg) transcript by an additional A20 insertion . Base substitutions of the major tRNA(Arg) at the discriminator position into pyrimidines led to a decrease by factors of three to four . These data show that the third letter of the anticodon G36 or U36 besides the second letter C35 and the A20 in the variable pocket is responsible for the arginine acceptor identity, to which the discriminator base A73 or G73 contributes in an auxiliary fashion . In contrast to the arginine system, the transcript with the wild-type tRNA(Lys) sequence showed only 140-fold lower lysine charging activity than the native tRNA(Lys), suggesting the involvement of base modifications in recognition . Replacement of the anticodon UUU with not only UCU and UAC but also UUA and UUC seriously affected the lysine acceptor activity, and those with GUU and UUG also decreased by factors of 17 and 5, respectively . Introduction of UUU into the anticodons conferred lysine charging activity upon both tRNA(Val) and tRNA(Arg) . Substitution of the discriminator base A73 by any of the other bases decreased the lysine acceptor activity by a factor of ten . These results indicate the involvements of all the three bases of the anticodon and A at the discriminator position in lysine specific aminoacylation.

J Appl Physiol, 1992 May, 72(5), 1895 - 901
Role of tissue hypoxia as the mechanism of lactic acidosis during E . coli endotoxemia; Hurtado FJ et al.; We compared the hemodynamic and metabolic alterations produced in rabbits by similar decreases in cardiac output created by inflating a balloon placed in the right ventricle (n = 6) with those produced by an intravenous bolus of Escherichia coli lipopolysaccharide (LPS; SEP group; n = 6) . We measured O2 consumption (VO2), O2 transport (TO2), and O2 extraction ratio (ERO2) for the whole animal and also for the left hindlimb . Both groups experienced similar decreases in cardiac output, systemic TO2, and VO2 and similar increases in ERO2 . For the hindlimb, TO2 was similar, but VO2 and ERO2 were lower for the SEP group 30 min after LPS administration (P less than 0.05); however, this difference disappeared during the remainder of the experiment . Arterial lactate concentration was greater (P less than 0.05) for the SEP group . There were no differences in skeletal muscle PO2, measured with a multiwire surface electrode, or in cardiac and skeletal muscle concentrations of high-energy phosphates . We hypothesize that a direct effect of LPS on cellular metabolism may have resulted in greater arterial lactate concentration for the SEP group.

Infect Immun, 1992 May, 60(5), 2092 - 5
Enteroaggregative Escherichia coli strains secrete a heat-labile toxin antigenically related to E . coli hemolysin; Baldwin TJ et al.; A protein toxin of approximately 120,000 Da secreted by nonhemolytic enteroaggregative Escherichia coli strains cross-reacted in Western blots (immunoblots) with antibodies raised against the C-terminal region of E . coli hemolysin . Treatment of HEp-2 cells with enteroaggregative E . coli or culture supernatants caused elevation of intracellular calcium and stimulated calcium-dependent protein phosphorylation.

Ital J Biochem, 1992 May-Jun, 41(3), 200 - 11
Fructose 1,6-bisphosphate-activated pyruvate kinase from E . coli: ligand promoted conformational changes; Speranza ML et al.; The ligand-dependent susceptibility to heat inactivation and to tryptic digestion and the intrinsic fluorescence of the fructose 1,6-bisphosphate-activated pyruvate kinase from Escherichia coli were investigated in the absence and in the presence of physiological ligands . With respect to the enzyme alone, binding of the allosteric activator fructose 1,6-bisphosphate makes the protein sensitive to tryptic attack and thermolabile, while binding of phosphoenolpyruvate and Mg2+, but not of either ligand separately, induces in the enzyme a highly thermostable conformation, the attainment of which does not require an ordered binding sequence of the two ligands . The apparent loosening of the enzyme structure induced by fructose bisphosphate suggests that the activation it exerts at low phosphoenolpyruvate concentration might be due to an increased accessibility of substrate to the active site.

Bioorg Khim, 1992 May, 18(5), 660 - 70
{Expression of a synthetic gene for human interleukin-4 in E . coli cells . Preparation of a biologically active protein}; Batchikova NV et al.; Expression E . coli plasmid were constructed in which the human interleukin-4 (hIL4) synthetic gene is controlled by tac promoter . The expression level of the gene depends on the distance between RBS and the initial codon ATG, with the maximal production in case of the nine base pair distance . The recombinant protein, accumulated in the inclusion bodies, was solubilized, renaturated, and purified to homogeneous, biologically active preparation, the yield being 2 mg/g wet cells.

Bioorg Khim, 1992 May, 18(5), 623 - 34
{p26--a calcium binding protein from photoreceptor cells in the bovine retina: primary structure and expression in E . coli}; Kutuzov MA et al.; The primary structure of the bovine retinal calcium binding protein P26 has been determined by the parallel analysis of the protein and the corresponding cDNA . This protein is identical to recovering and shares 59% homology with visinin, a cone specific calcium binding protein from chicken retina . P26 was expressed in E . coli as a fusion protein and, after purification by affinity chromatography on IgG-Sepharose 6, cleaved off with enteropeptidase.

Sci China B, 1992 May, 35(5), 585 - 91
Molecular cloning of a new variant of human papillomavirus type 6 isolated from a Chinese woman and expression of its L1 gene in E . coli--molecular cloning & expression of HPV6 gene; Shu LL et al.; A human papillomavirus genome DNA of 7.9 kb from a Chinese woman with genital condyloma acuminata was cloned in BamHI site of pAT153 . According to the results obtained from Southern blotting, restriction mapping as well as partial DNA sequencing, the isolated genome (HPV6BV) had obvious variance and was referred to as a new variant of HPV6 found in China the first time . HPV6BV L1 gene was successfully expressed in E . coli as a fusion protein with pUR288 . The beta-galactosidase/L1 fusion protein reacted with both beta-galactosidase antiserum and HPV antibody using Western blot technique . The E . coli-produced fusion protein, possessing HPV antigenicity, may provide a reagent for clinical diagnosis and epidemiological survey.

FEBS Lett, 1992 Apr 27, 301(3), 243 - 5
Expression of the N-terminal domain of dystrophin in E . coli and demonstration of binding to F-actin; Way M et al.; The N-terminal head domain of human dystrophin has been expressed in soluble form and high yield in E . coli, allowing us to test the previously unconfirmed assumption that dystrophin binds actin . DMD246, the first 246 amino acid residues of dystrophin, binds F-actin in a strongly co-operative manner with a Hill constant of 3.5, but does not bind G-actin . Dystrophin heads are thus functionally competent actin-binding proteins . This result opens the way to identifying critical residues in the actin-binding site and encourages us that the other domains of dystrophin might also be treated as functionally autonomous modules, accessible to a similar approach.

Biochem Biophys Res Commun, 1992 Apr 15, 184(1), 478 - 84
Identity determinants of E . coli threonine tRNA; Hasegawa T et al.; To investigate the identity determinants of E . coli threonine tRNA, various transcripts were prepared by in vitro transcription system with T7 RNA polymerase . Substitutions of the anticodon second letter G35 and the third letter U36 to other nucleotides led to a remarkable decrease of threonine charging activity . Charging experiments with a series of anticodon-deletion transcripts also suggest the importance of the G35U36 sequence . A mutation at either the G1-C72 or C2-G71 base pair in the acceptor stem seriously affected the threonine charging activity . These results indicate that the second and third positions of the anticodon and the first and second base pairs in the acceptor stem are the recognition sites of E . coli tRNA(THR) for threonyl-tRNA synthetase . Discriminator base, A73, is not involved in threonine charging activity.

Nucleic Acids Res, 1992 Apr 11, 20(7), 1663 - 8
DNA mismatch correction by Very Short Patch repair may have altered the abundance of oligonucleotides in the E . coli genome; Bhagwat AS et al.; A base mismatch correction process in E . coli K-12 called Very Short Patch (VSP) repair corrects T:G mismatches to C:G when found in certain sequence contexts . Two of the substrate mismatches (5'-CTWGG/3'-GGW'CC; W = A or T) occur in the context of cytosine methylation in DNA and reduce the mutagenic effects of 5-methylcytosine deamination to thymine . However, VSP repair is also known to repair T:G mismatches that are not expected to arise from 5-methylcytosine deamination (example--CTAG/GGT-C) . In these cases, if the original base pair were a T:A, VSP repair would cause a T to C transition . We have carried out Markov chain analysis of an E . coli sequence database to determine if repair at the latter class of sites has altered the abundance of the relevant tetranucleotides . The results are consistent with the prediction that VSP repair would tend to deplete the genome of the 'T' containing sequences (example--CTAG), while enriching it for the corresponding 'C' containing sequences (CCAG) . Further, they provide an explanation for the known scarcity of CTAG containing restriction enzyme sites among the genomes of enteric bacteria and identify VSP repair as a force in shaping the sequence composition of bacterial genomes.

Nucleic Acids Res, 1992 Apr 11, 20(7), 1657 - 62
Statistical evaluation and biological interpretation of non-random abundance in the E . coli K-12 genome of tetra- and pentanucleotide sequences related to VSP DNA mismatch repair; Merkl R et al.; The abundance of all tetra- and pentanucleotide sequences is calculated for a set of DNA sequence data comprising 767,393 nucleotides of the E . coli K-12 genome . Observed frequencies are compared to those expected from a Markov chain prediction algorithm . Systematic and extreme non-random representations are found for special sets of sequences . These are interpreted as arising from incorporation of a 2'-deoxyguanosine residue opposite thymidine during replication which, in special sequence contexts, leads to a T/G mismatch that is simultaneously substrate for two competing DNA mismatch repair systems: the mutHLS and the VSP pathway . Processing by the former leads to error correction, by the latter to mutation fixation . The significance of the latter process, as demonstrated here, makes it unlikely that VSP repair has evolved mainly as a mutation avoidance mechanism . It is proposed that in E . coli K-12, VSP repair, together with DNA cytosine methylation, constitutes a mutagenesis/recombination system capable of promoting gene-conversion-like unidirectional transfer of short stretches of DNA sequence.

Nucleic Acids Res, 1992 Apr 11, 20(7), 1579 - 85
Purification and characterization of the MspI DNA methyltransferase cloned and overexpressed in E . coli; Dubey AK et al.; The MspI restriction-modification system, which recognizes the sequence 5'-CCGG-3', has been previously cloned and sequenced (1) . We subcloned the methyltransferase gene (M.MspI) downstream of the ptac promoter in the multicopy vector pUC119 and overexpressed it in E . coli . Upon induction with IPTG, M.MspI constitutes more than 10% of cellular protein . A scheme has been devised to purify large amounts of biologically active M.MspI to apparent homogeneity from these overexpressing E . coli cells . Approximately 0.8 mg of pure M.MspI per gram of cells (wet weight) can be obtained . The apparent molecular weight of M.MspI is 49 kD, by SDS gel electrophoresis and 48-54 kD by gel filtration . At low concentrations (less than 0.4 mg/ml), the methyltransferase is a monomer in solution but at higher concentrations (greater than 3.0 mg/ml) it exists predominantly as a dimer . Polyclonal antibodies raised against M.MspI cross-react with the DNA-methyltransferases of several other restriction-modification systems.

Nucleic Acids Res, 1992 Apr 11, 20(7), 1553 - 8
Preferential binding of E.coli histone-like protein HU alpha to negatively supercoiled DNA; Shindo H et al.; Binding specificity of histone-like HU alpha protein to supercoiled DNA was examined by gel retardation assay and chemical probing with OsO4 . The latter method was proved to be a unique means for detecting torsional tension restrained in supercoiled plasmid in the presence of HU alpha . It was shown that HU alpha protein has preferential affinity to negatively supercoiled DNA relative to relaxed, nicked and linearized DNAs . There were two modes for binding of HU alpha to the supercoiled DNA: one was the binding associated with topological changes in DNA and the other was relatively strong binding, probably specific to certain particular structures of DNA . It was suggested that HU in vivo interacts preferentially with the regions deformed under torsional stress or with the metabolically active regions along DNA.

Nucleic Acids Res, 1992 Apr 11, 20(7), 1567 - 71
Mutations in E.coli 16s rRNA that enhance and decrease the activity of a suppressor tRNA; Prescott CD et al.; The in vivo expression of mutations constructed within helix 34 of 16S rRNA has been examined together with a nonsense tRNA suppressor for their action at stop codons . The data revealed two novel results: in contrast to previous findings, some of the rRNA mutations affected suppression at UAA and UAG nonsense codons . Secondly, both an increase and a decrease in the efficiency of the suppressor tRNA were induced by the mutations . This is the first report that rRNA mutations decreased the efficiency of a suppressor tRNA . The data are interpreted as there being competition between the two release factors (RF-1 and RF-2) for an overlapping domain and that helix 34 influences this interaction.

Dtsch Tierarztl Wochenschr, 1992 Apr, 99(4), 140 - 3
Influence of E . coli infection on the disposition kinetic of nalidixic acid in broiler chickens; Atef M et al.; The pharmacokinetic data of nalidixic acid were investigated in normal and E . coli infected chickens . The highest serum concentration were reached after 2 hours with t0.5 (ab) of (1.706 +/- 0.1 min in normal and 2.030 +/- 0.11 min in diseased) and (1.72 +/- 0.11 min in normal and 1.416 +/- 0.044 in diseased chickens) following oral and intramuscular administration, respectively . The elimination half-life t0.5 (beta) were (2.514 in normal and 2.35 hr in diseased) and (2.567 hr in normal and 2.672 hr in diseased) respectively . Following intravenous injection the kinetic of nalidixic acid followed two compartments open model with t0.5 of (6.27 and 9.15 hr), Vd (0.45 and 0.79 L/kg), Cltot (8.86 and 13.32 ml/kg/min) in normal and E . coli infected chickens, respectively . Administration of nalidixic acid twice daily for 5 successive days in a dose level of 25 mg/kg b . wt . by oral and intramuscular routes showed a cumulative behaviour.

Mol Cell Probes, 1992 Apr, 6(2), 93 - 9
DNA probe analysis of diarrhoeagenic Escherichia coli: detection of EAF-positive isolates of traditional enteropathogenic E . coli serotypes among Bangladeshi paediatric diarrhoea patients; Strockbine NA et al.; Escherichia coli isolates from all surveillance patients less than or equal to 20 months of age seen for diarrhoea at the Dhaka Clinical Treatment Facility of the International Centre for Diarrhoeal Disease Research, Bangladesh between March 1 and August 31, 1988, were collected and hybridized with DNA probes to assess the potential importance of diarrhoeagenic E . coli among paediatric patients in Bangladesh . Of 396 patients evaluated, 18% were infected with enteropathogenic E . coli (EPEC) adherence factor (EAF)-positive E . coli, 23% were infected with enterotoxigenic E . coli (ETEC), 9% were infected with Shiga-like toxin-positive E . coli, and 13% were infected with diffuse adhesiveness-positive E . coli . None were infected with enteroinvasive E . coli . Ten percent of patients were colonized with more than one type of potential diarrhoeagenic E . coli . The majority of EAF-positive isolates were of traditional EPEC O:H serotypes . Although this was not a case-control study, the large number of EPEC and ETEC, which are recognized enteric pathogens, suggests these organisms are important causes of diarrhoeal diseases in this pediatric population.

Protein Eng, 1992 Apr, 5(3), 273 - 8
Insertion of a disulfide-containing neurotoxin into E . coli alkaline phosphatase: the hybrid retains both biological activities; Gillet D et al.; We have inserted a disulfide-containing snake neurotoxin into the N-terminal end of Escherichia coli alkaline phosphatase, between residues +6 and +7 of the mature enzyme . For this purpose, we have designed a cloning and expression vector which allows insertion of foreign DNA between the corresponding codons, and visual selection of the desired recombinant clones upon recovery of phosphatase activity . The hybrid protein is exported to the bacterial periplasm, the alkaline phosphatase signal peptide is correctly processed, and both domains are functionally conformed . The phosphatase domain displays catalytic activity, and the inserted toxin is able to bind to its biological target, the nicotinic acetylcholine receptor . The hybrid molecule is remarkably stable and resistant to proteolysis . Crude periplasmic extract containing the hybrid can be used as a tracer-containing reagent in competitive enzymo-immuno and enzymo-receptor assays . We propose to use the system described in this paper for fast preparation of properly folded disulfide-containing enzymatic probes.

Hybridoma, 1992 Apr, 11(2), 191 - 201
High level expression in E . coli of an alternate reading frame of pS2 mRNA that encodes a mimotope of human breast epithelial mucin tandem repeat; Larocca D et al.; The high molecular weight mucin found in human milk fat globule and on the surface of mammary and other epithelial cells contains a 20 amino acid tandem repeat sequence that is highly immunogenic . We have immunoscreened lambda gt11 cDNA expression libraries from MCF7 cells and lactating breast tissue with 5 anti-mucin monoclonal antibodies . We isolated a group of cDNA clones that had the repeat sequence (HB11-2, HB11-6, HB11-10) and a group that had little or no homology with the repeat sequence (NP4, NP5, HB11-4) . A fusion protein produced by NP4 bound preferentially BrE2, while HB11-4 bound only BrE2 and BrE3, NP5 produced a fusion protein that bound only Mc5 and not the other MAbs . Sequencing of the NP5 cDNA revealed it to be distinct from the mucin sequence and instead to have 97% identity with the estrogen induced transcript, pS2 . An alternate reading frame was translated by the lambda gt11 fusion gene yielding a 44 amino acid protein having no homology with pS2 protein . Only a short region of homology (5 amino acids) with the breast mucin tandem repeat was found which was shown to be a mimotope for the Mc5 epitope on the breast mucin . High level expression of the NP5 cDNA was achieved by subcloning it into pEX2 . The NP5 fusion protein has been useful for developing an assay for the presence of mucin derived antigen in patient serum.

Mutat Res, 1992 Apr, 266(2), 205 - 13
Bio-antimutagenic activities of vitamin B6 in E . coli and mouse peripheral blood cells; Shimoi K et al.; Pyridoxal (PL) and pyridoxal 5'-phosphate (PLP) showed a marked bio-antimutagenic effect on UV-induced mutagenesis in E . coli B/r WP2, but not in the DNA excision repair-deficient strain WP2suvrA under the condition where no cellular toxicity was observed . No delay in the first cell division was seen on post-treatment with PL after UV irradiation . PL reduced not only UV- but 4-nitroquinoline-1-oxide-induced mutation, while it was ineffective in N-methyl-N'-nitro-N-nitrosoguanidine- or gamma-ray-treated cells . These results suggest that PL promotes DNA excision repair directly or indirectly and the decrease in the amount of unrepaired DNA damage might cause the reduction of UV-induced mutations in E . coli B/r WP2 . In addition to the above observation, PLP reduced the frequency of mitomycin C- (2 mg/kg, i.p.) induced micronuclei in mouse peripheral blood cells . Simultaneous or subsequent oral administration of PLP (25 mg/kg) decreased the frequency of micronucleated peripheral reticulocytes.

Protein Eng, 1992 Apr, 5(3), 279 - 83
Overproduction, preparation of monoclonal antibodies and purification of E . coli asparagine synthetase A; Hinchman SK et al.; In order to explore the structure--function relationship of the Escherichia coli asparagine synthetase A it was necessary to devise a system for overexpression of the gene and purification of the gene product . The E . coli asparagine synthetase A structural gene was fused to the 3' end of the human carbonic anhydrase II structural gene and overexpressed in E . coli . The gene product, a 66 kDa fusion protein, which exhibited asparagine synthetase activity, was purified in a single step by affinity chromatography and used as the antigen for the production of monoclonal antibodies . The monoclonal antibodies were screened by ELISA . Colonies were chosen which were positive for purified fusion protein and negative for purified human carbonic anhydrase II . The E . coli asparagine synthetase A gene was then overexpressed and the gene product was used without purification for the final screen . The antibodies selected were used for immunoaffinity chromatography to purify the recombinant overexpressed E . coli asparagine synthetase A . Thus, a procedure is now available so that asparagine synthetase A can be purified to homogeneity in a single step.

