Nucleic Acids Res, 1989 Jul 11, 17(13), 5057 - 69 Cosmid-derived map of E . coli strain BHB2600 in comparison to the map of strain W3110; Birkenbihl RP et al.; A physical map for the genome of E . coli K12 strain BHB2600 was constructed by use of 570 cloned DNA elements (CDEs) withdrawn from a cosmid library . Dot blot hybridisation was applied to establish contig interrelations with subsequent fine mapping achieved by analysis of EcoR1 restriction patterns on Southern blots . The derived map covers nearly 95% of the E . coli genome resulting in 12 minor gaps . It may be compared to the almost complete map for strain W3110 of Kohara et al . (1) . Except for one tiny gap (lpp,36.5') remaining gaps in BHB2600 do not coincide with those in W3110 so that both maps complement each other establishing an essentially complete clone represented map . Besides numerous minute differences (site and fragment gains and losses) both strains harbour at differing positions extended rearrangements flanked by mutually inverted repetitive elements, in our case insertion elements (IS1 and IS5). Nucleic Acids Res, 1989 Jul 11, 17(13), 4947 - 56 Uranyl mediated photofootprinting reveals strong E . coli RNA polymerase--DNA backbone contacts in the +10 region of the DeoP1 promoter open complex; Jeppesen C et al.; Employing a newly developed uranyl photofootprinting technique (Nielsen et al . (1988) FEBS Lett . 235, 122), we have analyzed the structure of the E . coli RNA polymerase deoP1 promoter open complex . The results show strong polymerase DNA backbone contacts in the -40, -10, and most notably in the +10 region . These results suggest that unwinding of the -12 to +3 region of the promoter in the open complex is mediated through polymerase DNA backbone contacts on both sides of this region . The pattern of bases that are hyperreactive towards KMnO4 or uranyl within the -12 to +3 region furthermore argues against a model in which this region is simply unwound and/or single stranded . The results indicate specific protein contacts and/or a fixed DNA conformation within the -12 to +3 region. FEBS Lett, 1989 Jul 3, 250(2), 201 - 4 Diacylglycerol breakdown in plasma membrane of rat intestinal epithelial cells . Effect of E . coli heat-stable toxin; Banik ND et al.; Rat intestinal epithelial cells were isolated and the activity of the enzyme diacyglycerol lipase (DG lipase, EC 3.1.1.3) was investigated . When cells were treated with Escherichia coli heat-stable toxin (ST) liberation of endogenous glycerol and fatty acids was observed . The enzyme responsible for this effect could be demonstrated to be a DG lipase by using specific substrates . It was found that the activity of DG lipase was increased 5-6-fold with the substrates diolein and 1,2-dioleyl-rac-glycerol and triolein being neutral lipid insensitive to DG lipase . ST had no direct effect on the DG lipase . The enzyme DG lipase was activated via a chain reaction due to the hydrolysis of phosphatidylinositol (PI) by the enzyme PI-specific phospholipase C stimulated by ST. Am J Physiol, 1989 Jul, 257(1 Pt 1), G118 - 23 Differences in jejunal and ileal response to E . coli enterotoxin: possible mechanisms; Cohen MB et al.; Escherichia coli, which produce heat-stable enterotoxin (STa), cause intestinal fluid secretion as a mechanism of diarrhea . To determine the factors that modulate the intestinal secretory response, we first compared the time course of the STa-induced secretion in ligated in situ loops of rat jejunum and ileum . We found that the jejunal secretory response was brief (less than or equal to 30 min) while the ileal response to STa was sustained (greater than or equal to 3 h) . We then compared the modification of purified STa that occurred in jejunum and ileum and found a close correlation between the continued presence of unmodified, authentic STa and continued fluid secretion in the ligated-loop model . At both sites alteration of STa was demonstrated by high-performance liquid chromatography profile and brush-border membrane binding activity . However, in the jejunum, the modification of STa was qualitatively different and quantitatively much greater . We conclude that the degree to which STa is inactivated or removed from the intestine correlates with the secretory response observed . Inactivation of STa may be a mechanism by which the host limits its secretory response. Biochem Int, 1989 Jul, 19(1), 163 - 72 Expression of the kinase domain of mouse protein kinase C in E . coli; Dietrich A et al.; The kinase domain of mouse protein kinase C type alpha has been expressed at high levels in E . coli . The protein has been purified 500-fold taking advantage of the fact that highly expressed fusion proteins precipitate out in the bacterial cell and can be solubilized in 7 M urea . The purified protein can be detected with an antibody generated against a PKC alpha derived synthetic peptide . The purified kinase domain exhibits no measurable kinase activity in a protamin phosphorylation assay . This could be an indication that post-translational modifications of the protein kinase domain which do not happen in bacteria are a requirement for functional enzyme activity or that the regulatory domain of protein kinase C is indispensable for kinase function. Comput Appl Biosci, 1989 Jul, 5(3), 211 - 8 An extension of the graph theoretical approach to predict the secondary structure of large RNAs: the complex of 16S and 23S rRNAs from E . coli as a case study; Thanaraj TA et al.; An algorithm using the graph theoretical approach to predict secondary structures of large nucleic acids is discussed . Reliability of prediction can be improved by incorporating available experimental data and sequence homology information . As a case study, this algorithm is applied to predict the secondary structure of the 16S-23S rRNA complex from E . coli . It was found that several structures of the complex can coexist . The computer program developed to predict the secondary structure of large RNAs can be run on IBM PC/AT compatible systems. Nucleic Acids Res, 1989 Jun 26, 17(12), 4799 - 815 A novel method for promoter search enhanced by function-specific subgrouping of promoters--developed and tested on E.coli system; Rozkot F et al.; A new method for evaluating some complex characteristics of the primary structure of E.coli promoters is proposed . The method, of nonparametric statistical significance, selects important conserved single-base positions in combination with 2-base coupling relations of identity and complementarity . The extended consensus of promoter characteristics thus obtained was used to scan unknown sequences for similarity with E.coli promoters . In terms of this method, a complete set of 244 E.coli promoters was shown to be structurally inconsistent . The set was then broken down into functionally homogeneous subsets of promoters to enhance the selectivity of the search for E.coli-specific promoter sequences, with a high significance level being attained. Cell, 1989 Jun 2, 57(5), 869 - 80 The interaction of E . coli IHF protein with its specific binding sites; Yang CC et al.; We have used two kinds of footprinting techniques, dimethylsulfate interference and hydroxyl radical protection, to explore the way that IHF recognizes its specific target sequences . Our results lead us to conclude that IHF recognizes DNA primarily through contacts with the minor groove, an unprecedented mode for a sequence-specific binding protein . We have also determined that, although IHF is a small protein that protects a large region of DNA, only a single IHF protomer is present at each binding site . IHF bends the DNA to which it binds . We have combined this fact plus our footprinting and stoichiometry data together with the crystal structure of a related protein, the nonspecific DNA binding protein HU, to propose a model for the way in which IHF binds to its DNA target. J Biomol Struct Dyn, 1989 Jun, 6(6), 1071 - 6 Preliminary crystallographic analysis of a proteolytically modified form of E . coli single stranded DNA binding protein; Ng JD et al.; A proteolytically modified form of the Escherichia coli single-stranded DNA-Binding protein (SSB) has been crystallized from 15% saturated sodium citrate . Crystals as large as 1.0 mm x 0.3 mm x 0.2 mm were obtained and these diffract beyond 3A resolution . X-ray photographic analysis demonstrated a rhombohedral unit cell of space group R3 with an equivalent triple centered hexagonal unit cell having dimensions of a = b = 62.9A and c = 264.3A . These crystals were judged to be adequate for a three dimensional structure determination. Mol Gen Mikrobiol Virusol, 1989 Jun, (6), 16 - 20 {Biosynthesis of the membrane protein of the acquired immunodeficiency syndrome virus (HIV) with a deleted hydrophobic region in E . coli cells}; Starov AI et al.; Effect of the structure of cloned env gene of HIV virus on the level of its expression has been studied . The deletion of the hydrophobic