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