Biochem Biophys Res Commun, 1992 Mar 31, 183(3), 1238 - 46
A lung type prostaglandin F synthase is expressed in bovine liver: cDNA sequence and expression in E . coli; Kuchinke W et al.; Using the cDNA of bovine lung prostaglandin F synthase (EC 1.1.1.2) as a probe, we isolated a clone from a bovine liver cDNA library which differed in only eleven nucleotides from the probe . The corresponding protein contained three amino acid substitutions, including a leucine residue which is conserved throughout all aldo-keto reductases . We inserted the liver cDNA into expression vector pUC19 and expressed the recombinant liver enzyme in E.coli . The purified liver enzyme reduced prostaglandin H2 as well as prostaglandin D2 and various carbonyl compounds . The high relative activity against prostaglandin H2 in combination with a high Km value for prostaglandin D2 identified this liver enzyme as a lung type prostaglandin F synthase . However, the binding constant for NADPH of the liver enzyme was 3.5 fold higher than that of lung prostaglandin F synthase.

Biochem Biophys Res Commun, 1992 Mar 31, 183(3), 925 - 30
Expression of MBP-HCV NS1/E2 fusion protein in E . coli and detection of anti-NS1/E2 antibody in type C chronic liver disease; Mita E et al.; To characterize the putative NS1/E2 (non-structural protein 1/envelope 2) domain of HCV (hepatitis C virus), we expressed the hydrophilic three-quarters of this domain in a form of MBP (maltose binding protein) fusions in Escherichia coli . When we checked the positive frequency of antibody to this fusion protein, 17% of patients with type C chronic liver disease had this antibody . However, they were all positive for HCV-RNA in sera . These results suggest that the appearance of anti-NS1/E2 antibody does not serve as evidence of viral clearance.

Biochim Biophys Acta, 1992 Mar 27, 1120(1), 87 - 96
Purification and cellular localization of wild type and mutated dihydrolipoyltransacetylases from Azotobacter vinelandii and Escherichia coli expressed in E . coli; Schulze E et al.; Wild type dihydrolipoyltransacetylase(E2p)-components from the pyruvate dehydrogenase complex of A . vinelandii or E . coli, and mutants of A . vinelandii E2p with stepwise deletions of the lipoyl domains or the alanine- and proline-rich region between the binding and the catalytic domain have been overexpressed in E . coli TG2 . The high expression of A . vinelandii wild type E2p (20% of cellular protein) and of a mutant enzyme with two lipoyl domains changed the properties of the inner bacterial membrane . This resulted in a solubilization of A . vinelandii E2p after degradation of the outer membrane by lysozyme without any contamination by E . coli pyruvate dehydrogenase complex (PDC) or other high-molecular-weight contaminants . The same effect could be detected for A . vinelandii E2o, an E2 which contains only one lipoyl domain, whereas almost no solubilization of A . vinelandii E2p with one lipoyl domain or of E2p consisting only of the binding and catalytic domain was found . Partial or complete deletion of the alanine- and proline-rich sequence between the binding and the catalytic domain did also decrease the solubilization of the E2p-mutants after lysozyme treatment . Immunocytochemical experiments on E . coli TG2 cells expressing A . vinelandii wild type E2p indicated that the enzyme was present as a soluble protein in the cytoplasm . In contrast, overexpressed A . vinelandii E2p with deletion of all three lipoyl domains and E . coli wild type E2p aggregated intracellularly . The solubilization by lysozyme is therefore ascribed to excluded volume effects leading to changes in the properties of the inner bacterial membrane.

FEBS Lett, 1992 Mar 23, 300(1), 18 - 20
The affinity of the Klenow fragment of E . coli DNA-polymerase 1 to primers containing bases noncomplementary to the template and hairpin-like elements; Ljach MV et al.; The Km and Vmax values for a set of primers: d(pT)n (pC) (pT)m (n = 3-9, m = 0-7) and d(pT)4 (pCpG)k (pT)4 (k = 1-5) have been estimated . Poly(dA) was used as a template . The number of complementary bases from the 3' end to a noncomplementary ones was shown to determine the efficiency of interaction of d(pT)n (pC) (pT)m with the Klenow fragment . Oligonucleotides d(pT)4 (pCpG)k (pT)4, in solution forming duplexes containing hairpin-like elements, show a higher affinity to the enzyme than control d(pT)4, d(pT)8 and d(pT)n (pC) (pT)m primers . For example, the Km value (1.1 nM) for d(pT)4 (pCpG)5 (pT)4 is about 14,000 and 200 times lower than those for d(pT)4 and d(pT)8, respectively . Possible reasons for such an abnormally high affinity of the above primers are discussed.

Biochem Biophys Res Commun, 1992 Mar 16, 183(2), 774 - 80
Reactivation of denatured fungal glucose 6-phosphate dehydrogenase and E . coli alkaline phosphatase with E . coli ribosome; Das B et al.; Fungal glucose 6-phosphate dehydrogenase and E . coli alkaline phosphatase were denatured either by physical or by chemical means . In vitro reconstitution of these denatured enzymes was assisted by 70S E . coli ribosome, as shown by the recovery of their catalytic competence . Almost total recovery of the activities of completely inactivated enzymes was obtained when 70S ribosome was present at about equimolar concentration with the enzyme molecules at 37C and 50C, respectively.

Cell, 1992 Mar 6, 68(5), 989 - 94
Requirement for E . coli NusG protein in factor-dependent transcription termination; Sullivan SL et al.; The 21 kd NusG protein is essential for E . coli viability . Cells depleted for NusG were defective for factor-dependent transcription termination . Rho-induced polarity in the gal operon and the Rho-dependent lambda tR1 and lambda tL1 terminators were suppressed in NusG-deficient cells . NusG depletion inactivated the phage HK022 Nun termination factor . In contrast, the factor-independent lambda tl terminator was fully active in NusG-depleted cells and could be suppressed by phage lambda N function.

Biochem Int, 1992 Mar, 26(3), 537 - 43
E . coli gpt gene expression effects on K562 human leukemia cell proliferation and erythroid differentiation altered by mycophenolic acid; Kamano H et al.; Inosine monophosphate dehydrogenase (IMPDH, EC 1.1.1.205) inhibitors including mycophenolic acid (MPA) were reported to induce K562 human leukemia cells to differentiate into erythroid cells . A shuttle vector plasmid pMSG containing E . coli xanthine-guanine phosphoribosyl transferase (Eco gpt) gene was transfected into K562 cells (K562-pMSG cells) to investigate the role of IMPDH in both K562 cell proliferation and erythroid differentiation . Eco gpt provides K562 cells with an additional salvage pathway for GMP production from xanthine . In the presence of xanthine, K562-pMSG cells continued to proliferate and did not differentiate to erythroid cells regardless of the presence of MPA, but they discontinued proliferating and differentiated into the erythroid lineage in the absence of xanthine . Proliferation and differentiation of control K562 cells into erythroid cells were suppressed by MPA regardless of the presence or absence of xanthine . Addition of guanosine maintained the proliferation and blocked the erythroid differentiation of K562 and K562-pMSG cells induced by MPA even in the absence of xanthine . These data indicate that a decrease in the activity of IMPDH and a subsequent decline in the concentration of guanine nucleotides caused by MPA resulted in the induction of the erythroid differentiation and discontinuation of the proliferation of K562 cells.

J Public Health Med, 1992 Mar, 14(1), 78 - 83
A restaurant-associated outbreak of E . coli O157 infection; Marsh J et al.; An outbreak of haemorrhagic colitis due to Escherichia coli O157 and associated with a restaurant in Lothian, occurred in September 1990 . There were 16 symptomatic cases, four of whom (all children) required dialysis . Notable features of the outbreak were the wide range of incubation periods (1-14 days), the occurrence of secondary spread through asymptomatic carriers and the prolonged period (at least seven days) during which the restaurant appears to have been the source of infection . Despite careful investigation, no single source within the restaurant was identified . The implications for public health are discussed.

EMBO J, 1992 Mar, 11(3), 1075 - 83
The E.coli fis promoter is subject to stringent control and autoregulation; Ninnemann O et al.; The DNA binding protein FIS is involved in processes like site specific DNA inversion, lambda excision and stimulation of stable RNA synthesis in Escherichia coli . The amount of FIS protein is subject to dramatic changes during growth . We demonstrate that fis is part of an operon with one ORF of unknown function preceding the fis gene . Regulation of fis synthesis occurs at the transcriptional level . Within 15 min after nutritional upshift a large burst of fis mRNA is produced which levels off when cells begin to grow . By mutational analysis using promoter-lacZ fusions we demonstrate that the fis promoter is autoregulated by FIS . Growth phase regulation of the fis promoter depends on the presence of a GC motif downstream of the -10 region . We show that the fis promoter is subject to stringent control and discuss this unusual feature with respect to the known and putative functions FIS serves in E . coli.

Bioorg Khim, 1992 Mar, 18(3), 391 - 7
{Recombinant interleukin-3 expression system in E . coli}; Lutsenko SV et al.; Plasmid pTOTE2IL3 (III) has been constructed, expressing an artificial human interleukin-3 (hIL3) gene under conditions of the induced protein biosynthesis . Levels of the recombinant protein synthesis have been compared in several E . coli strains containing expression plasmids pTE2IL3 (I) (constitutive biosynthesis) and (III) (induced biosynthesis) . Optimal combinations of the expression plasmids and the bacterial strains are of importance . A simple and effective method has been elaborated for isolation, purification and renaturation of the recombinant protein accumulated in inclusion bodies.

Appl Microbiol Biotechnol, 1992 Mar, 36(6), 745 - 7
Expression in E . coli of the gene encoding phenylalanine ammonia-lyase from Rhodosporidium toruloides; Orum H et al.; The active sites of the enzyme phenylalanine ammonia-lyase (Pal) from Rhodosporidium toruloides contains a dehydroalanine residue that is believed to be essential for catalytic activity . Furthermore, the dehydroalanine is believed to be added post-translationally as part of a prosthetic group covalently attached to the enzyme . Perhaps for this reason no attempts to produce Pal in foreign host cells have been reported . We have inserted the entire uninterupted pal gene from R . toruloides into the Escherichia coli expression vector pKK 223-3 . E . coli cells containing this vector synthesize a protein of the expected size, and extracts prepared from these cells contain a Pal-like activity . The potential implications of this finding are discussed.

Protein Sci, 1992 Mar, 1(3), 310 - 21
NMR structure of oxidized Escherichia coli glutaredoxin: comparison with reduced E . coli glutaredoxin and functionally related proteins; Xia TH et al.; The determination of the NMR structure of oxidized Escherichia coli glutaredoxin in aqueous solution is described, and comparisons of this structure with that of reduced E . coli glutaredoxin and the related proteins E . coli thioredoxin and T4 glutaredoxin are presented . Based on nearly complete sequence-specific 1H-NMR assignments, 804 nuclear Overhauser enhancement distance constraints and 74 dihedral angle constraints were obtained as the input for the structure calculations, for which the distance geometry program DIANA was used followed by simulated annealing with the program X-PLOR . The molecular architecture of oxidized glutaredoxin is made up of three helices and a four-stranded beta-sheet . The three-dimensional structures of oxidized and the recently described reduced glutaredoxin are very similar . Quantitative analysis of the exchange rates of 34 slowly exchanging amide protons from corresponding series of two-dimensional {15N,1H}-correlated spectra of oxidized and reduced glutaredoxin showed close agreement, indicating almost identical hydrogen-bonding patterns . Nonetheless, differences in local dynamics involving residues near the active site and the C-terminal alpha-helix were clearly manifested . Comparison of the structure of E . coli glutaredoxin with those of T4 glutaredoxin and E . coli thioredoxin showed that all three proteins have a similar overall polypeptide fold . An area of the protein surface at the active site containing Arg 8, Cys 11, Pro 12, Tyr 13, Ile 38, Thr 58, Val 59, Pro 60, Gly 71, Tyr 72, and Thr 73 is proposed as a possible site for interaction with other proteins, in particular ribonucleotide reductase . It was found that this area corresponds to previously proposed interaction sites in T4 glutaredoxin and E . coli thioredoxin . The solvent-accessible surface area at the active site of E . coli glutaredoxin showed a general trend to increase upon reduction . Only the sulfhydryl group of Cys 11 is exposed to the solvent, whereas that of Cys 14 is buried and solvent inaccessible.

Transgenic Res, 1992 Mar, 1(2), 63 - 70
Plant endogenous beta-glucuronidase activity: how to avoid interference with the use of the E . coli beta-glucuronidase as a reporter gene in transgenic plants; Alwen A et al.; We have detected a plant beta-glucuronidase activity, present in several tissues and organs of plant species belonging to different families . The fluorimetric beta-glucuronidase assay was used to partially characterize this activity in post-ribosomal supernatants of tobacco leaves . The tobacco activity is very stable at low temperatures, but quickly inactivated above 45 degrees C . It is relatively resistant to proteases and insensitive to -SH group reagents and to ionic conditions . It does not require, nor is it inhibited by, divalent cations . Although these properties are shared by the Escherichia coli beta-glucuronidase, the two activities can be distinguished by: (i) their different sensitivity to the specific inhibitor saccharic acid-1,4-lactone; (ii) their different thermal stability (iii) their different pH optima (5.0 for the plant activity and close to neutral for the bacterial enzyme) . Therefore, under appropriate experimental conditions, it should be possible to assay the E . coli beta-glucuronidase in transgenic plants without interference from the endogenous plant activity.

Biochim Biophys Acta, 1992 Feb 13, 1119(1), 74 - 80
Purification and properties of human glucose-6-phosphate dehydrogenase made in E . coli; Bautista JM et al.; The cDNA for the X-chromosome encoded human glucose-6-phosphate dehydrogenase (G6PD) has been expressed in E . coli and the enzyme purified to homogeneity, using a simple one-step fractionation on 2'5'-ADP-Sepharose . By selecting one of several different expression vectors and by optimizing culture conditions a yield of more than 10 mg of pure enzyme per liter of culture is obtained reproducibly . When the recombinant enzyme and authentic G6PD purified from normal human red cells were compared, they proved to be indistinguishable by the following criteria: electrophoretic mobility in both native and denaturing conditions, the Km values for glucose 6-phosphate and NADP and the Ki value for NADPH . The recombinant enzyme, unlike the red cell enzyme, retained 100% activity when stored at 4 degrees C for over 1 year.

Nucleic Acids Res, 1992 Feb 11, 20(3), 389 - 94
DNA containing a chemically reduced apurinic site is a high affinity ligand for the E . coli formamidopyrimidine-DNA glycosylase; Castaing B et al.; The E . coli Formamidopyrimidine-DNA Glycosylase (FPG protein), a monomeric DNA repair enzyme of 30.2 kDa, was purified to homogeneity in large quantities . The FPG protein excises imidazole ring-opened purines and 8-hydroxyguanine residues from DNA . Besides DNA glycosylase activity, the FPG protein is endowed with an EDTA-resistant activity which nicks DNA at apurinic/apyrimidic sites (AP sites) . In contrast, DNAs containing chemically reduced AP sites are not incised by the FPG protein . However, the DNA glycosylase activity of the FPG protein is strongly inhibited in the presence of a purified synthetic 24 base-pair double-stranded oligonucleotide which contains a single apurinic site transformed chemically through borohydride reduction into a ring-opened deoxyribose derivative . The ability of the FPG protein to form a complex with this synthetically modified DNA was studied by electrophoresis in non-denaturing polyacrylamide gels . The FPG protein specifically binds the double-stranded oligonucleotide containing an apurinic site previously reduced in the presence of sodium borohydride . The complex was identified as a single retardation band on non-denaturing polyacrylamide gel electrophoresis . Complex formation is reversible and an apparent dissociation constant, KDapp, of 2.6 x 10(-10) M was determined . In contrast, no such retardation band was obtained between the FPG protein and double-stranded DNA containing an intact apurinic site or single-stranded DNA containing either an intact or a reduced apurinic site.

FEBS Lett, 1992 Feb 3, 297(1-2), 179 - 82
X-ray studies reveal lanthanide binding sites at the A/B5 interface of E . coli heat labile enterotoxin; Sixma TK et al.; The crystal structure determination of heat labile enterotoxin (LT) bound to two different lanthanide ions, erbium and samarium, revealed two distinct ion binding sites in the interface of the A subunit and the B pentamer of the toxin . One of the interface sites is conserved in the very similar cholera toxin sequence . These sites may be potential calcium binding sites . Erbium and samarium binding causes a change in the structure of LT: a rotation of the A1 subunit of up to two degrees relative to the B pentamer.

Int J Biochem, 1992 Feb, 24(2), 235 - 42
Isolation, biochemical characterization and crystallization of the p15gag proteinase of myeloblastosis associated virus expressed in E . coli; Pichova I et al.; 1 . The p15gag proteinase responsible for the processing of the polyprotein precursor of the myeloblastosis associated virus was obtained by a recombinant technique in an E . coli expression system . The massive expression of the intentionally truncated precursor (Pr25lac-delta gag) was accompanied by its structurally correct processing . 2 . Three procedures for the purification of the recombinant proteinase from both the cytoplasmic fraction and the inclusion bodies were developed . 3 . The purified proteinase was compared with the authentic proteinase isolated from MAV virions by N-terminal sequence analysis and amino acid analysis, molecular weight determination, reverse-phase HPLC and FPLC elution profiles, electrophoretic mobility and isoelectric point determination, and activity assays with proteins and synthetic substrates . The identity of both enzymes was shown . 3 . Contrary to reported data, the amino acid sequence of the p15gag proteinase differs from the sequence of the homologous Rous sarcoma virus proteinase in one residue only, as follows from cDNA sequencing . 4 . Crystallization of the proteinase from a citrate-phosphate buffer at pH 5.6 afforded hexagonal crystals which diffracted well as 2.3 A without deterioration.

Biochem Int, 1992 Feb, 26(1), 43 - 50
Chemiluminescence response of phagocytes in E . coli induced experimental ascending pyelonephritis; Gupta A et al.; The mechanism of tissue injury at the cellular level by following the chemiluminescence response of various phagocytes in E . coli induced experimental pyelonephritis in mice was investigated . There was a marked increase in the capacity of various phagocytic cells viz; renal neutrophils and macrophages peritoneal macrophages, blood monocytes and neutrophils to produce reactive oxygens species through the respiratory burst activity as monitored by the chemiluminescence response . The chemiluminescence response was observed to be increased significantly (p less than 0.001) with increasing days post infection in all phagocytic cells . However, the quantity of total reactive oxygen species produced per million cells was much more in the renal and peritoneal macrophages as compared to blood monocytes and neutrophils . The peak chemiluminescence response time was observed to be decreased from 4 to 2 minutes with the progression of the diseases . The implications of these findings have been discussed.

Bioessays, 1992 Feb, 14(2), 105 - 11
Accessory protein function in the DNA polymerase III holoenzyme from E . coli; O'Donnell M; DNA polymerases which duplicate cellular chromosomes are multiprotein complexes . The individual functions of the many proteins required to duplicate a chromosome are not fully understood . The multiprotein complex which duplicates the Escherichia coli chromosome, DNA polymerase III holoenzyme (holoenzyme), contains a DNA polymerase subunit and nine accessory proteins . This report summarizes our current understanding of the individual functions of the accessory proteins within the holoenzyme, lending insight into why a chromosomal replicase needs such a complex structure.