region from the env gene has been shown to increase considerably the biosynthesis of antigen-specific proteins in Escherichia coli cells . At the same time, the low level of expression of the gene fragment the transcription of which is controlled by a powerful lac-promoter suggests the presence of toxic regions of nonhydrophobic nature in the synthesized antigen. Wei Sheng Wu Xue Bao, 1989 Jun, 29(3), 228 - 31 {Restriction map of E . coli shuttle plasmid (p# GTE5) with secretive function}; Chen YX et al.; The shuttle plasmid (p# GTE5) DNA with secretive function was extracted by the alkali lysozyme method from E . coli RRI strain . Its molecular weight is 4.5 Md and DNA size is 6.9 Kb . Restriction fragments of plasmid was obtained by single and double enzymes complete digestion using five different restriction endonucleases . The restriction map of shuttle plasmid (p# GTE5) was established for the enzymes EcoRI, BglII, pstI, PvuII, and TaqI. FEMS Microbiol Lett, 1989 Jun, 50(3), 345 - 9 Detection and identification of pathotypes of verocytotoxigenic Escherichia coli isolated from weaned piglets using gene probes for seven E . coli toxins; Mainil J et al.; Seventy verocytotoxigenic (VTEC) and sixty-three non VTEC haemolytic Escherichia coli isolated from recently weaned piglets were examined by the colony hybridization assay using gene probes for three verocytotoxins: Edema disease principle (EDP) and Shiga-like toxins I and II (SLTI and SLTII) . The results with the EDP and SLTII probes were identical . All VTEC hybridized with these two probes, while non VTEC did not . All 133 E . coli were negative for the SLTI probe . Hybridization of the plasmid content of 14 VTEC did not show any evidence for plasmid localization of the genes coding for the EDP . The 70 VTEC were also assayed with gene probes for heat-stable (STaP, STb) and heat-labile (LT, LTIIa) enterotoxins . Only the STb probe was hybridized by 36 of them . Most STb-positive isolates belonged to serotype O141: K85 biotypes 9 and 13 PC. Zentralbl Hyg Umweltmed, 1989 Jun, 188(3-4), 380 - 4 UV disinfecting experiments with E.coli and actinometric determination of the irradiation intensity; Zemke V et al.; The UV disinfection of E . coli was carried out and additionally an actinometric determination of the irradiation intensity was made . This kind of proceeding renders it possible--in regard to the time of exposure--to set up a dose-effect-relation for the UV disinfection of water. Mutat Res, 1989 Jun, 212(2), 269 - 74 Modifying action of gamma-radiation in mutagenesis of E . coli WU36-10 induced by ethylene oxide, ethyl methanesulfonate and methyl methanesulfonate; Kolman A et al.; The influence of gamma-radiation pre-exposure on ethylene oxide, ethyl methanesulfonate and methyl methanesulfonate mutagenesis in Escherichia coli WU36-10 was studied . Pretreatment with gamma-radiation resulted, in the case of subsequent treatment with ethylene oxide and ethyl methanesulfonate, in a decrease of the frequency of leu+ revertants, and in the case of subsequent treatment with methyl methanesulfonate, in an increase of this mutation frequency. Brain Res Mol Brain Res, 1989 Jun, 5(4), 271 - 8 Grafting genetically modified cells into the rat brain: characteristics of E . coli beta-galactosidase as a reporter gene; Shimohama S et al.; The utility of grafting genetically modified cells to the mammalian brain was examined using the E . coli beta-galactosidase gene (lacZ) as a reporter gene in retroviral infection . Following implantation of the infected cells to the brain, lacZ continued to be expressed in vivo and could be detected easily with enzyme histochemistry . However, beta-galactosidase-positive cells were also observed in control grafts which had not been infected with the virus . This false-positive staining was found to be endogenous lysosomal activity associated with macrophage infiltration presumably induced by the damage associated with grafting . The E . coli gene product was distinguished from cellular lysosomal beta-galactosidase by using immunohistochemical staining with an antibody specific for E . coli beta-galactosidase . With this antibody, retrovirus-infected cells could be distinguished in the brain, and no false positives were observed in non-infected cells . We conclude that E . coli beta-galactosidase is a useful reporter gene for determining the fate of implanted cells to the brain if appropriate caution is taken to distinguish it from cellular beta-galactosidase