Acta Chem Scand, 1992 Feb, 46(2), 186 - 93
Synthesis of deoxy derivatives of lactose and their hydrolysis by beta-galactosidase from E . coli; Bock K et al.; Methyl 2-deoxy-alpha-lactoside, methyl 3-deoxy-beta-lactoside, 1,5-anhydro-4-O-beta-D-galactopyranosyl-D-glucitol and the 2-deoxy and 2,3-dideoxy derivatives of 1,5-anhydro-4-O-beta-D-galactopyranosyl-D-glucitol have been synthesized by deoxygenation of lactose derivatives at appropriate positions . Cyclohexyl beta-D-galactopyranoside has also been synthesized . All derivatives proved to be substrates for the enzyme beta-galactosidase from E . coli, but the rate of hydrolysis of the substrate analogues was strongly dependent on the nature of the aglycone.

Zhonghua Wai Ke Za Zhi, 1992 Feb, 30(2), 115 - 6, 126
{E coli-induced idiopathic portal hypertension in rabbits . An experimental study}; Zhang MJ; An experimental model of portal hypertension was induced in healthy rabbits by intraportal injection of E coli or a mixture of E coli and rabbit anti-E coli sera . In all established models, there were significant elevation of portal vein pressure, increase of spleen weight, decrease of whole blood cells count, infiltration of inflammatory cells and fibrosis in intrahepatic portal area, decrease of the number and discontinuation of intrahepatic portal vein branch on portography with gross normal appearance of the liver . Hence this model was similar to that seen in human idiopathic portal hypertension (IPH) in many aspects . We believe that bacterial toxin and the abnormal immune reaction due to repeated stimulation of bacterial antigen may play a role in the pathogenesis of human IPH.

Nature, 1992 Jan 23, 355(6358), 318 - 25
The structure of the E . coli recA protein monomer and polymer; Story RM et al.; The crystal structure of the recA protein from Escherichia coli at 2.3-A resolution reveals a major domain that binds ADP and probably single- and double-stranded DNA . Two smaller subdomains at the N and C termini protrude from the protein and respectively stabilize a 6(1) helical polymer of protein subunits and interpolymer bundles . This polymer structure closely resembles that of recA/DNA filaments determined by electron microscopy . Mutations in recA protein that enhance coprotease, DNA-binding and/or strand-exchange activity can be explained if the interpolymer interactions in the crystal reflect a regulatory mechanism in vivo.

Arch Virol, 1992, 122(3-4), 391 - 7
Reactivity of a recombinant rubella E1 antigen expressed in E . coli; Londesborough P et al.; The E1 nucleic acid sequence of rubella virus strain Judith (RJ) has been cloned into an E . coli expression vector LB03 . The reactivity of the expressed unglycosylated antigen (E1J) was compared with its glycosylated counterpart in native virus (RJ) using rabbit and human sera . Rabbit antisera raised against RJ and E1J reacted differently with wild type, RJ (laboratory strain) and RA27/3 (vaccine virus) strains in a kinetic neutralisation test . Reciprocally, human post RA27/3 vaccination sera were also found to differ from post infection or post re-infection sera in their reactivity with RJ and E1J antigens . Our observations suggest that E1, in the conformation adopted in the RA27/3 virion may have unique antigenic properties.

Zhonghua Yu Fang Yi Xue Za Zhi, 1992 Jan, 26(1), 23 - 4
{The effects of chlorine disinfection on the resistance of E . coli in water}; Wu ZC et al.; Under defined conditions E . coli were subjected to repeated chlorine disinfections 10 times . The survival E . coli at 30 s (A10), and the survival E . coli at 10 min (B10) had no difference in resistance to chlorine to their original strain (A0) . However, the compound E . coli (C10) survived at various contact time showed an increased resistance than their original strain (A10), the degree of increased resistance varying with different conditions of disinfection . E . coli C1(0) lost its increased resistance after it has been passaged 10 times on nutrient agar.

Schweiz Arch Tierheilkd, 1992, 134(1), 31 - 7
{Detection with nonradioactively labeled probes of the toxin genes of different E . coli pathotypes from swine}; Boss P et al.; We tested hemolytic E . coli from 86 pigs with edema disease or colidiarrhea . They were tested serologically and with nonradioactive digoxigenin-dUTP labelled probes for the presence of enterotoxin or Shiga-like-toxin genes . By slide-agglutination we detected 38 cases with E . coli O149:K88, 28 with E . coli O139:82B and 20 with E . coli O141 . E . coli of serogroup O149:K88 isolated from diarrheic pigs, reacted with the probes for LT and STb genes . Edema disease E . coli O139:82B reacted with the SLTII probe . E . coli O141, isolated from colidiarrhea or edema disease showed a diversity of toxin gene patterns . All the E . coli O141 from diarrheic pigs reacted with the probes for LT and STap in addition to SLTII . No strains isolated from pigs with edema disease possessed any of these enterotoxin genes . Gene probe technique confirmed the serological method as useful tool for diagnosing E . coli O149:K88 and O139:82B as ETEC or VTEC, respectively . On the other hand only the demonstration of toxin genes with probes could explain the pathological findings in the pigs shedding E . coli of serogroup O141.

Yi Chuan Xue Bao, 1992, 19(2), 186 - 91
{Effect of ribosome binding site sequence on the expression level of HBcAg in E . coli}; Li M et al.; The recombinant (pKL series) Plasmids showing different levels of HBcAg antigenicity in ELISA were constructed by inserting the HBcAg gene which was randomly deleted at the non-coding region by Bal-31 exonuclease into plasmid vector pKK223-3 containing tac promoter and SD sequence . SDS-PAGE and Western blot experiments indicated that molecular weight of the HBcAg protein generated by these positive clones was 21000D . Three plasmids with high, moderate and low HBcAg expression level were sequenced and the distance between SD sequence and HBcAg gene ATG codon was 12, 13 and 19bp respectively . Computer analysis of secondary structure of the ribosome binding sites on RNA transcripts also revealed energy and structure differences between the low and high level expression plasmids, suggesting the importance of this distance and the mRNA structure to gene expression.

J Comp Physiol {B}, 1992, 162(4), 327 - 30
Effect of ambient temperature and E . coli endotoxin upon the plasma iron level in wild house mice in winter season; Tegowska E et al.; The effects of ambient temperatures of 10 degrees C and 30 degrees C and of E . coli endotoxin on brain temperature and plasma iron level were investigated in unrestrained wild house mice, Mus musculus . In control animals (i.p . saline-injected) exposed to cold environment the brain temperature decreased and plasma iron levels were lower than those observed under thermoneutral conditions (30 degrees C) . Animals injected i.p . with endotoxin (0.5 micrograms.kg-1) and placed at 30 degrees C showed a drop in plasma iron level during the fever episode . The results provide strong evidence for a relationship between brain temperature and plasma iron level in control mice under thermoneutral conditions, and show that during cold exposure or after injection of endotoxin, there is no linear correlation between brain temperature and plasma iron . Moreover, it was found that cold stress influences plasma iron level and that this influence is not mediated by changes in brain temperature.

Folia Microbiol (Praha), 1992, 37(1), 24 - 30
Immunological quantification of RecA protein in cell extracts of E . coli after exposure to chemical mutagens or UV radiation; Fridrichova I et al.; Increased synthesis of RecA protein is induced in E . coli cells after their damage, the rate of synthesis being dependent on the extent of DNA alterations . The level of the RecA protein was determined in E . coli cell extracts after damage induced by NQO, MNNG, MMC, NAL or UV radiation, using competitive enzyme-linked immunosorbent assay (ELISA) . Purified E . coli RecA protein and rabbit monospecific polyclonal antibodies against it were prepared for the quantitative assay . The level of the RecA protein was increased after treatment with all mutagens . Contrary to other induced proteins, the synthesis of the RecA protein increased within 30 min after damage with UV radiation at a relatively slow rate . The ELISA method made it possible to determine 0.5-50 ng of the RecA protein in bacterial extracts . The method can be employed as an auxiliary test for DNA damage determination and also in studied concerning the role of the RecA protein in repair processes.

Biull Eksp Biol Med, 1992 Jan, 113(1), 70 - 2
{Expression of human LSB 32/67 KD gene in E . coli and analysis of its interactions with laminin}; Siianova EIu; Earlier we have cloned cDNA coding for a polypeptide that reacts with monoclonal antibodies specific for some cytoskeleton structures . This gene is homologous to the laminin receptor 67 KD . However, cDNA suffices only for a polypeptide of 32 Da, far smaller than the 67 rDa laminin receptor . We have constructed a vector that produces the fusion protein LBP-TrpE in the bacterial strain CAG-456 . Our studies show that hybrid protein LBP-TrpE is able to interact with laminin . This result was confirmed by using following methods: immunoblotting, "ELISA" and affinity chromatography on laminin.

C R Acad Sci III, 1992, 315(6), 221 - 4
{Recombinant colorimetric antibodies: genetic construction and production in E . coli}; Ducancel F et al.; We constructed an expression vector comprising a transcription unit composed of (1) the gene of alkaline phosphatase from E . coli in which we inserted a DNA fragment encoding the VH and CH domains of an IgG2A; (2) a DNA fragment encoding the light chain of the same immunoglobulin . Bacteria, E . coli were transformed with the plasmid, and hence it produced a periplasmic and chimaeric protein having both the alkaline phosphatase enzymatic activity and the immunoglobulin binding function . The hybrid protein mimics a bivalent antibody whose Fc part is replaced by a dimer of alkaline phosphatase . Recombinant colorimetric antibodies offer interesting potentialities for future developments in the field of immuno-enzymatic diagnosis.

Hum Mutat, 1992, 1(2), 113 - 23
Screening for mutations by expressing patient cDNA segments in E . coli: homocystinuria due to cystathionine beta-synthase deficiency; Kozich V et al.; Deficiency of cystathionine beta-synthase (CBS) causes the most common form of inherited homocystinuria . We developed a simple CBS expression system in E . coli to screen for pathogenic mutations in affected individuals . Portions of patient cDNAs were amplified by PCR and used to replace the corresponding segments of normal human CBS cDNA in the bacterial expression plasmid pHCS3 . Hybrid CBS was expressed in E . coli and the segments of patient's cDNA which extinguished CBS activity were sequenced to identify the mutation . The first study of a pyridoxine-responsive patient using this screen revealed that of the clones which contained either the middle or the 3'-portion of his cDNA, about half were devoid of catalytic activity . Subsequent sequencing of the affected segments confirmed a compound heterozygosity for a maternal T833-->C transition (I278T) and for a paternal A-->C transversion in the intron 11 splice acceptor . The latter mutation leads to an in-frame deletion of exon 12 (nt 1224-1358, amino acids W408 to G453) . This bacterial expression system proved to be a rapid screening method for localizing pathogenic mutations in CBS, allowing us to sequence the affected portions of mutant cDNA within 7-10 days of harvesting cultured fibroblasts.

J Nutr Sci Vitaminol (Tokyo), 1992, Spec No, 216 - 9
Aspartate aminotransferase of E . coli: effects of site-directed mutagenesis on substrate recognition; Kagamiyama H; R292 is crucial for both the binding and the catalysis of the transamination reaction of dicarboxylic acid substrates . Substitution of R292 to uncharged residues greatly enhanced the catalytic efficiency of transamination of neutral amino acids without any effect on the binding . Residues at position 292 may not be involved in recognition of the neutral side chain . The indole ring of W140 not only regulates the rotational movement of the coenzyme ring during catalysis, but it also may be involved in binding the carboxyl side chain of dicarboxylic substrates . The phenol group of Y70 is essential for the stabilization of the transition states with all substrates . Benzene ring at position 70 is necessary to recognize the glutamate-2-oxoglutarate substrate pair.

Med Dosw Mikrobiol, 1992, 44(1-2), 13 - 9
{occurrence of mannose- resistant adhesins MRHA in E.Coli strains isolated from children with diarrhea}; Andziak J; The study was aimed at determination of the frequency of occurrence of mannose-resistant adhesins in E . coli strains isolated from children with diarrhoea . It was also of interest whether their presence is associated with the serological type or other virulence factors . The material used in this study consisted of 1022 strains of E . coli (EPEC, ETEC and EIEC) and 3431 isolates from sick children and 960 from healthy children (non-EPEC-ETEC-EIEC) . Enterotoxigenicity and entero-invasiveness of strains was evaluated by biological tests performed on animals and in tissue culture . Production of MRHA adhesins was determined by the test of mannose-resistant active hemagglutination, and of colonization factors antigens CFA by application of agglutination and agar gel immunodiffusion tests . Most frequently MRHA adhesins were produced by ETEC strains-80% of strains . All of them appeared to be a colonization factor antigen CFA/I . EPEC strains produced various MRHA adhesins only by 12.6% of strains . Production of MRHA adhesins by EIEC strains was not detected . Frequency of occurrence of MRHA adhesins in E . coli strains which were non-EPEC-ETEC-EIEC was dependent from the isolation source . MRHA adhesins were most frequently found in strains isolated from sporadic cases of light diarrhoea in ambulatory treated children (49%), much less among isolates from children hospitalized because of severe diarrhoea (33%), and from healthy children in 9% of isolates only . These results may indicate the potential role of MRHA adhesins in pathogenesis of diarrhoea in children.

C R Acad Sci III, 1992, 315(11), 403 - 7
Repression of the E . coli lactose operon by cooperation between two individually unproductive "half-operator" sites; Amouyal M et al.; Two remote and weak lac regressor binding sites can be used jointly to repress the synthesis of beta-galactosidase in E . coli, while they cannot separately . When this result is discussed in reference to the various modes of cooperation between the sites, it supports with a new approach a model implying the simultaneous binding of lac repressor to both sites with the formation of a DNA loop . In connection with this point, we present a new strategy to detect cooperative interactions in vivo, based on the asymmetry of the DNA binding site, formally equivalent here to a half-site, heterodimerization of the protein, and influence of orientation of the sites on repression at short and long distance.

Nucleic Acids Symp Ser, 1992, (27), 143 - 4
In vitro study of E . coli tRNA identity elements; Tamura T et al.; Various tRNA transcripts were constructed to study the identity elements of E . coli tRNAs (Arg, Lys, Ala, Trp, Thr, Gly, Ser, Asn, Cys, His) . Anticodon are involved in the identity elements in these tRNA species except the case of tRNA(Ala) and tRNA(Ser) . Especially, the second and third positions of the anticodon are the recognition sites of E . coli tRNA(Arg), tRNA(Lys) and tRNA(Thr) for their cognate aminoacyl-tRNA synthetases . Discriminator base is an identity determinant of the above examined tRNAs except tRNA(Thr) and tRNA(Ser) . In some cases, acceptor stem (Thr, Gly, His) and variable pocket (Arg, Ala) are considered to be the recognition elements.

Arch Virol Suppl, 1992, 4, 119 - 21
HBc and HBe specificity of monoclonal antibodies against complete and truncated HBc proteins from E . coli; Korec E et al.; We have prepared and used monoclonal antibodies against various populations of full-length and truncated hepatitis B core proteins in order to distinguish between epitopes of HBcAg and HBeAg . Our results show that various epitopes are specific for the different proteins . Certain epitopes, however, are ubiquitous to HBc/e proteins and these are probably exposed on the surface of HBc particles.

Biochem Biophys Res Commun, 1991 Dec 31, 181(3), 1572 - 9
Tandem translation of E . coli initiation factor IF2 beta: purification and characterization in vitro of two active forms; Nyengaard NR et al.; Two forms of E . coli initiation factor IF2, IF2 alpha and IF2 beta, have been known for several years . Both forms are products of the gene infB with translational initiation at codon 1 (AUG) and codon 158 (GUG) in the same reading frame . In this work we demonstrate that IF2 beta exists in two forms, IF2 beta and IF2 beta' with initiation codons 158 (GUG) and 165 (AUG) and molecular masses of 79.7 kDa and 78.8 kDa respectively . We have recently described a fast purification method for IF2 alpha, using an FPLC procedure consisting of ion-exchange liquid chromatography on Q Sepharose HP, Mono Q and Mono S . After the Mono Q step, an apparently homogeneous IF2 beta was observed when analyzed by SDS-PAGE . However the chromatography on Mono S results in the elution of two peaks containing IF2 beta . The N-terminal amino acid sequence of the two proteins identified the first peak to be IF2 beta and the second as a protein which we term IF2 beta' starting seven residues downstream at the AUG codon 165 . The activity in vitro of the two purified forms of IF2 beta was tested by measuring the stimulation of binding of the initiator fMet-tRNA(fMet) to 70S ribosomes in the presence of GTP and poly(A,U,G) as messenger-RNA . In this assay no difference in activity is detected.

Carbohydr Res, 1991 Dec 30, 222, 245 - 53
Structure of the capsular polysaccharide (K98 antigen) of E . coli O7:K98:H6; Hahne M et al.; The capsular polysaccharide (K98 antigen) of E . coli O7:K98:H6 contains rhamnose, glucuronic acid, and acetate in the molar ratios 3:1:0.6 . Methylation analysis, oligosaccharide analysis, and 1D- and 2D-n.m.r . spectroscopy revealed the polysaccharide to be a glucuronic acid-substituted rhamnan with the structure {formula; see text} Of the 3-linked rhamnose residues, approximately 60% are O-acetylated at position 2.

Nature, 1991 Dec 19-26, 354(6354), 506 - 10
Formation and resolution of recombination intermediates by E . coli RecA and RuvC proteins; Dunderdale HJ et al.; The recombination of DNA molecules has been reconstituted in vitro using two purified enzymes from Escherichia coli . RecA protein catalyses homologous pairing and strand exchange reactions to form intermediate DNA structures that are acted upon by RuvC . The newly identified RuvC protein resolves the intermediates by specific endonucleolytic cleavage to produce recombinant DNA molecules.

FEBS Lett, 1991 Dec 16, 295(1-3), 171 - 5
A plant metallothionein produced in E . coli; Kille P et al.; A metallothionein cDNA was generated from pea (Pisum sativum L.) roots, amplified by PCR and inserted into a plasmid for expression in E . coli . Purification of the resultant product generated 3 pools of cadmium-containing material after DEAE-cellulose chromatography . The amino acid composition of each was in excellent agreement with that predicted for pea metallothionein . A cadmium content of approximately 6 g.atoms per mole of protein was estimated . N-terminal sequence analysis revealed that the recombinant molecule had been proteolysed within the extended region linking the 2 cysteine-rich (putative) metal-binding regions . The significance of these findings in terms of the protein folding/targeting of the molecule are considered.

FEBS Lett, 1991 Dec 16, 295(1-3), 227 - 9
Transcript hairpin structures are not required for RNA polymerase pausing in the gene encoding the E . coli RNase P RNA, M1 RNA; Ramamoorthy R et al.; Strong pauses at nucleotides +118 and +121 relative to the transcriptional start occur during in vitro transcription of the E . coli rnpB gene encoding the catalytic M1 RNA subunit of Ribonuclease P . These pauses are immediately downstream of 2 phylogenetically conserved stem-loop structures in the RNA . In the present work, single-base changes which disrupted Watson-Crick base-pairing in the hairpins were introduced into rnpB . Transcription studies in vitro with these modified templates revealed that none of the nucleotide changes predicted to increase or decrease the stability of the first hairpin significantly affected the pause half-lives . A mutation which disrupted the second hairpin increased the pause half-life 2-fold . The data suggest that the upstream stem and loop structures in the transcript are not involved in the pausing event.

FEBS Lett, 1991 Dec 16, 295(1-3), 123 - 6
The active site structure of E . coli HPII catalase . Evidence favoring coordination of a tyrosinate proximal ligand to the chlorin iron; Dawson JH et al.; E . coli produces 2 catalases known as HPI and HPII . While the heme prosthetic group of the HPII catalase has been established to be a dihydroporphyrin or chlorin, the identity of the proximal ligand to the iron has not been addressed . The magnetic circular dichroism (MCD) spectrum of native ferric HPII catalase is very similar to those of a 5-coordinate phenolate-ligated ferric chlorin complex, a model for tyrosinate proximal ligation, as well as of chlorin-reconstituted ferric horseradish peroxidase, a model for 5-coordinate histidine ligation . However, further MCD comparisons of chlorin-reconstituted myoglobin with parallel ligand-bound adducts of the catalase clearly rule out histidine ligation in the latter, leaving tyrosinate as the best candidate for the proximal ligand.

Bioorg Khim, 1991 Dec, 17(12), 1649 - 54
{Isolation of recombinant interleukin-3 produced by E . coli}; Lutsenko SV et al.; A synthetic gene coding for human interleukin-3 (hIL3) was cloned in the plasmid pTE2IL3, the gene expression being controlled by the phage fd PVIII promotor and the phage T7 gene 10 translational enhancer . Under constitutive biosynthesis conditions in E . coli, the accumulation of recombinant hIL3 (in the inclusion bodies) was up to 30-40% of the total cell protein . An effective procedure of the hIL3 isolation is suggested . The hIL3 was solubilized in 5 M guanidinium chloride, renaturated and purified to homogeneity by a single chromatographic step . The protein's yield was 34 mg/g wet cells . The isolated hIL3 showed a specific biological activity.

Immunology, 1991 Dec, 74(4), 680 - 4
Conglutinin exhibits a complement-dependent enhancement of the respiratory burst of phagocytes stimulated by E . coli; Friis P et al.; Conglutinin is a mammalian C-type lectin which shows anti-bacterial activity when tested in vivo and in vitro . This study concerns the effect of conglutinin on the respiratory burst of murine spleen cells, using a chemiluminescence assay for measurement of generated reactive oxygen metabolites . Conglutinin enhances, in a dose-dependent manner, the respiratory burst of spleen cells stimulated with serum-opsonized Escherichia coli . The enhancement was only demonstrable in the presence of a functional complement system . The conglutinin-mediated enhancement of the respiratory burst was inhibited in the presence of a N-acetyl-D-glucosamine, D-mannose and N-acetyl-D-mannosamine, monosaccharides reported to inhibit conglutinin-binding to zymosan and the complement factor iC3b . On the other hand, N-acetyl-D-galactosamine was non-inhibitory.

Circ Shock, 1991 Dec, 35(4), 215 - 22
Total body blood volume redistribution in porcine E . coli septic shock: effect of volume loading, dobutamine, and norepinephrine; Schneider AJ et al.; The purpose of the present study was to determine the effect of volume loading alone (CONTR) vs . volume loading in combination with dobutamine (DOBU) or norepinephrine (NOR) on total body blood volume distribution in septic shock . After instrumentation, injection of in vitro labelled 99mTc red blood cells, and baseline measurements, anesthetized, ventilated pigs (n = 21) received 3-4.10(8).kg-1 live E . coli bacteria intravenously . Images of thorax, abdomen, and hindlimb were obtained by using a gamma camera simultaneously with hemodynamic measurements . E . coli infusion resulted in a decrease in arterial pressure, ventricular filling pressures, and cardiac output with a concomitant increase in pulmonary arterial pressure . Blood volume was redistributed from the heart, lungs, spleen, abdomen, and leg to the liver . After randomization, the CONTR group (I, n = 5) was subjected to volume loading, and treatment groups (each n = 8) received volume loading in combination with DOBU (group II, 5-10 micrograms/kg/min) or NOR (group III, 0.25-0.50 micrograms/kg/min) . As compared to volume loading alone, DOBU and NOR increased cardiac output but only NOR restored arterial pressure . Volume loading increased blood volume in all regions studied; however, it was unequally distributed amongst organs with a preference for the liver . Neither DOBU nor NOR influenced the partitioning of the infused volume between organs . However, NOR prevented pooling of blood in the leg.

Biochimie, 1991 Dec, 73(12), 1473 - 9
Regulation of the heat shock response in E coli: involvement of positive and negative cis-acting elements in translation control of sigma 32 synthesis; Nagai H et al.; When cells of E coli are transferred from 30 to 42 degrees C, the cellular level of sigma 32 (rpoH gene product) increases transiently, resulting in increased transcription of a set of heat shock genes . Both increased synthesis and increased stability of sigma 32 contribute to transient accumulation of sigma 32 . Evidence suggests that synthesis of sigma 32 is enhanced primarily at the level of rpoH translation . We have constructed and examined the expression of deletion derivatives of rpoH-lacZ gene fusion at 30 degrees C and after shift to 42 degrees C . It was revealed that two cis-acting sequences within the rpoH coding region are involved in thermal regulation of fusion protein synthesis . One region immediately downstream of the initiation codon is required for high level expression, whereas the other internal region is involved in repression at low temperature . Thus, these regions act as positive and negative cis-elements in thermal regulation of rpoH translation . The rpoH mRNA secondary structure model suggesting an interplay between the two regions has been proposed to account for the temperature-induced sigma 32 synthesis as a primary cellular response to the heat shock stress.

Nucleic Acids Res, 1991 Nov 25, 19(22), 6197 - 203
High yield purification of active transcription factor IIIA expressed in E . coli; Del Rio S et al.; Transcription factor IIIA (TFIIIA), a sequence-specific DNA-binding protein from Xenopus laevis, is a zinc finger protein required for transcription of 5S rRNA genes by RNA polymerase III . We describe the purification and characterization of recombinant TFIIIA (recTFIIIA) expressed in E . coli . RecTFIIIA was purified to greater than 95% homogeneity at a yield of 2-3 milligrams per liter of bacterial culture . This purified protein protects the internal control region of a 5S rRNA gene from DNase I digestion, yielding footprints on both strands identical to those produced by the ovarian protein (ovaTFIIIA) . Quantitative analysis of binding data from gel retardation assays yielded a KD of about 0.4 nM for TFIIIA from either source . Using a quantitative TFIIIA-dependent in vitro transcription assay, we found that recTFIIIA is equivalent to ovaTFIIIA in supporting transcription of 5S rRNA genes . We conclude that recTFIIIA is functionally indistinguishable from the protein purified from Xenopus ovaries, and can be readily obtained in pure form and large quantity.

Nucleic Acids Res, 1991 Nov 11, 19(21), 5915 - 22
The N-terminal part of the E.coli DNA binding protein FIS is essential for stimulating site-specific DNA inversion but is not required for specific DNA binding; Koch C et al.; FIS protein is involved in several different cellular processes stimulating site-specific recombination in phages Mu and lambda as well as transcription of stable RNA operons in E.coli . We have performed a mutational analysis of fis and provide genetic and biochemical evidence that a truncated version of FIS lacking the N-terminal region is sufficient for specific DNA binding and for stimulating lambda excision . These mutants also retain their ability to autoregulate fis gene expression . Such mutant proteins, however, cannot stimulate the enhancer dependent DNA inversion reaction.

FEBS Lett, 1991 Nov 4, 292(1-2), 48 - 52
Expression of the pea gene PSMTA in E . coli . Metal-binding properties of the expressed protein; Tommey AM et al.; The pea (Pisum sativum L.) gene PSMTA has an ORF encoding a predicted protein with sequence similarity to class I metallothioneins (MTS) . To examine the metal-binding properties of the PSMTA protein it has been expressed in E . coli as a carboxyterminal extension of glutathione-S-transferase (GST) . Metal ions were associated with the expressed protein when purified from lysates of E . coli grown in metal supplemented media . The pH of half-dissociation of Zn, Cd and Cu ions from the recombinant fusion protein was determined to be 5.35, 3.95 and 1.45 respectively, compared with equivalent estimates of 4.50, 3.00 and 1.80 for equine renal MT.

J Med Microbiol, 1991 Nov, 35(5), 278 - 83
Identification of enteropathogenic Escherichia coli isolated in Britain as enteroaggregative or as members of a subclass of attaching-and-effacing E . coli not hybridising with the EPEC adherence-factor probe; Scotland SM et al.; Strains of Escherichia coli from sporadic cases of diarrhoea and belonging to serotypes O44:H18, O55:H7, O111ab:H21, O111ab:H25 or O126:H27 were examined for virulence properties . With the exception of O111ab:H25 these are considered to be classical enteropathogenic E . coli (EPEC) serotypes . The strains had been isolated in Britain from the faeces of children less than 3 years old . Of the serotypes examined, 7 of 13 O44:H18 strains, all of 10 O111ab:H21 strains and 13 of 21 O126:H27 strains belonged to the enteroaggregative class of E . coli (EAggEC) that attached to HEp-2 cells in the characteristic aggregative pattern and hybridised with the EAggEC probe . They also caused mannose-resistant haemagglutination of rat erythrocytes, a property which may be a useful marker for their identification . Strains of O44:H18 with similar properties were also isolated from three small outbreaks in Britain, one of which involved elderly patients . EAggEC have not been considered previously as aetiological agents of diarrhoea in developed countries and have rarely been reported as belonging to EPEC serotypes . All 15 O55:H7 strains and seven of eight O111ab:H25 strains were also considered to be potentially diarrhoeagenic as they gave localised attachment (LA) to HEp-2 cells that resulted in a positive fluorescence actin-staining test . This test is considered to correlate with the attaching-and-effacing virulence mechanisms of EPEC in vivo.(ABSTRACT TRUNCATED AT 250 WORDS)

Biofizika, 1991 Nov-Dec, 36(6), 1037 - 42
{Use of the principle of reverse problems for detecting processes, occurring in cells suspended in culture . II . Distribution of glutathione, SH groups, and optical density in the E . coli cell cycle}; Kazarian LS et al.; A method was proposed for calculating the content of intracellular components during the cell cycle of an individual cell . The principle of reverse problems was used in the mathematical model proposed . The model allowed us to calculate changes of intracellular parameters of an individual cell from corresponding parameters measured in the whole culture . Optical density, total SH-group and glutathione content in synchronous culture of E . coli were the parameters studied . The proposed method may be applied for both synchronous and asynchronous cell cultures.

Neuron, 1991 Nov, 7(5), 703 - 16
The dopamine beta-hydroxylase gene promoter directs expression of E . coli lacZ to sympathetic and other neurons in adult transgenic mice; Mercer EH et al.; Dopamine beta-hydroxylase (DBH) catalyzes the final step in the biosynthesis of norepinephrine, the principal classic neurotransmitter of peripheral sympathetic neurons . We have shown that 5.8 kb of 5' upstream region from a cloned human DBH gene promoter is sufficient to direct expression of the E . coli lacZ gene in transgenic mice to neurons of the locus ceruleus and other classic noradrenergic brain stem nuclei, sympathetic ganglion neurons, and adrenal chromaffin cells . lacZ expression was also observed in neurons of the enteric system, the retina, some sensory and all cranial parasympathetic ganglia, and some diencephalic and telencephalic brain nuclei . The expression pattern of the transgene in DBH-immunonegative sites overlapped with many sites where expression of tyrosine hydroxylase or phenylethanolamine N-methyltransferase, two other catecholamine biosynthetic enzymes, has been reported.

Nucleic Acids Res, 1991 Oct 25, 19(20), 5519 - 23
Isolation of cDNA clones encoding a human apurinic/apyrimidinic endonuclease that corrects DNA repair and mutagenesis defects in E . coli xth (exonuclease III) mutants; Robson CN et al.; Apurinic/apyrimidinic (AP) sites in cellular DNA are considered to be both cytotoxic and mutagenic, and can arise spontaneously or following exposure to DNA damaging agents . We have isolated cDNA clones which encode an endonuclease, designated HAP1 (human AP endonuclease 1), that catalyses the initial step in AP site repair in human cells . The predicted HAP1 protein has an Mr of 35,500 and shows striking sequence similarity (93% identity) to BAP 1, a bovine AP endonuclease enzyme . Significant sequence homology to two bacterial DNA repair enzymes, E . coli exonuclease III and S . pneumoniae ExoA proteins, and to Drosophila Rrp1 protein is also apparent . We have expressed the HAP1 cDNA in E . coli mutants lacking exonuclease III (xth), endonuclease IV (nfo), or both AP endonucleases . The HAP1 protein can substitute for exonuclease III, but not for endonuclease IV, in respect of some, but not all, DNA repair and mutagenesis functions . Moreover, a dut xth (ts) double mutant, which is nonviable at 42 degrees C due to an accumulation of unrepaired AP sites following excision of uracil from DNA, was rescued by expression of the HAP1 cDNA . These results indicate that AP endonucleases show remarkable conservation of both primary sequence and function . We would predict that the HAP1 protein is important in human cells for protection against the toxic and mutagenic effects of DNA damaging agents.

Nature, 1991 Oct 24, 353(6346), 776 - 8
The vsr gene product of E . coli K-12 is a strand- and sequence-specific DNA mismatch endonuclease; Hennecke F et al.; In Escherichia coli K-12, the Dcm methyltransferase catalyses methylation of the inner cytosine residue in the sequence CCA/TGG . Hydrolytic deamination of 5-methylcytosine bases in DNA leads to thymine residues, and hence to T/G mismatches, pre-mutagenic DNA lesions consisting of two natural DNA constituents and thus devoid of an obvious marker of the damaged DNA strand . These mismatches are corrected by the VSP repair pathway, which is characterized by very short patches of DNA repair synthesis . It depends on genes vsr and polA and is strongly stimulated by mutL and mutS . The vsr gene product (Vsr; Mr 18,000) was purified and characterized as a DNA mismatch endonuclease, a unique and hitherto unknown type of enzyme . Vsr endonuclease nicks double-stranded DNA within the sequence CTA/TGN or NTA/TGG next to the underlined thymidine residue, which is mismatched to 2'-deoxyguanosine . The incision is mismatch-dependent and strand-specific . These results illustrate how Vsr endonuclease initiates VSP mismatch repair.

Nucleic Acids Res, 1991 Oct 11, 19(19), 5379 - 83
Probing the activation of the replicative origin of broad host-range plasmid R1162 with Tus, the E.coli anti-helicase protein; Zhou HS et al.; The E.coli Tus protein is an anti-helicase involved in the termination of chromosome replication . The binding site for this protein, ter, was cloned into derivatives of the broad host-range plasmid R1162 . The ter site caused the orientation-specific termination of plasmid replication fork movement in cell extracts containing Tus . Plasmids were constructed so that two sites for initiation of R1162 replication flanked the iteron-containing domain of the origin . In these plasmids, the site next to the AT-rich region within the iteron-containing domain was more active . In addition, when ter was placed between the more active site and the iterons, initiation of replication from this site was specifically inhibited . The data support a model for entry of the essential, plasmid-encoded helicase at one side of the direct repeats, and for its movement primarily in one direction away from these repeats to activate the initiation sites for DNA replication.

Nucleic Acids Res, 1991 Oct 11, 19(19), 5281 - 3
Ribosomes containing the C1054-deletion mutation in E . coli 16S rRNA act as suppressors at all three nonsense codons; Prescott C et al.; It was established some time ago that the deletion of base C1054 in E . coli 16S rRNA specifically affects UGA-dependent termination of translation . Based on this observation, a model for the termination event was proposed in which the UGA nonsense codon on the mRNA base-pairs with a complementary motif in 'helix 34' of the 16S rRNA, thus potentially providing a recognition signal for the binding of the release factor . This model has been re-examined here and evidence is presented which demonstrates that ribosomes containing the C1054 delta mutation enhance the activity of suppressors of both UAG and UAA termination codons introduced into the host . The results do not support the nonsense codon-16S rRNA base pairing model, and rather imply a more general involvement of 'helix 34' in the translation termination reactions.

Hepatogastroenterology, 1991 Oct, 38(5), 388 - 90
Effect of bile on liver function tests in experimental E . coli peritonitis in the rat; Andersson R et al.; Hepatic dysfunction is a frequent finding in sepsis and peritonitis . In the present study, hepatic function in experimental peritonitis in the rat was determined by measuring serum levels of bilirubin, alkaline phosphatase (ALP), glutamic-oxaloacetic transaminase (GOT) and glutamic-pyruvic transaminase (GPT), together with antipyrine (AP) clearance as a determinant of microsomal function . Peritonitis was induced by intraperitoneal injection of 3 x 10(8) colony-forming units of E . coli together with either 1.0 ml bile or saline . E . coli + bile peritonitis rats had significantly elevated levels of bilirubin, ALP, GOT and GPT as compared with both controls and rats with peritonitis induced by E . coli alone . The derangements gradually increased with time over the 10-hour period studied . In contrast, no reduction of AP clearance was observed in the peritonitis models . On the contrary, AP clearance was enhanced at 10 hours after induction of peritonitis by E . coli alone . In conclusion, hepatic dysfunction as revealed by routine laboratory tests is seen early in experimental peritonitis in the rat, but this is not accompanied by a reduced AP clearance rate.

FEBS Lett, 1991 Sep 23, 290(1-2), 173 - 6
Substrate antagonism in the kinetic mechanism of E . coli phosphofructokinase-1; Deville-Bonne D et al.; In the presence of its allosteric activator GDP, the major phosphofructokinase-1 from Escherichia coli K12 follows Michaelis-Menten kinetics . The kinetic behavior observed at steady-state using different concentrations of the substrates ATP and fructose-6-phosphate and the pattern of inhibition by the substrate analogs adenylyl-(beta, gamma-methylene)-diphosphonate and D-arabinose-5-phosphate are consistent with a random sequential mechanism in rapid equilibrium, rather than with an ordered binding as was suggested earlier . However, ATP and fructose-6-phosphate do not bind independently to the same active site, since the apparent affinity for one substrate is decreased about 20-fold when the other substrate is already bound . The antagonism between ATP and fructose-6-phosphate shows that a negative interaction occurs during the reaction with E . coli phosphofructokinase-1 which must be considered in addition to its allosteric properties.

Eur J Pharmacol, 1991 Sep 17, 202(2), 201 - 11
Segment-specific effects of the heat-stable enterotoxin of E . coli on electrolyte transport in the rat colon; Nobles M et al.; The heat-stable enterotoxin of E . coli (STa) induced an increase in short-circuit current (Isc) in the rat colon . The maximal increase in Isc was about three times larger in the proximal than the distal colon . The action of STa was mimicked by 8-Br-cyclic GMP . Unidirectional flux measurements revealed that STa decreased Na+ and Cl- absorption in the distal colon, while it decreased Na+ absorption and activated Cl- secretion in the proximal colon . In the distal, but not in the proximal colon, indomethacin inhibited the action of STa and of 8-Br-cyclic GMP . Inhibition by indomethacin could be overcome by addition of prostaglandin E2 or forskolin, but not by addition of a non-hydrolysable analogue of cyclic AMP, suggesting an action of STa on cyclic AMP hydrolysis . Amrinone and trequinsin, two inhibitors of cyclic GMP-inhibited phosphodiesterases, mimicked the action of STa on Isc and inhibited the response to a subsequent administration of the toxin indicating the modulation of a cyclic GMP-inhibited phosphodiesterase by STa in the distal colon . The results give evidence for different intracellular action sites of STa in the two parts of the rat colon.