by immunocytochemical procedures. Nucleic Acids Res, 1989 May 25, 17(10), 3855 - 63 Effect of the sequence-dependent structure of the 17 bp AT spacer on the strength of consensuslike E.coli promoters in vivo; Lozinski T et al.; Three E.coli promoters with the consensus sequences in the -35 and -10 regions and the 17 bp spacer made of random, heteronomous, and of both these classes of AT DNA simultaneously were constructed and cloned into plasmid pDS3 . Electrophoretic gel mobilities of restriction fragments containing these promoters indicated that bending of the latter was proportional to the number of heteronomous AT DNA tracts . The strength of these promoters in vivo measured in relation to an internal transcriptional standard was shown to correlate well with gel mobilities of the respective restriction fragments and to decrease with the number of potential DNA bending sites encoded in the promoter structure. Zhonghua Min Guo Wei Sheng Wu Ji Mian Yi Xue Za Zhi, 1989 May, 22(2), 145 - 9 {Detection of heat-labile enterotoxin produced by E . coli with reversed passive latex agglutination}; Wang FI et al.; From June 1987 to March 1988, 103 E . coli strains were collected from non-foodborne samples, foodborne samples, FDA (USA) etc . and analyzed for heat-labile E . coli enterotoxin (LT) by reversed passive latex agglutination (RPLA) . The results showed that 84 strains were LT-positive, 19 strains were LT-negative . Forty six strains of enteropathogenic E . coli isolated from foodborne samples showed that 34 strains were LT-positive, 12 strains were negative . Four enterotoxigenic E . coli strains from FDA (USA) used as positive controls, can produce heat-labile E . coli enterotoxin and give positive results. Nippon Seikeigeka Gakkai Zasshi, 1989 May, 63(5), 569 - 79 {Production of rheumatoid factor-like substance and arthritic findings in knees of mice immunized with E . coli 0:14}; Itoh J; Four strains of mice (A . SW, B10 . D2, C57BL/6, DBA/1J) were immunized by subcutaneous injections of heat-killed E . coli 0: 14 . After single or multiple immunizations, histologic sections of knee joints and a serum rheumatoid factor-like substance (RFLS) at different time intervals were studied . Immunized A . SW . mice developed hyperplasia of synovial lining cells (25 out of 49 knees) and pannus (9 out of 49 knees) which was significantly higher rate than control . A serum rheumatoid factor-like substance (RFLS) was detected in B10 . D2 and C57BL/6 mice by using RAHA test and enzyme-linked immunosorbent assay . Serum IgM-RFLS was positive in all B10 . D2 mice (11 of 11 mice) immunized for 24 weeks . The incidence of serum IgM-RFLS positive mice in C57BL/6 immunized for 40 weeks (3 of 4 mice) was significantly higher than in control (p less than 0.05) . Production of serum RFLS was not recognized in a single-immunized group of B10 . D2 mice on statistical evaluation . These findings provided evidence that arthritis was developed in multiple-immunized groups of A . SW mice and RFLS was produced in multiple-immunized groups of B10 . D2 and C57BL/6 mice . In addition, these findings suggested that multiple-immunization was necessary for the production of RFLS. Trends Genet, 1989 May, 5(5), 139 - 43 The great GATC: DNA methylation in E . coli; Barras F et al.; In Escherichia coli the methylation of the adenine in the sequence 5'-GATC-3' is catalysed by the dam gene product, a DNA adenine methylase . We review the proposed roles for this methylation, and the sequence it modifies, in mismatch repair, DNA-protein interaction, gene expression, the initiation of chromosome replication, chromosome segregation, chromosome structure and the occurrence of mutational hotspots. Antibiot Khimioter, 1989 May, 34(5), 370 - 4 {Evolutionary changes in the structural and regulatory regions of aminoglycoside-3'-phosphotransferase type I gene of E . coli}; Vakulenko SB et al.; To investigate the evolutionary relationships between the aph(3') genes from different plasmids, the nucleotide sequence of the aph(3') gene from the E . coli R plasmid was determined and compared with the known aph(3') genes of Tn903 and Tn4352 . Three point mutations in the structural part of the cloned aph(3') gene caused amino acid changes in the enzyme molecule at positions 19, 27 and 48 beginning from the start codon . The structural part of the gene was followed by two stop codons and a long DNA region containing no nucleotide sequences homologous to the sequences of Tn903 