Clin Ther, 1991 Sep-Oct, 13(5), 627 - 36
Impact of transfer from animal-source insulins to biosynthetic human insulin (rDNA E coli) in patients with diabetes mellitus; Garber AJ et al.; Six hundred forty-eight patients (50.5% men; 49.5% women) with diabetes mellitus on animal-source insulin therapy for at least five years were studied . In this patient population, approximately 68.7% had Type I insulin-dependent diabetes mellitus and 31.3% had Type II noninsulin-dependent diabetes mellitus, nonetheless requiring insulin therapy . Patients were voluntarily transferred from animal-source insulin to biosynthetic human insulin derived by recombinant DNA technology from genetically altered Escherichia coli {human insulin (rDNAE coli)} and were monitored regularly thereafter . At a mean interval of 14 months after transfer to human insulin (rDNAE coli), these patients had gained 0.8 kg in body weight (P less than 0.01) . There was a significant decline in systolic (P less than 0.01) but not in diastolic blood pressure . Insulin requirements while on animal-source insulin averaged 47.6 +/- 22.9 U/day (mean +/- SD); this requirement was not significantly different after transfer to human insulin (rDNAE coli) (47.0 +/- 21.2 U/day) . The distribution of regular and modified insulin types prescribed did not change after patients were transferred from animal-source insulin to human insulin (rDNAE coli) . However, a significant increase in the number of insulin injections from 1.79 +/- 0.59 to 1.96 +/- 0.61 injections/day was observed (P less than 0.001) . Fasting glucose levels declined significantly from 202 +/- 87 mg/dl on animal-source insulin to 178 +/- 66 mg/dl on human insulin (rDNAE coli) (P less than 0.001) . Postprandial glucose levels (at two hours) also declined from 227 +/- 83 mg/dl to 212 +/- 80 mg/dl . Glycosylated hemoglobin (HbA1c) decreased from 9.57 +/- 2.01% while taking animal insulin to 8.97 +/- 2.00% on human insulin (rDNAE coli) (P less than 0.001) Serum cholesterol and triglyceride levels insulin (rDNAE coli) . Serum high-density lipoprotein cholesterol (HDL-cholesterol) levels increased from 54.2 +/- 15.1 mg/dl on animal insulin to 57.2 +/- 15.5 mg/dl on human insulin (rDNAE coli) (P less than 0.001) . These data demonstrate that transfer of patients from animal-source insulins to human insulin (rDNAE coli) was associated with: (1) an improvement in glycemic control parameters; (2) a slight increase in the number of insulin injections in some patients, but no overall alteration in insulin requirements; and (3) no adverse trends in indicators of cardiovascular risks, such as serum lipids . Indeed, overall cardiovascular risk may have declined not only as a result of improvement in glycemic control, but also owing to a reduction in systolic blood pressure and an elevation in HDL-cholesterol levels.

FEBS Lett, 1991 Sep 9, 289(2), 231 - 4
15N NMR studies of the conformation of E . coli dihydrofolate reductase in complex with folate or methotrexate; Huang FY et al.; We have employed 15N NMR to characterize the conformations of Escherichia coli dihydrofolate reductase (ECDHFR) in complex with {5-15N}folate or {5-15N}methotrexate (MTX) . Two 15N resonances were observed for DHFR/MTX binary complex . The relative population of these two conformations is pH dependent . Addition of NADP+ or NADPH results in the disappearance of the low field resonance . In contrast, only one conformation was observed for both the DHFR/folate and DHFR/folate/NADP+ complexes . However, the 15N chemical shift of {5-15N}folate in the binary DHFR/folate complex is 7.28 ppm upfield from that of the ternary complex, suggesting the possible loss of a hydrogen bonding to N5 of folate in the ternary complex.

J Radiat Res (Tokyo), 1991 Sep, 32(3), 286 - 95
RBE of HTO to 60Co gamma-rays for cell killing of a radioresistant E . coli harboring plasmid; Yamamoto O et al.; Radioresistant E . coli TGl harboring pUC 18 plasmid which was Ampicillin-resistant was exposed to 60Co gamma-rays or 3H beta-rays in saline to determine whether the relative biological effectiveness of 3H beta-rays is higher than one . After exposure to 60Co gamma-rays at a dose rate of 0.465 Gy/min, the D0 by colony formation was 145 Gy in the presence of Ampicillin in or absence from the agar medium; whereas, the D0 was calculated as 118 Gy with and without Ampicillin after exposure to 3H beta-rays at a dose rate of 0.431 Gy/min . The relative biological effectiveness established for 3H beta-rays to 60Co gamma-rays was 1.23 . The reason for the higher effectiveness of 3H beta-rays as compared to the reference 60Co gamma-rays is discussed in terms of nascent 0 production.

Prikl Biokhim Mikrobiol, 1991 Sep-Oct, 27(5), 731 - 7
{Comparative study of E . coli strains producing amino acids}; Astaurova OB et al.; Transduction of the locus of stability to high threonine concentrations (Thrr) into E . coli str M1 and C600 resulted in enhancements of the amino acid production and retardation of the culture development . Besides the mutation caused increase of the specific activity of glutamate synthase, aspartate kinase and homoserine dehydrogenase . The cells of the mutant strains had poorly developed walls and were smaller than those of the parent strains.

Kansenshogaku Zasshi, 1991 Sep, 65(9), 1188 - 93
{A case report of siblings with hemolytic uremic syndrome from whose stool E . coli O157:H7 was isolated}; Andou M et al.; We experienced siblings of hemolytic uremic syndrome which occurred following diarrhea and bloody stool . They were immediately treated with dipyridamole and aspirin, and recovered from hemolytic uremic syndrome in about two weeks . E . coli O157:H7 (verotoxin producing E . coli) which is recently thought to be related to the pathogenesis of hemolytic uremic syndrome was isolated in their stool cultures on admission . As far as we know, this is the first case in Japan from which stool E . coli O157:H7 was detected . Moreover, we reported clinical effectiveness of genomic investigation utilizing polymerase chain reaction method for verotoxin coding region in a rapid diagnosis of this bacterial infection.

Mutat Res, 1991 Sep-Oct, 250(1-2), 73 - 7
Protective effects of sodium selenite on killing and mutation by N-methyl-N'-nitro-N-nitrosoguanidine in E . coli; Sato M et al.; Sodium selenite was found to protect Escherichia coli cells against killing and mutagenic effects of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) . Such protective effects were not observed when cells were treated with N-methyl-N-nitrosourea (MNU) . The protection by sodium selenite was not controlled by the ada gene, which is responsible for the repair of alkylated damage in DNA . A reduction of the amount of glutathione was found when cells were treated with sodium selenite, and glutathione is known to be involved in the methylation of DNA by MNNG, not by MNU . Reduced methylation by MNNG due to the reduction of the amount of glutathione caused by abundant sodium selenite was suggested to be the mechanism of protection.

Biochem Int, 1991 Sep, 25(2), 381 - 6
Activity of chloramphenicol acetyltransferase overproduced in E . coli with wild-type and mutant GroEL; Kim HB et al.; The homo-oligomeric protein chloramphenicol acetyltransferase (CAT) has previously been shown to interact with a chaperone GroEL in vitro, suggesting a possible involvement of GroEL in CAT assembly . CAT was overproduced to various levels in the presence and absence of GroEL overproduction, and in groEL mutants . CAT was accumulated to 9-45% of total cellular protein in a fully soluble form, without formation of inclusion bodies . In all cases, even with groEL mutants, CAT specific activity was shown proportional to the amount of protein produced, indicating the formation of active trimer CAT structure does not need GroEL in Escherichia coli.

J Biomol NMR, 1991 Sep, 1(3), 247 - 55
Binding of metal ions to E . coli RNase HI observed by 1H-15N heteronuclear 2D NMR; Oda Y et al.; The divalent metal ion binding site and binding constant of ribonuclease HI from Escherichia coli were investigated by observing chemical shift changes using 1H-15N heteronuclear NMR . Chemical shift changes were monitored during the titration of the enzyme with salts of the divalent cations . The enzyme was uniformly labeled by 15N, which facilitated the monitoring of the chemical shift change of each cross peak between the backbone amide proton and the amide 15N . The chemical shifts of several amide groups were affected upon the addition of a divalent metal ion: Mg2+, Ca2+, or Ba2+ . These amide groups resided close to the active site, consistent with the previous X-ray crystallographic studies . From the titration analysis, a single divalent ion binding site was observed with a weak binding constant (KD = 2-4 mM for the current divalent ions).

Bioorg Khim, 1991 Sep, 17(9), 1201 - 12
{Cloning and expression of the 3C-proteinase gene from the poliomyelitis virus in E . coli cells}; Sablina EP et al.; Expression of the 3C protease gene of poliovirus type 1 (Mahoney) in E . coli cells using various vectors was studied . The 3C gene was shown to be expressed effectively upon its cloning in HindII/HindII (bases 5240 to 6770) and in HindII/HindIII (bases 5240 to 6056) fragments of poliovirus cDNA in pTTQ8 vector containing tac-promoter and lacI-repressor gene . Products of processing at the N-terminal 3C protease Gln-Gly site and polypeptides formed upon translation from an alternative methionine, which was coded by bases 5516-5518 of poliovirus cDNA, were found among virus-specific proteins . Processing at the C-terminal 3C protease Gln-Gly site was not observed.

Nucleic Acids Res, 1991 Aug 25, 19(16), 4473 - 8
Human uracil-DNA glycosylase complements E . coli ung mutants; Olsen LC et al.; We have previously isolated a cDNA encoding a human uracil-DNA glycosylase which is closely related to the bacterial and yeast enzymes . In vitro expression of this cDNA produced a protein with an apparent molecular weight of 34 K in agreement with the size predicted from the sequence data . The in vitro expressed protein exhibited uracil-DNA glycosylase activity . The close resemblance between the human and the bacterial enzyme raised the possibility that the human enzyme may be able to complement E . coli ung mutants . In order to test this hypothesis, the human uracil-DNA glycosylase cDNA was established in a bacterial expression vector . Expression of the human enzyme as a LacZ alpha-humUNG fusion protein was then studied in E . coli ung mutants . E . coli cells lacking uracil-DNA glycosylase activity exhibit a weak mutator phenotype and they are permissive for growth of phages with uracil-containing DNA . Here we show that the expression of human uracil-DNA glycosylase in E . coli can restore the wild type phenotype of ung mutants . These results demonstrate that the evolutionary conservation of the uracil-DNA glycosylase structure is also reflected in the conservation of the mechanism for removal of uracil from DNA.

FEMS Microbiol Lett, 1991 Aug 15, 66(3), 345 - 51
Molecular cloning and expression in Escherichia coli K-12 of chromosomal genes determining the O antigen of an E . coli O2: K1 strain; Neal BL et al.; The rfb gene cluster which determines the biosynthesis of the O2 O antigen has been cloned from an Escherichia coli O2: K1 strain isolated from a case of septicaemia in chickens . The region required for expression of the O antigen in E . coli K-12 was localised to a 10.7 to 14.15-kb segment which was shown to be chromosomal in origin with a close linkage to the gnd and his genetic loci.

Nucleic Acids Res, 1991 Aug 11, 19(15), 4241 - 6
Assessment of inhomogeneities in an E . coli physical map; Karlin S et al.; A statistical method based on r-fragments, sums of distances between (r + 1) consecutive restriction enzyme sites, is introduced for detecting nonrandomness in the distribution or too markers in sequence data . The technique is applicable whenever large numbers of markers are available and will detect clumping, excessive dispersion or too much evenness of spacing of the markers . It is particularly adapted to varying the scale on which inhomogeneities can be detected, from nearest neighbor interactions to more distant interactions . The r-fragment procedure is applied primarily to the Kohara et al . (1) physical map of E . coli . Other applications to DAM methylation sites in E . coli and NotI sites in human chromosome 21 are presented . Restriction sites for the eight enzymes used in (1) appear to be randomly distributed, although at widely differing densities . These conclusions are substantially in agreement with the analysis of Churchill et al . (3) . Extreme variability in the density of the eight restriction enzyme sites cannot be explained by variability in mono-, di- or trinucleotide frequencies.

Nature, 1991 Aug 8, 352(6335), 544 - 7
Preferential DNA secondary structure mutagenesis in the lagging strand of replication in E . coli; Trinh TQ et al.; When present in single-stranded DNA, palindromic or quasi-palindromic sequences have the potential to form complex secondary structures, including hairpins, which may facilitate interstrand misalignment of direct repeats and be responsible for diverse types of replication-based mutations, including deletions, additions, frameshifts and duplications . In regions of palindromic symmetry, specific deletion events may involve the formation of a hairpin or other DNA secondary structures which can stabilize the misalignment of direct repeats . One model suggests that these deletions occur during DNA replication by slippage of the template strand and misalignment with the progeny strand . The concurrent DNA replication model, involving an asymmetric dimeric DNA polymerase III complex which replicates the leading and lagging strands, has significant implications for mutagenesis . The intermittent looping of the lagging strand template, and the fact that the lagging strand template may contain a region of single-stranded DNA the length of an Okazaki fragment, provides an opportunity for DNA secondary-structure formation and misalignment . Here we report our design of a palindromic fragment to create an 'asymmetric palindromic insert' in the chloramphenicol acetyltransferase gene of plasmid pBR325 . The frequency with which the insert was deleted in Escherichia coli depends on the orientation of the gene in the plasmid . Our results suggest that replication-dependent deletion between direct repeats may occur preferentially in the lagging strand.

FEBS Lett, 1991 Aug 5, 287(1-2), 129 - 32
High-level expression of enzymatically active bovine leukemia virus proteinase in E . coli; Andreansky M et al.; An E . coli plasmid expressing efficiently an artificial precursor of bovine leukemia virus (BLV) proteinase under transcriptional control of the phage T7 promoter was constructed . The expression product accumulates in the induced E . coli cells in the form of insoluble cytoplasmic inclusions . Solubilization of the inclusions and a refolding step yield almost pure and completely self-processed proteinase . Purification to homogeneity was achieved by ion-exchange chromatography and reverse-phase HPLC . On a preparative scale, a high yield of enzymatically active proteinase was obtained . An initial study using a series of synthetic peptide substrates shows a distinct substrate specificity of BLV proteinase.

Nippon Geka Gakkai Zasshi, 1991 Aug, 92(8), 913 - 20
{Analysis of mechanism of lung edema in E . coli injected septic rat model--in relation to tumor necrosis factor (TNF) produced from liver, spleen, and alveolar macrophages}; Inoue S et al.; After injection of live E . coli, TNF in blood and culture supernatant of the isolated macrophages from the lung, liver, and spleen, were measured, and possible relationship between their TNF levels and lung edema was examined . The blood TNF activity increased significantly until 3h after the injection in the lethal group (p less than 0.01) . The TNF activity of alveolar macrophages showed no changes even in the lethal group . The TNF activities of the liver and spleen macrophages decreased significantly in the lethal group (p less than 0.01-0.05), while those in the sublethal group that didn't induce lung edema and showed no significant decrease . The blood leukocyte count decreased until 6h after the injection in the both sublethal and lethal groups, but there was no significant difference between the two groups . The lung weight difference of the lethal group was significantly higher than that in the control group 12h after injection (p less than 0.05) . Therefore, the elevated blood TNF activities in our study may be not elicited from alveolar macrophages but mainly from liver and spleen macrophages . There may be a relationship between the lung edema and the elevated blood TNF activity in the lethal groups.

Kansenshogaku Zasshi, 1991 Aug, 65(8), 905 - 8
{Isolation of vero toxin-producing Escherichia coli (enterohemorrhagic E . coli) O111:H- from 2 cases diagnosed as appendicitis}; Uchimura M et al.; Vero toxin-producing E . coli O111:H- was isolated from 2 cases of patients with severe abdominal pain and bloody diarrhea and were diagnosed as appendicitis . E . coli from both cases produced both VT1 and VT2.

Immunology, 1991 Aug, 73(4), 394 - 7
Expression of a dietary protein in E . coli renders it strongly antigenic to gut lymphoid tissue; Dahlgren UI et al.; Bacteria that colonize the intestinal mucosa elicit a strong mucosal immune response, whereas food antigens such as ovalbumin are very weakly immunogenic to the gut-associated lymphoid tissue . This may either be due to special physico-chemical properties of bacterial substances versus proteins from animals and plants, or to stimulating properties of the bacteria on, e.g., antigen presentation, rendering all substances contained within bacteria antigenic . To test these hypotheses, ovalbumin was expressed in wild-type Escherichia coli and germ-free female rats were colonized with this strain . The systemic and mucosal antibody response of these rats was compared with that of rats given large amounts of dietary ovalbumin . Biliary IgA antibodies, which reflect the local IgA antibody production in the intestine, were only found in the rats colonized with ovalbumin-synthesizing E . coli . IgG antibodies in the bile were also only seen in these rats . We conclude that mucosal immunogenicity depends on the context in which a protein is presented to the gut-associated lymphoid tissue, rather than to special antigenic characteristics of the protein in itself.

Acta Virol, 1991 Aug, 35(4), 391 - 5
Expression of bovine leukaemia virus antigens fused to MS2 polymerase in E . coli; Ulrich R et al.; The main core protein and segments of envelope proteins of bovine leukaemia virus fused to MS2 polymerase were expressed in E . coli . The synthesis rate varied between 3 and 25% of the total cellular proteins . BLV-MS2 polymerase fusion proteins were detected immunologically using rabbit anti-BLV sera, monoclonal antibodies and a serum of a BLV-infected cow.

Nature, 1991 Jul 18, 352(6332), 258 - 60
Anticodon and acceptor stem nucleotides in tRNA(Gln) are major recognition elements for E . coli glutaminyl-tRNA synthetase; Jahn M et al.; The correct attachment of amino acids to their corresponding (cognate) transfer RNA catalysed by aminoacyl-tRNA synthetases is a key factor in ensuring the fidelity of protein biosynthesis . Previous studies have demonstrated that the interaction of Escherichia coli tRNA(Gln) with glutaminyl-tRNA synthetase (GlnRS) provides an excellent system to study this highly specific recognition process, also referred to as 'tRNA identity' . Accurate acylation of tRNA depends mainly on two principles: a set of nucleotides in the tRNA molecule (identity elements) responsible for proper discrimination by aminoacyl-tRNA synthetases and competition between different synthetases for tRNAs . Elements of glutamine identity are located in the anticodon and in the acceptor stem region, including the discriminator base . We report here the production of more than 20 tRNA(2Gln) mutants at positions likely to be involved in tRNA discrimination by the enzyme . Unmodified tRNA, containing the wild-type anticodon and U or G at its 5'-terminus, can be aminocylated by GlnRS with similar kinetic parameters to native tRNA(2Gln) . By in vitro aminoacylation the mutant tRNAs showed decreases of up to 3 x 10(5)-fold in the specificity constant (kcat/KM)14 with the major contribution of kcat . Despite these large changes, some of these mutant tRNAs are efficient amber suppressors in vivo . Our results show that strong elements for glutamine identity reside in the anticodon region and in positions 2 and 3 of the acceptor stem, and that the contribution of different identity elements to the overall discrimination varies significantly . We discuss our data in the light of the crystal structure of the GlnRS:tRNA(Gln) complex.

FEBS Lett, 1991 Jul 8, 285(1), 55 - 8
A catalytic role for threonine-12 of E . coli asparaginase II as established by site-directed mutagenesis; Harms E et al.; A threonine-12 to alanine mutant of E . coli asparaginase II (EC 3.5.1.1) has less than 0.01% of the activity of wild-type enzyme . Both tertiary and quaternary structure of the enzyme are essentially unaffected by the mutation; thus the activity loss seems to be the result of a direct impairment of catalytic function . As aspartate is still bound by the mutant enzyme, Thr-12 appears not be involved in substrate binding.

EMBO J, 1991 Jul, 10(7), 1749 - 57
One of three transmembrane stretches is sufficient for the functioning of the SecE protein, a membrane component of the E . coli secretion machinery; Schatz PJ et al.; The E . coli secE (prlG) gene codes for an integral cytoplasmic membrane protein which is part of the cell's secretory machinery . A deletion of nearly the entire gene renders the cell dependent on the presence of a complementing secE+ plasmid, indicating that the SecE protein is essential for growth . Deletions which remove carboxy-terminal sequences or substantial amounts near the amino-terminus of SecE can still complement the lethal deletion . This deletion analysis suggests that the essential domain of the SecE protein includes only a single one of its three hydrophobic membrane-spanning segments . Two of three dominant prlG signal sequence suppressors map to this segment . Consistent with the insensitivity of SecE to major structural changes, several cold-sensitive mutations cause lethality not because of any change in the protein, but because of a reduction in its level of expression . Our results suggest that higher levels of the protein are needed at the lower temperature . These findings are discussed in terms of the interactions between various components of the secretory machinery.

Biotechniques . 1991 Jul;11(1):32, 34.
A rapid and complete 4-step protocol for obtaining nucleotide sequence from E . coli genomic DNA from overnight cultures; Seetharam S et al.; We describe a rapid and complete 4-step protocol for obtaining DNA sequence from E . coli genomic DNA starting from overnight cultures . These steps are as follows: isolation and purification of genomic DNA; PCR amplification of the desired region; purification of the amplified DNA product; and finally, direct sequencing using the dideoxy chain termination procedure with simple modifications to improve signal intensity . Clean DNA sequence can be obtained from a large number of strains in a matter of days, using easily available kit-type reagents . This protocol should be generally applicable to genomic DNA from other species.

Biochimie, 1991 Jul-Aug, 73(7-8), 983 - 9
Superexpression and fast purification of E coli initiation factor IF2; Mortensen KK et al.; For the production of large quantities of E coli initiation factor IF2 we have constructed an improved overexpression system . The gene infB was cloned into the thermo-inducible runaway plasmid pCP40 {1} and subsequently transformed into the E coli strain C600{pcI857} . In this system the expression of infB is under the control of the strong promoter lambda PL and the cells carry the plasmid pcI857, which contains a thermosensible lambda cI repressor . Overexpression of IF2, which is approximately 30 times higher than the expression in wild-type-cells, is induced at 42 degrees C and continues for 2 h at 37 degrees C . From these cells pure and active IF2 was obtained using a novel 3-step FPLC-procedure consisting of ion-exchange liquid chromatography on Q-sepharose HP, MonoQ and MonoS . In approximately 8 h, 5 mg of pure and active IF2 can be obtained from 10 g overproducing cells . This corresponds to 5 mg of IF2 per litre of medium . The purification was monitored by Western immunoblotting and the activity of the purified factor was tested by measuring the stimulation of binding of the initiator fMet-tRNA(Met)f to 70S ribosomes in the presence of GTP and poly(A,U,G) as messenger RNA . Compared with previous methods our purification procedure avoids the use of materials such as DEAE-cellulose and phosphocellulose which have relatively poor flow rates . In addition to the higher flow capacity of Q-sepharose HP, this new matrix can be loaded with an S30 supernatant.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochimie, 1991 Jul-Aug, 73(7-8), 971 - 81
Probing the initiation complex formation on E coli ribosomes using short complementary DNA oligomers; Weller J et al.; Interactions between Escherichia coli 16S rRNA sequences (as components of 30S ribosomal subunits or tight-couple 70S ribosomes) with the ligands poly(U), poly(AGU), tRNAPhe, tRNAfMet, and the initiation factors have been studied . The ligands were employed as competitors for selected sites on 16S rRNA known to be accessible for hybridization to cDNA oligomers, regions 517-528, 1397-1404, and 1534-1542 . The binding of cDNAs 1534-1541 and 1398-1403 decreased in the presence of the ligand pair poly(U)/tRNAPhe . Only the binding of cDNA 1534-1541 was affected by poly(AGU), while none of the complementary DNA oligomer binding was affected by tRNAPhe or tRNAfMet alone . The poly(AGU)/tRNAfMet ligand pair caused an additional decline in the binding of cDNA 1534-1541, relative to that caused by poly(AGU) alone, but the ligand pair did not affect the binding of the cDNA oligomers 517-528 or 1398-1403 . The inclusion of the initiation factors did not significantly alter the binding level decreases observed for cDNA 1534-1541 in the presence of mRNAs or tRNA . At the 517-528 and 1398-1403 regions, the inclusion of the initiation factors, in either the presence or absence of the other ligands, caused a large decrease in the binding of the cDNA oligomers . The oligomers complementary to 16S bases 517-528 and 1398-1403 did not bind to tight-couple or reassociated 70S ribosomes . The data are discussed in terms of the decoding site hypothesis, and in terms of the mRNA alignment mechanism proposed by Trifonov {1}.

Enzyme Microb Technol, 1991 Jul, 13(7), 554 - 64
Cultivation of recombinant E . coli and production of fusion protein in 60-1 bubble column and airlift tower loop reactors; Kracke-Helm HA et al.; E . coli K12 with multicopy plasmid (lambda PR-promoter and temperature-sensitive lambda cI 857 repressor) was cultivated in 60-l bubble column and airlift tower loop reactors . The medium composition, cell concentration, and intracellulary enzyme activity were monitored on-line during batch, fed-batch, and continuous cultivations . The specific growth rates, cell mass yield coefficients, plasmid stabilities, productivities of the amount of active fusion protein (beta-galactosidase activity), concentrations and yields of acetic acid, and volumetric oxygen transfer coefficient were evaluated for different medium compositions and cultivation conditions . The enzyme activity was also monitored during the temperature induction . The results evaluated in the 60-l bubble column and airlift tower loop reactors are compared with those evaluated in a 1-1 stirred-tank reactor.

Biochem Biophys Res Commun, 1991 Jun 14, 177(2), 619 - 23
Identity determinants of E . coli tRNA(Val); Tamura K et al.; In order to study the identity elements of valine tRNA, various transcripts of E . coli valine tRNA mutants were constructed . Both mutations at the second letter A35 of the anticodon and at the discriminator base A73 seriously damaged valine charging activity . Mutations at either the G3-C70 or U4-A69 base pairs in the acceptor stem also affected the activity . Only one nucleotide substitution of the second letter G35 of the anticodon with A35 brought an 18% valine charging activity into alanine tRNA, which acquired an almost full charging activity with valine by introducing an additional change at those two base pairs in the acceptor stem . These results indicate that the second letter A35 of the anticodon, discriminator base and acceptor stem are involved in recognition by valyl-tRNA synthetase.

Cell, 1991 Jun 14, 65(6), 1015 - 22
Bipartite functional map of the E . coli RNA polymerase alpha subunit: involvement of the C-terminal region in transcription activation by cAMP-CRP; Igarashi K et al.; The alpha subunit of Escherichia coli RNA polymerase plays a major role in the subunit assembly . Carboxyterminal deletion derivatives lacking 73 or 94 amino acid residues were assembled in vitro into enzyme molecules . Core enzymes consisting of these C-terminal-truncated alpha subunits were as active in RNA synthesis as native core enzyme . By the addition of sigma 70 subunit, these mutant enzymes initiated transcription from certain promoters . The mutant RNA polymerases, however, did not show cAMP-CRP activated transcription . These results demonstrate that the N-terminal region of the alpha subunit is involved in the formation of active enzyme molecule, while the C-terminal region plays an essential role in response to transcription activation by cAMP-CRP.

Nucleic Acids Res, 1991 Jun 11, 19(11), 2889 - 92
High level heterologous expression in E . coli using the anaerobically-activated nirB promoter; Oxer MD et al.; The anaerobically-regulated nirB promoter was used to express heterologous genes in Escherichia coli . Under anaerobic conditions the promoter was able to express tetanus toxin fragment C at approximately 20% total cell protein (tcp) and the Bordetella pertussis antigen pertactin at greater than 30% tcp . These levels are comparable to those obtained for the same products using the tac promoter . The nirB promoter is very well regulated, giving almost two orders of magnitude increase in fragment C on complete removal of oxygen . The use of this anaerobically-induced promoter in the production of recombinant proteins in E . coli is discussed.

J Biochem (Tokyo), 1991 Jun, 109(6), 799 - 802
Yeast gene which suppresses the defect in protein export of a secY mutant of E . coli; Sakaguchi M et al.; To find factors participating in protein translocation in yeast, we screened a yeast genomic library for genes which, when introduced into Escherichia coli, suppressed secY24, a temperature sensitive mutation of an essential integral membrane protein (SecY) required for protein export . We isolated and characterized a gene (YSY6) which improved the translocation of the OmpA protein in mutant strain IQ85(secY24) . It could also suppress another mutant {rplO215(Am)}, in which the level of expression of the SecY protein is decreased at high-temperature . The YSY6 gene encodes a small amphiphilic peptide consisting of 65 amino acids, which can be expressed in E . coli cells.

Zhonghua Jie He He Hu Xi Za Zhi, 1991 Jun, 14(3), 143 - 5, 189
{The observation of changes in plasma PGI2 and TXA2 levels and the therapeutic effect of tetramethylpyrazine in E . coli induced acute lung injury in rabbits}; Li B; Thirty two rabbits were equally divided at random into 4 groups: A . control; B . E.coli; C . E.coli + ibuprofen; D . E.coli + tetramethylpyrazine . The plasma concentration of 6-keto-PGF1 alpha and TXB2, arterial blood gas as well as platelet aggregability were measured and the pathological changes of lung tissue were observed . The results suggest that TXA2 and PGI2 do take part in the pathogenesis of acute lung injury; that PGI2 may serve as an indicator in the evaluation of the degree of injury in the pulmonary endothelial cells and may also contribute to septic shock; and that both tetramethylpyrazine and ibuprofen possess therapeutic effects on the amelioration of acute lung injury, and the former is rather stronger than the latter in the inhibitory effect on granulocytic sequestration within the lung.

J Neurosci Res, 1991 Jun, 29(2), 251 - 60
Synthesis, purification, and characterization of human ciliary neuronotrophic factor from E . coli; Negro A et al.; The cDNA for human ciliary neuronotrophic factor (CNTF) has been cloned into an expression vector under the control of the T7 promoter . The BL21 strain of E . coli was transformed with this vector . Human CNTF accounted for about 30% of the total bacterial protein after induction with isopropyl-B-D-thiogalactopyranoside . This human CNTF was purified to homogeneity from inclusion bodies by a combination of ion exchange chromatography and reverse-phase high performance liquid chromatography . The amino-terminal amino acid sequence of the purified protein was identical to the deduced amino acid sequence; however, the methionyl residue has been removed . On SDS-PAGE gels, human CNTF displayed a molecular weight of about 24 kDa, in accord with its deduced molecular mass; a pI of 5.8 indicates the acidic nature of the molecule . A proposed structure for human CNTF includes major alpha helical regions . The ED50 of purified human CNTF was approximately 30 pM, using cultured embryonic day 10 chicken dorsal root ganglion neurons; no activity was observed with neurons from embryonic day 8 ganglia . Polyclonal antibodies prepared against both a synthetic peptide of CNTF and the entire human CNTF protein recognized a single 24 kDa band on Western blots, corresponding to human CNTF . However, only the antibodies against intact CNTF blocked its biological activity . This represents the first molecular expression and purification of human CNTF.

Hua Xi Yi Ke Da Xue Xue Bao, 1991 Jun, 22(2), 211 - 2
{Determination and comparison of beta-glucuronidase activity among strains of B . fragilis and E . coli}; Fan X et al.; We determined and compared the beta-glucuronidase (beta-G) activity among strains of B . fragilis and E . coli under the optimum condition . Results showed that the mean beta-G activity of strain B . fragilis ATCC25285 was 94.7u . B . fragilis ATCC25285 strain was selected as the model strain to establish the animal model of bilirubin gallstones because its beta-G activity was obviously higher than that of B . fragilis CDC14462 and E . coli 3362 (O157K88) strain.

Bioorg Khim, 1991 Jun, 17(6), 779 - 88
{Effective synthesis and cloning of the human interleukin-2 gene and its analog: expression of the interleukin-2 gene in E . coli cells}; Daniliuk NK et al.; Artificial DNA fragments encoding human interleukin-2 (133 a.a.) and its analogue (deletion of 14 C-terminal a.a.) were prepared by means of the DNA polymerase I mediated extension of synthetic polynucleotides having short overlapping sequences at their 3'-ends . The fragments were cloned in specially designed pFH-type plasmids and then excised by the FokI and other restriction endonucleases to yield the subfragments with the structurally predetermined 5'-unique cohesive ends . The complete synthetic gene was constructed by one or two-step ligation . The expressed IL-2 was tested by analysing the T-cell proliferation activity of E.coli crude lisates containing the pEXIL2 expression plasmid.

Biochimie, 1991 Jun, 73(6), 713 - 8
FIS-induced bending of a region upstream of the promoter activates transcription of the E coli thrU(tufB) operon; Verbeek H et al.; The upstream activator sequence (UAS) of the thrU(tufB) operon, which is the target of the trans-activating protein FIS, has a bent structure . Here we show that the center of bending lies around position -95, between the two FIS-binding regions . Studies with fis+ and fis- cells show that FIS-induced bending of the UAS plays a major role in the trans-activation of the thrU(tufB) operon . This has been concluded from the finding that insertions of small DNA segments, comprising less than one or two complete helix turns, in the junction of the UAS and the RNA polymerase-binding site reduce transcription significantly . Partial restoration of transcriptional activity occurs when one or more full helix turns are inserted . These data are in line with but do not prove that a direct interaction between FIS and RNA polymerase is involved in trans-activation . A role of bending per se resulting from FIS/DNA interaction cannot be excluded.

Biochimie, 1991 Jun, 73(6), 699 - 712
Analysis of sequence elements important for the synthesis and control of ribosomal RNA in E coli; Zacharias M et al.; The regulation of the synthesis of ribosomal RNA is a key problem for the understanding of bacterial growth . Many different regulatory mechanisms involving cis and trans acting components participate in a concerted way to achieve the very efficient, flexible and coordinated production of this class of molecules . We have studied three different sequence regions within a ribosomal RNA transcription unit which are believed to control different stages of ribosomal RNA expression . In the first part of the study the function of AT-rich sequences upstream of the -35 hexamer of rRNA promoter P1 in the activation of rRNA transcription was analyzed . We confirm that a sequence dependent bend upstream of P1 is responsible for the high promoter activity . Experiments employing linker scanning mutations demonstrated that the distance as well as the angular orientation of the bent DNA is crucial for the degree of activation . In addition, the effect of the trans activating protein Fis on the transcription initiation of promoter P1 was investigated . We can show, using the abortive initiation assay, that the predominant effect of Fis is due to an increase in the affinity of RNA polymerase for the promoter (binding constant KB) while the isomerisation rate (kf) from a closed to an open RNA polymerase promoter complex is not altered significantly . We also describe the characterization of sequence determinants important for stringent regulation and growth rate control . Evidence is provided that the discriminator motif GCGC is a necessary but not sufficient element for both types of control . Furthermore we show that not simply a particular DNA primary structure but the higher order conformation of the complete promoter region is recognized and triggers the two regulatory mechanisms, both of which are apparently mediated by the effector molecule guanosine tetraphosphate (ppGpp) . Finally, we have carried out a systematic mutational analysis of the rrnB leader region preceding the structural gene for 16S RNA . We could demonstrate that highly conserved sequence elements within the rrnB leader, which were believed to be involved in transcription antitermination have post-transcriptional functions . We present evidence that these sequence elements direct the biogenesis of active ribosomal particles.

Biochimie, 1991 Jun, 73(6), 647 - 55
Genetic engineering and overexpression of ribosomal L12 protein genes from three different archaebacteria in E coli; Kopke AK et al.; Genes coding for ribosomal protein L12 from Methanococcus vannielii (Mva), Halobacterium halobium (Hha) and Sulfolobus solfataricus (Sso) have been subcloned in the polylinker region of pUC19 . An efficient Shine-Dalgarno sequence has been attached to the 5' end of the genes, and two ochre stop codons have been created at their 3' ends, where necessary . In addition, mutants of the MvaL12 and HhaL12 genes were constructed, which coded for a cysteine residue at the C-terminus of the protein . The constructs were transferred together with the pUC19 polylinker as gene cartridges into different expression vectors . These constructed plasmids were transformed in the appropriate E coli hosts and tested for expression . Two systems were found to work efficiently for overexpression, namely the pKK223-3 vector featuring a tac promoter, and the pT7-5 vector featuring a T7-promoter . The over-expressed proteins were purified to homogeneity; their purity was investigated by one and two-dimensional gel systems, amino acid analysis and N-terminal protein sequencing for 10 steps or more . The amount of protein purified from E coli test cultures bearing the expression plasmids was always more than 2.5 mg/l of medium used.

FEBS Lett, 1991 May 20, 283(1), 127 - 30
19F NMR evidence for interactions between the c-AMP binding sites on the c-AMP receptor protein from E . coli; Hinds MG et al.; The 19F NMR spectra of 3-fluorotyrosine containing c-AMP receptor protein (CRP) from E . coli have been recorded in the presence of increasing amounts of c-AMP . One of the signals (from Tyr B) shifts upfield by 0.6 ppm in the presence of excess c-AMP and shows both slow and fast exchange behaviour during the titration . This is evidence for interactions between the two c-AMP binding sites on the CRP dimer leading to different dissociation rate constants (less than or equal to 75 s-1; greater than or equal to 350 s-1) for complexes containing one and two c-AMP molecules.

Nucleic Acids Res, 1991 May 11, 19(9), 2417 - 21
High level heterologous expression in E . coli using mutant forms of the lac promoter; Makoff AJ et al.; Single base deletions in the lac promoter which reduced the 18bp spacing between the -35 and -10 homology regions to 17bp, increased the strength of the promoter . A single base substitution (T----G) in the -35 region to generate the consensus sequence TTG-ACA increased the strength further and no longer required a 17bp spacing . The mutated lac promoter was as powerful as a shorter form of the tac promoter which lacked two AT-rich regions upstream of the -35 region, and expressed the P69 surface antigen (pertactin) of Bordetella pertussis to 30-40% total cell protein and tetanus toxin fragment C to 16-20% total cell protein.

FEBS Lett, 1991 May 6, 282(2), 268 - 72
Chloroplast ribosomal protein L15, like L1, L13 and L21, is significantly larger than its E . coli homologue; Johnson CH et al.; The purification and identification by peptide sequence and immunological data of the spinach chloroplast homologue of E . coli L15 is presented . A significant increase in its mass over the E . coli counterpart is shown and is accounted for, in part, by a sequenced 18-residue N-terminal extension . A still larger C-terminal extension or internal insertion(s) is inferred . The migration position of the L15 in a 2D gel pattern of spinach chloroplast 50S subunit proteins is shown . Lack of sequence identity with the known chloroplast genomic data confirms the nuclear coding of this protein, and the N-terminal sequence given here provides the transit peptide cleavage site of the cytoplasmic precursor.

Mutat Res, 1991 May, 254(3), 281 - 8
Site-specific mutagenesis using a gapped duplex vector: a study of translesion synthesis past 8-oxodeoxyguanosine in E . coli; Moriya M et al.; We have constructed a gapped plasmid vector in which a single defined lesion is introduced, site-specifically, within a single-strand region . Efficiency of translesional synthesis is determined by the number of colonies recovered following transformation of E . coli . The nucleotide sequence of progeny plasmids in the gapped region of the vector reflects incorporation of bases opposite and near the lesion . The analysis detects non-mutagenic as well as mutagenic events . This system was used to establish the mutagenic potential of 2'-deoxy-7,8-dihydro-8-oxoguanosine (8-oxodG), a lesion produced by the action of active oxygen species on DNA . The presence of 8-oxodG did not affect the number of transformants recovered . Most transformants (greater than 99%) contained G:C pairs at the site of the lesion; however, a limited number of targeted G----T transversions were observed in the presence and absence of SOS induction . Base substitutions neighboring the lesion, reported for an in vitro system, were not observed . We conclude that the 8-oxodG lesion in DNA is weakly mutagenic in E . coli.

Mutat Res, 1991 May, 254(3), 225 - 30
Characterization of O6-methylguanine-DNA methyltransferase in transgenic mice introduced with the E . coli ada gene; Nakatsuru Y et al.; The characteristics of O6-methylguanine-DNA methyltransferase (O6-MTase) produced in transgenic mice, in which the introduced E . coli ada gene was expressed under the control of the metallothionein promoter, were investigated . Liver extracts from transgenic homozygotes showed approximately 3 times the control activity, a marked increase of up to about 8 times the non-transgenic control levels being observed 10 h after zinc treatment . Examination of the substrate specificity of the enzyme revealed that the activity in the transgenic mice is due to the introduced foreign gene . The enzyme possessed methylphosphotriester-DNA methyltransferase as well as O6-MTase, characteristic of the E . coli Ada protein . Comparison of differences in biological response between transgenic and non-transgenic mice after treatment with the alkylating carcinogen methylnitrosourea (MNU) at various doses revealed transgenic mice to be more capable of repairing O6-MTase activity, only showing signs of exhaustion at very high levels of exposure . In non-transgenic mice, on the other hand, the basal level of O6-MTase was low, and the activity was hardly detectable when the animals were treated with MNU.

Br J Pharmacol, 1991 May, 103(1), 1172 - 8
The effect of E . coli STa enterotoxin on the absorption of weakly dissociable anti-malarial drugs from rat intestine in vivo; Rawlings JM et al.; 1 . The effect of E . coli heat stable (STa) enterotoxin on the absorption of radiolabelled anti-malarial weak bases and their appearance in peripheral blood was assessed in vivo by a recirculation procedure in rat intestinal loops . 2 . Enterotoxin increased the jejunal disappearance of quinine (P less than 0.05), trimethoprim (P less than 0.05), proguanil (P less than 0.05) and chloroquine (P less than 0.001) and left pyrimethamine disappearance unaltered . Peripheral blood levels of trimethoprim (P less than 0.02) and proguanil (P less than 0.05) were higher after STa exposure . 3 . In the ileum, enterotoxin increased the luminal disappearance (P less than 0.05) and peripheral blood appearance (P less than 0.001) of chloroquine . The luminal disappearance rate of trimethoprim was reduced (P less than 0.05) and that of pyrimethamine unchanged . 4 . The increased jejunal absorption of the anti-malarial drugs occurred despite STa causing a reduction in the amount of net fluid absorption . It seems likely that the enhanced absorption with STa exposure is related to the effect of STa on the microclimate pH . An elevation in the microclimate pH would increase the amount of undissociated weak base available for non-ionic diffusion . 5 . The favourable elevation of microclimate pH by STa seemed to be outweighted by the reduced fluid absorption in the ileum . Only chloroquine still showed enhanced absorption in the ileum and this may have been because unlike the other antimalarial drugs, chloroquine has two dissociable groups likely to be affected by the mucosal surface pH changes.

Biochimie, 1991 May, 73(5), 607 - 10
Fast preparative separation of 'native' core E coli 30S ribosomal proteins; Cachia C et al.; We have developed an ion-exchange high performance liquid chromatographic method for preparative separation of 'core' proteins from E coli 30S ribosomal subunits, extracted with salt under non-denaturing conditions . This method yields individual proteins in pure and native form at high concentrations, (5 to 25 mg/ml) suitable for direct use in 1D-, 2D- or 3D-NMR studies.

Biokhimiia, 1991 May, 56(5), 930 - 4
{Quaternary structure of uridine phosphorylase from E . coli K-12}; Tsuprun VL et al.; Uridine phosphorylase was isolated from E . coli K-12 cells in a homogeneous state . The molecular mass of the enzyme as determined by gel filtration corresponds, approximately, to a hexamer made up of 27.5 kDa monomers . Evidence for the hexameric structure of uridine phosphorylase was obtained by electron microscopy with numerical treatment of the images . The six monomers within the enzyme molecule are arranged in two layers, three monomers in each, at the apices of a triangular antiprism with a point group symmetry of 32.

J Rheumatol, 1991 May, 18(5), 705 - 8
Adsorption with a soluble E . coli antigen fraction improves the specificity of ELISA tests for Lyme disease; Fawcett PT et al.; We reported that preadsorption of patient serum with heat killed E . coli increased the specificity of ELISA for antibodies to Borrelia burgdorferi . That procedure required extra specimen handling and a preincubation . We report the use of a soluble E . coli antigen fraction that is included in serum diluent, eliminating additional steps . Sera from 220 individuals were tested for antibodies to B . burgdorferi . Twenty sera were obtained from patients with Lyme disease and 200 sera were from a population that included healthy controls and patients with different inflammatory conditions (viral infections and various rheumatic disorders) . Testing was performed using either a standard serum diluent or one containing soluble E . coli antigen fraction . Results demonstrate that inclusion of soluble E . coli antigen fraction in serum diluent increased assay specificity from 88% for the standard protocol to 98%, with no change in test sensitivity.

Mutat Res, 1991 May, 248(1), 1 - 15
A bacterial position effect: when the F factor in E . coli K12 is integrated in cis to a chromosomal gene that is flanked by IS1 repeats the elements are activated so that amplification and other regulatory changes that affect the gene can occur; Clugston CK et al.; In Escherichia coli K12 the argF gene is located within Tn2901, a genomic unit of approx . 12 kb that is flanked by IS1 elements in direct repeat . When strains in which the F factor is integrated in cis to Tn2901 are subjected to the appropriate selection, regulatory changes that result in over-production of the argF-encoded ornithine transcarbamylase occur at relatively high frequencies . When amplification occurs, the F factor is required only for the initial exchange between the IS1 elements, homologous recombination between tandem repeats of Tn2901 being independent of the F status of the cell . While amplification of Tn2901 is frequent in some Hfr strains, in others regulatory changes that do not involve amplification are predominant . The transfer region of the F factor does not contribute to the position effect.

J Inorg Biochem, 1991 May 1, 42(2), 87 - 96
Mechanistic studies on E . coli DNA topoisomerase I: divalent ion effects; Domanico PL et al.; E . coli DNA topoisomerase I catalyzes the hydrolysis of short, single stranded oligodeoxynucleotides . It also forms a covalent protein-DNA complex with negatively supercoiled DNA in the absence of Mg2+ but requires Mg2+ for the relaxation of negatively supercoiled DNA . In this paper we investigate the effects of various divalent metals on catalysis . For the relaxation reaction, maximum enzyme activity plateaus after 2.5 mM Mg2+ . However, the rate of cleavage of short oligodeoxynucleotide increased linearly between 0 and 15 mM Mg2+ . In the oligodeoxynucleotide cleavage reaction, Ca2+, Mn2+, Co2+, and Zn2+ inhibit enzymatic activity . When these metals are coincubated with Mg2+ at equimolar concentrations, the normal effect of Mg2+ is not detectable . Of these metals, only Ca2+ can be substituted for Mg2+ as a metal cofactor in the relaxation reaction . And when Mg2+ is coincubated with Mn2+, Co2+, or Zn2+ at equimolar concentrations, the normal effect of Mg2+ on relaxation is not detectable . We propose that Mg2+ allows the protein-DNA complex to assume a conformation necessary for strand passage and enhance the rate of enzyme turnover.

Biochem Biophys Res Commun, 1991 Apr 30, 176(2), 682 - 9
Nuclear overhauser effect studies of the conformations of Mg(alpha, beta-methylene)ATP bound to E . coli isoleucyl-tRNA synthetase; Williams JS et al.; Internuclear distances obtained from transferred nuclear Overhauser effects were used in combination with distance geometry calculations to define the E . coli isoleucyl-tRNA synthetase bound conformation of Mg(alpha, beta-methylene)ATP both in the absence and in the presence of the cognate and noncognate amino acids L-isoleucine and L-valine, respectively . A single nucleotide structure having an anti adenine-ribose glycosidic torsional angle of -114 degrees was found to satisfy the experimental distance constraints . The nearly identical anti glycosidic torsional angles observed in all three complexes demonstrate that the conformation of the adenosine moiety of the enzyme-bound nucleotide is not sensitive to the presence or to the nature of the amino acid bound at the aminoacyladenylate site . In addition, the acceptable range of Mg(alpha, beta-methylene)ATP conformations bound to the E . coli isoleucyl-tRNA synthetase was found to be nearly identical to that previously determined for the E . coli methionyl-tRNA synthetase (Williams and Rosevear (1991) J . Biol . Chem . 266, 2089-2098) . Thus, the predicted structural homology between the isoleucyl- and methionyl-tRNA synthetases, both members of the same class of synthetases on the basis of common consensus sequences, is further supported by consensus enzyme-bound nucleotide conformations.

FEBS Lett, 1991 Apr 22, 282(1), 157 - 60
Production of a full length Tat protein in E . coli and its purification; Armengaud J et al.; A full length tat gene was constructed by a combination of polymerase chain reaction (PCR) for the first exon and chemical synthesis for the second exon . This gene was expressed in E . coli under the control of the strongly regulated araB promoter, either directly or fused to a secretion signal encoding sequence . We then defined a rapid, three-step procedure for the purification of the Tat protein.

Biochem Biophys Res Commun, 1991 Apr 15, 176(1), 371 - 7
A novel method for the purification of porcine phospholipase A2 expressed in E . coli; Bhat KM et al.; Porcine phospholipaseA2 expressed in E . coli as a fusion protein was isolated, renatured and specifically cleaved by trypsin as described in (1) . Active phospholipaseA2, was purified to homogeneity on a column of PBE-94 over a pH region 7.4-4.5 . Using this method, several phospholipase A2 mutant enzymes have now been purified in a single step and all behaved identically during chromatofocusing . The method will therefore be extremely useful not only for those interested in understanding the structure-function relationships of phospholipaseA2 but also for preparing the enzyme in large quantities for industrial and pharmaceutical purposes.

Nucleic Acids Res, 1991 Apr 11, 19(7), 1593 - 9
Neural network optimization for E . coli promoter prediction; Demeler B et al.; Methods for optimizing the prediction of Escherichia coli RNA polymerase promoter sequences by neural networks are presented . A neural network was trained on a set of 80 known promoter sequences combined with different numbers of random sequences . The conserved -10 region and -35 region of the promoter sequences and a combination of these regions were used in three independent training sets . The prediction accuracy of the resulting weight matrix was tested against a separate set of 30 known promoter sequences and 1500 random sequences . The effects of the network's topology, the extent of training, the number of random sequences in the training set and the effects of different data representations were examined and optimized . Accuracies of 100% on the promoter test set and 98.4% on the random test set were achieved with the optimal parameters.

Mol Immunol, 1991 Apr-May, 28(4-5), 523 - 31
Peptide/antibody recognition: synthetic peptides derived from the E . coli tryptophan synthase beta 2 subunit interact with high affinity with an anti-beta 2 monoclonal antibody; Larvor MP et al.; Recent studies have shown that the antigenic determinant recognized by a monoclonal antibody (mAb 164-2) elicited against the beta 2 subunit of E . coli tryptophan synthase is localized between residues 276 and 297 of this protein . In order to delineate more precisely the epitope recognized by this antibody, peptides ranging in length from 11 to 29 amino acids and belonging to this region were synthesized, and their interactions with the antibody are described in this paper . The smallest peptide recognized with a high affinity by antibody 164-2 contains 11 residues (273-283) . This peptide is recognized by antibody 164-2 with an affinity (KD = 7.5 x 10(-9) M) very close to that of the native beta 2 subunit, suggesting a high structural similarity of the epitope inside the protein and in the isolated peptide . The corresponding sequence of beta 2 is located in a region protruding from the protein surface that contains a beta-turn as unique element of secondary structure in the crystallographic model . The absence of interaction between antibody 164-2 and the octapeptide lacking the three residues at the C-terminal end of peptide 11 suggests that the beta-turn is important in the recognition by the antibody . Kinetic studies were performed to find out whether or not the binding of the antibody to the peptide involves any conformational adaptation . The dissociation equilibrium constant (KD), the dissociation rate constant (koff) and the association rate constant (kon) were measured for eight peptide/antibody complexes . The values obtained were compatible with a one-step reaction, suggesting that no important conformational adaptation is involved in the formation of the peptide/antibody complexes . Furthermore, it has been shown that differences in affinity of antibody 164-2 for the various peptides were mainly due to differences in the dissociation rate constants (koff) and not in the association rate constants (kon) . The exceptional location of the epitope in the native protein and the unusually high affinity of the 11-residue peptide for mAb 164-2, makes this peptide a good model for studying the interaction between an antibody and a continuous epitope of a protein.

Biochimie, 1991 Apr, 73(4), 449 - 56
Repression of the E coli recA gene requires at least two LexA protein monomers; Thliveris AT et al.; To analyze the DNA binding domain of E coli LexA repressor and to test whether the repressor binds as a dimer to DNA, negative dominant lexA mutations affecting the binding domain have been isolated . A large number of amino acid substitutions between amino acid positions 39 and 46 were introduced using cassette mutagenesis . Mutants defective in DNA binding were identified and then examined for dominance to lexA+ . A number of substitutions weakened repressor function partially, whereas other substitutions led to a repressor with no demonstrable activity and a defective dominant phenotype . Since the LexA binding site has dyad symmetry, we infer that this dominance results from interaction of monomers of wild-type LexA protein with mutant monomers and that an oligomeric form of repressor binds to operator . The binding of LexA protein to operator DNA was investigated further using a mutant protein, LexA408, which recognizes a symmetrically altered operator mutant but not wild-type operator . A mixture of mutant LexA408 and LexA+ proteins, but neither individual protein, bound to a hybrid recA operator consisting of mutant and wild-type operator half sites . These results suggest that at least 1 LexA protein monomer interacts with each operator half site . We discuss the role of LexA oligomer formation in binding of LexA to operator DNA.

Biochimie, 1991 Apr, 73(4), 433 - 5
SOS-inducible DNA polymerase II of E coli is homologous to replicative DNA polymerase of eukaryotes; Shinagawa H et al.; The polB gene of Escherichia coli encodes DNA polymerase II whose role in vivo is not defined . The polB gene has been cloned and shown to be identical to a DNA damage-inducible gene dinA which is regulated by the LexA repressor . Nucleotide sequencing of polB reveals that E coli DNA polymerase II is highly homologous to replicative DNA polymerases of eukaryotes which include human DNA polymerase alpha and Saccharomyces cerevisiae DNA polymerases I, II and III . The polB gene is not required for growth, UV-repair and UV-mutagenesis.

Hum Genet, 1991 Apr, 86(6), 545 - 51
Molecular characterization of medium-chain acyl-CoA dehydrogenase (MCAD) deficiency: identification of a lys329 to glu mutation in the MCAD gene, and expression of inactive mutant enzyme protein in E . coli; Gregersen N et al.; A series of experiments has established the molecular defect in the medium-chain acyl-coenzyme A (CoA) dehydrogenase (MCAD) gene in a family with MCAD deficiency . Demonstration of intra-mitochondrial mature MCAD indistinguishable in size (42.5-kDa) from control MCAD, and of mRNA with the correct size of 2.4 kb, indicated a point-mutation in the coding region of the MCAD gene to be disease-causing . Consequently, cloning and DNA sequencing of polymerase chain reaction (PCR) amplified complementary DNA (cDNA) from messenger RNA of fibroblasts from the patient and family members were performed . All clones sequenced from the patient exhibited a single base substitution from adenine (A) to guanine (G) at position 985 in the MCAD cDNA as the only consistent base-variation compared with control cDNA . In contrast, the parents contained cDNA with the normal and the mutated sequence, revealing their obligate carrier status . Allelic homozygosity in the patient and heterozygosity for the mutation in the parents were established by a modified PCR reaction, introducing a cleavage site for the restriction endonuclease NcoI into amplified genomic DNA containing G985 . The same assay consistently revealed A985 in genomic DNA from 26 control individuals . The A to G mutation was introduced into an E . coli expression vector producing mutant MCAD, which was demonstrated to be inactive, probably because of the inability to form active tetrameric MCAD . All the experiments are consistent with the contention that the G985 mutation, resulting in a lysine to glutamate shift at position 329 in the MCAD polypeptide chain, is the genetic cause of MCAD deficiency in this family . We found the same mutation in homozygous form in 11 out of 12 other patients with verified MCAD deficiency.

Biotechniques, 1991 Apr, 10(4), 474, 476 - 7
Rapid identification of specific genes in E . coli by hybridization to membranes containing the ordered set of phage clones; Noda A et al.; A simplified protocol for the hybridization of labeled oligonucleotides and other radiolabeled DNAs to membranes is described and used for the detection of specific recombinant phage in the ordered miniset collection representing virtually the entire E . coli genome.

J Biochem Biophys Methods, 1991 Apr, 22(3), 233 - 41
Differential precipitation and zinc chelate chromatography purification of biologically active HTLV-I Tax1 expressed in E . coli; Lindholm PF et al.; A protocol which involves sequential ammonium sulfate precipitation and zinc chelate chromatography to purify the HTLV-I Tax1 protein expressed in E . coli is described . The final Tax1 product is greater than 90% pure and the yield is approximately 1 mg per liter of liquid culture . The purified Tax1 protein is biologically active in indirect in vitro DNA binding assays and cellular NF-kB induction experiments.

Enferm Infecc Microbiol Clin, 1991 Apr, 9(4), 223 - 5
{Detection of strains of E . coli producing STa toxin in the feces of patients with diarrhea using a competitive ELISA system}; Reina J et al.; We evaluate the presence of heat-stable STa toxin producers E . coli strains from stools of patients with acute diarrhoea . As detection method, we used a commercially-available enzyme-linked competitive assay (ELISA) . Over a three months period, 279 E . coli strains were analyzed . Only two (0.71%) were STa toxin producers . Both isolates are from pediatric cases (2 and 4 years of age), without any epidemiologic relationship between them . Therefore, we conclude that this infection could be considered as a non-epidemic community-acquired diarrhoeal disease.

Mol Gen Genet, 1991 Apr, 226(1-2), 318 - 20
Completion of the IS map in E . coli: IS186 positions on the E . coli K12 chromosome; Birkenbihl RP et al.; Insertion sites of the transposable element IS186 were physically mapped in the genome of E . coli K12 strain BHB2600 . This strain maintains four IS186 copies of which three, assigned to 0.3, 14.1 and 51.8 map min., share common map positions with the three IS186 copies in strains W3110 and HB101 . The fourth, unique IS copy in BHB2600 maps at 49.3 min . The IS186 data complete the BHB2600 map for all chromosomal sites of known K12-associated IS types.

Zhong Xi Yi Jie He Za Zhi, 1991 Apr, 11(4), 229 - 30, 198
{Effect of promoting biliation mixture on E . coli lipopolysaccharide under electron microscopy}; Qin MF et al.; With electron microscopy this article observed the effect of promoting biliation mixture (PBM) and polymyxin B on E . coli lipopolysaccharide (LPS) . The result showed that PBM could breakdown the typical structure of E . coli LPS with only short sections or partially disaggregate it . The morphology changes were similar to the effect of polymyxin B . It would appear that the loss of endotoxicity caused by PBM may be due to the loss of structural integrity of the E . coli LPS . The result may also give some evidence for the clinical effects of PBM theoretically.

Biokhimiia, 1991 Apr, 56(4), 687 - 93
{Hybridase cleavage of RNA . III . Use of UV-spectroscopy for studying the kinetic properties of E . coli RNAase properties}; Krynetskaia NF et al.; A one-step procedure for estimating the activity of ribonuclease H from E . coli has been developed . This method is based on continuous registration of the increment in the UV adsorption of the substrate in the course of the enzymatic reaction . The heteroduplex Am.dT20 (m = 18-24) was found to be the optimal substrate for the enzyme . A comparative analysis of the rates of the enzymatic reaction as determined by UV spectroscopy and ion-pair HPLC was carried out . The kinetic parameters of the Am hydrolysis in Am.dT20 catalyzed by E . coli RNase have been determined for the first time (Km = 44 +/- 11 nM, Vmax = 0.0363 +/- 0.0053 E) . The method sensitivity is 0.01-0.05 E which makes it possible to determine the RNAse H within the concentration range of 0.5-2.5 u./ml.

Infect Immun, 1991 Apr, 59(4), 1569 - 71
Escherichia coli O128 strains from infants with diarrhea commonly show localized adhesion and positivity in the fluorescent-actin staining test but do not hybridize with an enteropathogenic E . coli adherence factor probe; Scotland SM et al.; Twenty-nine strains of Escherichia coli O128 isolated from infants with diarrhea that did not produce heat-stable enterotoxin, heat-labile enterotoxins, or Vero cytotoxin showed localized attachment to HEp-2 cells (LA) . Only four strains hybridized with the enteropathogenic E . coli adherence factor (EAF) probe . One of the 25 LA+ EAF- strains attached to 72% of cells, while a plasmid-negative variant attached to 0.5% of cells . LA+ EAF- and LA+ EAF+ strains gave a positive fluorescent-actin staining test that correlates with the ability to cause attaching and effacing lesions in the intestine . The use of the EAF probe alone to detect LA+ strains is inadequate for epidemiological studies.

Biokhimiia, 1991 Apr, 56(4), 621 - 7
{Oxidative damage to E . coli membranes caused by superoxide radicals}; Sharonov BP et al.; The effects of bacterial membranes of active oxygen species photochemically generated by riboflavin-histidine systems were studied . According to SDS-PAAG data, the formation of high molecular weight protein aggregates and the appearance of fluorochromes whose fluorescence is seen in the longwave length region of the spectrum (lambda excit = 350 nm, lambda emis = 400-500 nm) and which are bound to the proteins, are suggestive of membrane oxidation consisting in the chemical modification of protein components . The presence in E . coli membranes of endogenous photosensitizers which upon illumination with visible light induce the oxidation of membrane proteins, was established.

Aust J Biotechnol, 1991 Apr, 5(2), 78 - 80, 86
A single step method for the solubilization and refolding of recombinant protein from E . coli inclusion bodies; Crivelli E et al.; Expression of recombinant porcine growth hormone (rpGH) in E . coli cells resulted in the accumulation of the rpGH within inclusion bodies (IBs) . The IBs were solubilized and the rpGH refolded in a single step using a cationic surfactant, N-cetyl pyridinium chloride (CPC; C21H38ClN) in the absence of reducing agents . No additional dialysis or rapid dilution steps of the solubilizing agent were required to obtain a 30% yield of refolded and oxidized rpGH monomer at protein concentrations of up to 15-20 mg/mL . In contrast, the refolding in vitro of rpGH in the absence of CPC resulted in the formation of significant amounts of higher molecular weight aggregate at the expense of the biologically active monomer . The fluorescence spectrum of the purified refolded rpGH was indistinguishable from that of biologically active, pituitary derived porcine GH and reverse-phase HPLC analysis of the purified rpGH showed similar retention times to that of pituitary GH . It is likely that the use of cetylpyridinium chloride is generally applicable to the simplified high yield recovery of biologically active recombinant proteins from IBs.

FEBS Lett, 1991 Mar 25, 280(2), 195 - 8
Alpha-haemolysin from E . coli . Purification and self-aggregation properties; Ostolaza H et al.; An improved, straightforward purification procedure for E.coli alpha-haemolysin has been developed . The protein exists in the form of large aggregates, held together mainly by hydrophobic forces . In the presence of urea or other chaotropic agents, the size of the aggregates decreases, while the specific activity is increased.

Cell, 1991 Mar 22, 64(6), 1145 - 53
The E . coli cell cycle and the plasmid R1 replication cycle in the absence of the DnaA protein; Bernander R et al.; In E . coli strain EC::71CW chromosome replication is under the control of the R1 miniplasmid pOU71 . A dnaA850::Tn10 derivative of EC::71CW was viable, which confirmed that R1 can replicate in the absence of the DnaA protein . The frequency of initiation of replication was, however, lowered and cell division was severely disturbed due to underreplication of the chromosome . Both replication and cell division could be restored to normal by increasing the production of RepA, the rate-limiting protein for initiation of replication from the integrated R1 origin . Therefore, the RepA protein seems to compensate for the absence of DnaA in the initiation of replication and assembly of replisomes . The role of the DnaA protein in the initiation of DNA replication, and as an overall regulator of the chromosome replication and cell division cycles of E . coli, is discussed in view of these results.

Alcohol Clin Exp Res, 1991 Mar, 15(2), 249 - 54
Acute ethanol intoxication suppresses E . coli lipopolysaccharide enhanced glucose utilization by hepatic nonparenchymal cells; D'Souza NB et al.; During infection or endotoxemia, the immune system is activated and its energy needs increase . Alcohol (ETOH) intoxication on the other hand suppresses the immune system and increases susceptibility to infection . Since the liver is the primary site both for metabolism of ETOH and detoxification of bacterial lipopolysaccharides (LPS), this investigation was directed at studying the effect of acute ETOH intoxication on the LPS-induced enhancement of in vivo glucose utilization in different types of hepatic cells . Rats were given an intravenous (IV) injection of ETOH followed by a constant infusion for 7 hr to maintain blood alcohol levels at about 175 mg/dl . E . coli LPS was administered IV at 4 hr and in vivo glucose utilization by the different types of liver cells was estimated 3 hr later using the 14C-2-deoxyglucose technique . Hepatocytes (HP), Kupffer (KC), and endothelial cells (EC), as well as the sequestered polymorphonuclear leukocytes (PMN), were separated from the liver by collagenase-pronase digestion followed by centrifugal elutriation and Ficoll-Hypaque density gradient centrifugation . The number of PMN in the liver was increased several-fold 3 hr after LPS administration . The presence of ETOH did not inhibit the LPS-induced neutrophil migration into the liver . ETOH depressed the LPS-induced increase in glucose uptake in both EC and KC by 50 to 80%, respectively . It also reduced the LPS-induced increase of plasma tumor necrosis factor activity by 80% . ETOH alone did not produce any significant changes in the parameters studied.(ABSTRACT TRUNCATED AT 250 WORDS)

Mutat Res, 1991 Mar, 247(1), 87 - 96
Evaluation of the DNA-repair host-mediated assay . III . Relationship between metabolic activation of dimethylnitrosamine and organ-specific differential lethality induced in E . coli indicator strains; Zeilmaker MJ et al.; In the present study the sensitivity of differential lethality as an endpoint for monitoring the presence of organ-specific genotoxic factors within the DNA-repair host-mediated assay (HMA) was determined . The induction of differential lethality in chemically exposed animals was assessed by measuring the recovery ratio Q, i.e., the relative survival of a repair-deficient E . coli K-12 derivative in comparison with its repair-proficient counterpart . Using untreated animals the interindividual fluctuation of the recovery ratio Q was first quantified and then used to determine the level below which it could be considered indicative of chemically induced differential lethality . This Q value was found to be 0.65 or lower . Using this criterion, a significant decrease of the Q value was observed in mice exposed to DMNA at a dose level as low as 15-30 mumole/kg, i.p . Inter-organ transport (liver----extrahepatic organs) of indicator bacteria was studied in reconstruction experiments using the direct-acting methylating agent MNU . These studies showed that inter-organ transport of indicator bacteria did not interfere with MNU-induced differential lethality . Time-related experiments were used to study the effects of inter-organ transport of genotoxic DMNA metabolites . In these studies significant, time-related differences were found in the induction of differential lethality in various organs of mice treated with DMNA . At a dose level of 200 mumole/kg (i.p.) genotoxic factors appeared within 25 min after administration in the liver . In the lungs and kidneys such factors appeared at a substantially slower rate, e.g., 20-120 min after DMNA administration . In persistence experiments differential lethality reached a maximum 30 min after DMNA treatment . No residual effects were detected 60 min after the injection of the carcinogen . These experiments showed that DMNA-derived genotoxic factors diffused from the liver into the bloodstream . The diffusion of these reactive species followed by their transport via the bloodstream to the lungs accounted for maximally 50% of differential lethality observed in bacteria recovered from the latter organ . In contrast, no indications were found for the transport of genotoxic DMNA metabolites from the liver via the bloodstream to the spleen and the kidneys . These results show that organ-specific effects observed in the DNA-repair HMA procedure after DMNA exposure can be primarily attributed to in situ metabolism, rather than diffusion of genotoxic metabolites from the liver to extrahepatic organs.

Virus Res, 1991 Mar, 19(1), 1 - 15
Antigenicity of hantavirus nucleocapsid proteins expressed in E . coli; Gott P et al.; DNA clones representing the small genomic segment of Nephropathia epidemica virus strain Hallnas B1 (NEV) and Hantaan virus strain 76-118 (HTV) encoding their nucleocapsid proteins were inserted into the E . coli vector pIN-III-ompA for secretion of proteins into the periplasmic space . The complete HTV and NEV nucleocapsid proteins and two truncated versions of the NEV nucleocapsid proteins were expressed as fusion proteins . Unexpectedly, all products accumulated as insoluble aggregates . Most of the ompA signal peptide remained uncleaved . However, nucleocapsid fusion proteins could be purified from the insoluble fraction by extraction with 8 M urea followed by separation on SDS-PAGE and electroelution . Rabbits were immunized with the eluted proteins and the resulting antibodies reacted specifically with authentic viral nucleocapsid proteins of HTV and NEV . The recombinant nucleocapsid proteins were found to react specifically with various hantavirus-immune sera, but not with human control sera, indicating their suitability as potential diagnostic antigens . This is the first report on the expression of a protein of a NEV serotype strain of hantaviruses by use of recombinant DNA techniques.

Cesk Epidemiol Mikrobiol Imunol, 1991 Mar, 40(2), 65 - 8
{A simple method for detection of P fimbriae in uropathogenic strains of E . coli}; Cejkova J et al.; The PF test of the Finnish firm Orion is used for detection of P fimbriae in E . coli strains . Its principle involves the establishment of a chemically defined receptor for these fimbriae on latex particles which can then be agglutinated by the appropriate strains . By means of this test the authors examined 453 uropathogenic E . coli strains . P fimbriae were detected in 121 strains, i.e . in 26.7% . Twenty-five strains agglutinated not only latex particles coated with receptor but also non-coated control particles . The test was thus falsely positive in 5.5% of the cases . Fifty-five strains, where by means of the PF test fimbriae were detected, were examined by the method of mannoresistent haemagglutination of human erythrocytes on a slide . The erythrocytes agglutinated in the presence of d-mannose of all 55 strains, and thus the results agreed in 100% . This suggests that P fimbriae can be detected by the haemagglutination method, but it must be taken into account that this method can, contrary to the PF test, detect also other adhesins . The PF test is very quick, simple and its principle ensures a high reliability.

Nucleic Acids Res, 1991 Feb 25, 19(4), 795 - 800
The effects of leader peptide sequence and length on attenuation control of the trp operon of E.coli; Roesser JR et al.; We have examined the effects of changing the length and codon content of the trp leader peptide coding region on expression of the trp operon of Escherichia coli, it had previously been shown that coupling of transcription and translation in the trp leader region is essential for both basal level control and tryptophan starvation control of transcription attenuation in this operon . We have found that increasing the length of the leader peptide coding region by 55 codons allowed normal basal level control and normal tryptophan starvation control . As expected, the presence of a nonsense codon early in the leader peptide coding region decreased basal expression and eliminated starvation control . Introducing tandem rare codons had no effect on basal level expression, but eliminated the tryptophan starvation response . Frameshifting at tandem rare codons was tested as the most likely explanation for loss of the tryptophan starvation response, but the results were inconclusive.

Nucleic Acids Res, 1991 Feb 25, 19(4), 801 - 8
Neighbourhood of the central fold of the tRNA molecule bound to the E . coli ribosome--affinity labeling studies with modified tRNAs carrying photoreactive probes attached to the dihydrouridine loop; Podkowinski J et al.; The neighbourhood of the dihydrouridine loop of tRNA molecule bound to E . coli ribosome has been studied by affinity labeling, using modified tRNAs carrying photoreactive azidonitrophenyl probes attached to the 3-(3-amino-3-carboxypropyl)-uridine located at position 20:1 of Lupin methionine elongator tRNA . The maximum distance between the pyrimidine ring and the azido group estimated for the two probes employed in this study is 10-11 A and 18-19 A, respectively . Cross-linking of the uncharged, modified tRNAs has been studied with poly(A, U, G) as a message, under conditions directing uncharged tRNAs preferentially to the ribosomal P-site . Modified tRNAs bind covalently to both ribosomal subunits with high yields upon irradiation of the respective non-covalent complexes . Proteins S7, L33 and L1 have been consistently found cross-linked to tRNAs modified with both probes, and S5 and L5 to tRNA modified with the longer probe . Surprisingly, an S5-tRNA cross-linking product is reproducibly found in a protein fraction prepared from the purified 50S subunit . Cross-linking to rRNAs is significant only for the longer probe and is stimulated 2-4 fold in the presence of poly(A,U,G) . The cross-linking sites are located between nucleotides 1302 and 1398 in 16S rRNA and between nucleotides 2281 and 2358 in 23S rRNA.

J Chromatogr, 1991 Feb 22, 539(2), 343 - 53
Purification of E . coli 30S ribosomal proteins by high-performance liquid chromatography under non-denaturing conditions; Cachia C et al.; High-performance ion-exchange chromatography was applied to the separation of proteins from the 30S ribosomal subunit under non-denaturing conditions . It was shown that a single chromatographic step only allows the purification of nine proteins . To increase the number of separated proteins, a prefractionation step was added that depends on the physical characteristics of the proteins to be purified . Sixteen out of 21 proteins could be purified by using prefractionation (gel permeation and lithium chloride salt washing) . This method is well suited to preparing fresh samples on demand for optical studies owing to the simplicity of the buffers used and the amounts of proteins recovered in the eluted peaks (0.05-0.1 mg/ml).

Nucleic Acids Res, 1991 Feb 11, 19(3), 637 - 47
Mapping sequenced E.coli genes by computer: software, strategies and examples; Rudd KE et al.; Methods are presented for organizing and integrating DNA sequence data, restriction maps, and genetic maps for the same organism but from a variety of sources (databases, publications, personal communications) . Proper software tools are essential for successful organization of such diverse data into an ordered, cohesive body of information, and a suite of novel software to support this endeavor is described . Though these tools automate much of the task, a variety of strategies is needed to cope with recalcitrant cases . We describe such strategies and illustrate their application with numerous examples . These strategies have allowed us to order, analyze, and display over one megabase of E . coli DNA sequence information . The integration task often exposes inconsistencies in the available data, perhaps caused by strain polymorphisms or human oversight, necessitating the application of sound biological judgment . The examples illustrate both the level of expertise required of the database curator and the knowledge gained as apparent inconsistencies are resolved . The software and mapping methods are applicable to the study of any genome for which a high resolution restriction map is available . They were developed to support a weakly coordinated sequencing effort involving many laboratories, but would also be useful for highly orchestrated sequencing projects.

FEBS Lett, 1991 Feb 11, 279(1), 49 - 51
Physico-chemical properties of actin cleaved with bacterial protease from E . coli A2 strain; Khaitlina SYu et al.; The 36 kDa fragment of actin molecule obtained with the protease from E . coli A2 strain {(1988) FEBS Lett . 228, 172} was shown to begin with Val-43 and retain the COOH-terminal amino acid residues of the parent molecule . The E . coli protease split actin preserves the NH2-terminal part of the polypeptide chain as well as the native conformation of actin molecule . However, the E . coli protease split actin failed to polymerize in 0.1 M KCl, suggesting that integrity of actin molecule between Gly-42 and Val-43 is crucial for actin polymerization.

Res Microbiol, 1991 Feb-Apr, 142(2-3), 189 - 94
Mutants defective in chromosome partitioning in E . coli; Hiraga S et al.; Recent experimental results suggest that replicated daughter chromosomes (nucleoids) in Escherichia coli move non-progressively and abruptly at an early stage of the D (division) period from midcell toward the cell quarter positions, which will become the centres of the daughter cells . The chromosome positioning at the quarter positions was found to be controlled by the muk gene products . In muk mutants, normal size anucleate cells are spontaneously produced during cell division . The mukA gene is identical to the tolC gene encoding an outer membrane protein . The mukB gene codes for a 177-kDa protein . The amino acid sequence of the MukB protein deduced for the nucleotide sequence suggests that the MukB protein has five characteristic secondary structure domains: an amino-terminal globular domain containing a consensus sequence binding with ATP or another nucleotide . The central region of the protein consists of two alpha-helical coiled-coil domains and one globular domain . A carboxyl-terminal globular domain is rich in cysteine and positively charged residues arginine and lysine . Two MukB protein molecules might form a homodimer in the coiled-coil regions . The predicted secondary structure of the MukB protein suggests that the protein provides the force required for the positioning of nucleoids from midcell toward the cell quarters . The mukC and mukD genes are located at 88 and 41 min of the chromosome map, respectively.

Res Microbiol, 1991 Feb-Apr, 142(2-3), 127 - 30
E . coli minichromosome replication: regulation of initiation at oriC; Crooke E et al.; The initiation of Escherichia coli DNA replication is a highly regulated event with many parameters exerting positive and negative effects . The activity of the dnaA protein (the initiator protein) is profoundly influenced by the tight binding of the adenine nucleotides ATP and ADP . Further regulation of dnaA protein activity may occur through dnaA protein-cell membrane associations . A replicatively inactive form of dnaA protein is found aggregated with phospholipids; enzymatic treatment of the aggregates with phospholipase A2 or dnaK protein liberates dnaA protein with restored replication activity . Proper DNA structure is essential for replication . The energy stored in the DNA's supercoiling is crucial for dnaA protein's ability to initiate replication . Under conditions where strand-opening by dnaA protein is inhibited, such as low free superhelicity, an R-loop formed by RNA polymerase activates the origin at a distance by aiding strand-opening . A novel protein has been identified as a specific inhibitor of the initiation of DNA replication . This 33-kDa protein binds to the AT rich region of oriC and inhibits strand-opening by dnaA protein.

Mol Biol Rep, 1991 Feb, 15(1), 9 - 18
Small acidic peptides are bound to E . coli DNA; Felici F et al.; Low molecular weight peptides have been isolated by alkali extraction from deproteinized DNA of E . coli cells grown in the presence of radioactive glutamic acid or orthophosphate . The labeled peptides, purified by gel filtration chromatography on Sephadex G25 and G10, contain prevailingly glutamic acid, aspartic acid, glycine, serine and alanine . Electrophoretic studies at different pH show that some peptide fractions contain a phosphoric residue . The N-terminus of the phosphorylated peptides is apparently blocked and they were able to bind to DNA in the presence of Mg2+ ions . Moreover the acidic peptides extracted from E . coli DNA show a sharp activity in the control of lambda phage DNA transcription 'in vitro'.

J Virol Methods, 1991 Feb-Mar, 31(2-3), 301 - 14
Synthesis of fusion proteins of Epstein-Barr virus nuclear antigens in E . coli and their antigenicity; Inoue N et al.; Expression and yield in E . coli of a panel of fusion proteins containing various domains of Epstein-Barr virus nuclear antigens, EBNA-1, EBNA-2, EBNA-3, EBNA-4 and EBNA-6, were scrutinized . The antigenicity of the EBNA fusion proteins against human sera was examined . Monospecific antisera to the different EBNA domains were produced by immunizing guinea pigs and rabbits . An EBNA-6 fusion polypeptide was useful for separating anti-EBNA-6 antibody from human sera by immunoaffinity purification . The applications of the fusion proteins to clinical diagnosis are discussed.

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