or Tn4352 . Both the cloned aph(3') gene and Tn4352 were limited on the left by the spacer sequence and the insertion sequence IS176 . Twenty one base pairs deletion abolished the -35 sequence of the promoter suggested for the aph(3') gene of Tn4352 and resulted in formation of a fusion promoter utilizing the -35 box of IS176 and the -10 box of the aph(3') gene . The distance between the -35 and -10 sequences changed from 18 to 17 bp . Changes in the cloned aph(3') gene and the flanking DNA regions resulted in formation of a new promoter and loss of the right IS176 element. Biofizika, 1989 May-Jun, 34(3), 464 - 7 {The effect of dimethylsulfoxide and thiourea on the water diffusion permeability of the E . coli membrane}; Sakharov BV et al.; It is shown that diffusive water permeability of E . coli cell membranes at 4-24 degrees C is in the range from 16.6 to 35.0 microM.s-1, that is close to erythrocyte membrane water permeability, but higher than that of lipid bilayers . Cryoprotectants (DMSO and thiourea) at the concentration of 1.0 M considerably decrease water bacterial membrane permeability . 1.5- and 2-fold, respectively . The obtained results are discussed in relation to two possible water transport ways through pores of the protein nature or lipid bilayer damages. Immunol Lett, 1989 May, 21(2), 157 - 63 Expression of Shiga toxin epitopes in E . coli immunological characterization; McEwen J et al.; Synthetic oligodeoxynucleotides encoding for two peptides corresponding to residues 9-21 and 19-31 in the amino acid sequence of the B subunit of Shiga toxin were prepared and inserted into pTOZ plasmid, in phase with the lacZ gene . The resultant vectors were used for transfection of E . coli . The bacteria containing the recombinant DNA expressed the respective peptides in the form of fusion proteins with beta-galactosidase . The N-terminal region of the Shiga toxin B subunit, containing the two peptides, was previously identified as a relevant epitope of the toxin, leading to the induction of neutralizing antibodies . The present study demonstrates that the bacterial extracts containing the fusion proteins as the products of the recombinant vectors, when used for immunization of rabbits, elicited a humoral immune response which was specific toward the respective peptides . Furthermore, the antibodies cross-reacted with the intact Shiga toxin. Biokhimiia, 1989 May, 54(5), 870 - 6 {Synthesis in E . coli cells of short RNA encoded in plasmids}; Dontsova OA et al.; The synthesis of 5S rRNA and 4.5S RNA in E . coli HB 101 cells harbouring plasmids pKK 5-1 and pKK 247-2 was studied . The plasmids were derived from pBK 322 and contained genes coding for 5S rRNA and 4.5S RNA with regulatory elements of an rRNA transcription operon rrn B . When the cells were grown on enriched or minimal media (2 and 0.3 duplications per hour), the synthesis of both 5S rRNA and 4.5S RNA was proportional to the gene dosage and was greater in the plasmid than in the host strain . Such RNA accumulation did not change the cell growth parameters and was thus not toxic for the cells . At high growth rates, the RNA synthesis in the cells became excessive, and the processing system was upset with the accumulation of RNA precursors . The fact confirms the hypothesis, according to which the whole rRNA operon is essential for its own feedback regulation. Nucleic Acids Res, 1989 Apr 25, 17(8), 3179 - 97 Effect of nucleotide sequences directly downstream from the AUG on the expression of bovine somatotropin in E . coli; Tomich CS et al.; We have studied the expression of bovine somatotropin (BSt) to gain more understanding of various factors affecting translation in E . coli . The unmodified cDNA coding for mature bovine somatotropin does not produce significant amounts of BSt in E . coli using a pBR322-derived vector . However, a translation fusion with 16 codons from trpLE in front of BSt cDNA results in greater than 20% of total cell protein as the fusion product . Analysis of transcription by measuring the rate and integrity of the mRNA confirms that a post-transcriptional event is responsible for the poor expression of the BSt cDNA . There are two potential stem-loop structures in the 5' region of the mRNA which may interfere with translation . To study their effect on translation, lacZ fusions and oligonucleotide mutagenesis were carried out . The results demonstrate that the secondary structure involving the initiation codon blocks translation initiation . Removal of this stem-loop results in a 100-fold increase in BSt expression . However, the expression level is still low, amounting to only 0.5-1% of total cell protein . High level expression can be obtained by replacement of the beginning sequence of BSt cDNA with trpLE codons . These results suggest that in addition to the secondary structure, the nucleotide sequence or amino acid context within the beginning of BSt is incompatible with one of the steps in translation initiation. Nucleic Acids Res, 1989 Apr 25, 17(8), 2933 - 45 In vivo gene expression directed by synthetic promoter constructions restricted to the -10 and -35 consensus hexamers of E . coli; Jacquet MA et al.; Two synthetic DNA sequences, carrying no other known E . coli promoter element than the consensus hexamers (CH) TTGACA (CH-35) and TATAAT CH(-10), spaced by 17 bp, were inserted in pBR329, in a position enabling transcription of the complete Cmr gene . The region upstream of the Cmr transcription start was carefully cleared of w.t . promoter elements (full deletion of the wild type (w.t.) Cmr promoter upstream +2 and large portion of an upstream coding sequence) . Both synthetic promoters, which differ only by the sequences of the spacers (non consensus, constrained in AT or GC) support in vivo high level Cmr gene expression . The GC rich spacer is associated with transcription start at the usual +1 position, but with the AT rich spacer, transcription starts at several places, mainly in CH(-10) . Rearranged promoter sequences derived from the synthetic ones upon transformation with partly ligated plasmids, yield new insights on the role of the standard CH pair, the size of the spacer and the sequence downstream of CH(-10). FEBS Lett, 1989 Apr 24, 247(2), 361 - 6 Structural consequences of a one atom mutation on aspartate transcarbamylase from E . coli; Cherfils J et al.; Tyr-240 of the catalytic chain of aspartate transcarbamylase from E . coli has been substituted by Phe using site-directed mutagenesis . The regulatory mechanisms of the mutant enzyme have been shown to be slightly less effective than the wild-type enzyme . A study of the structural consequences of the mutation using solution X-ray scattering and computer simulations is reported here . No significant change from the wild-type enzyme is detectable in the quaternary structure . Simulations suggest that the only effect of the mutation is an increased mobility of the mutated side chain. J Biomol Struct Dyn, 1989 Apr, 6(5), 971 - 84 Use of lead(II) to probe the structure of large RNA's . Conformation of the 3' terminal domain of E . coli 16S rRNA and its involvement in building the tRNA binding sites; Gornicki P et al.; The present work shows that lead(II) can be used as a convenient structure probe to map the conformation of large RNA's and to follow discrete conformational changes at different functional states . We have investigated the conformation of the 3' domain of the E . coli 16S rRNA (nucleotides 1295-1542) in its naked form, in the 30S subunit and in the 70S ribosome . Our study clearly shows a preferential affinity of Pb(II) for interhelical and loop regions and suggests a high sensitivity for dynamic and flexible regions . Within 30S subunits, some cleavages are strongly decreased as the result of protein-induced protection, while others are enhanced suggesting local conformational adjustments . These rearrangements occur at functionally strategic regions of the RNA centered around nucleotides 1337, 1400, 1500 and near the 3' end of the RNA . The association of 30S and 50S subunits causes further protections at several nucleotides and some enhanced reactivities that can be interpreted in terms of subunits interface and allosteric transitions . The binding of E . coli tRNA-Phe to the 70S ribosome results in message-independent (positions 1337 and 1397) and message-dependent (1399-1400, 1491-1492 and 1505) protections . A third class of protection (1344-1345, 1393-1395, 1403-1409, 1412-1414, 1504, 1506-1507 and 1517-1519) is observed in message-directed 30S subunits, which are induced by both tRNA binding and 50S subunit association . This extensive reduction of reactivity most probably reflects an allosteric transition rather than a direct shielding. Wei Sheng Wu Xue Bao, 1989 Apr, 29(2), 113 - 6 {Subcloning of K88ac antigen gene of enterotoigenic E . coli and the restriction map of recombinant plasmid}; Zhang LY et al.; We had reported a recombinant E . coli RR1(pNZ8801) which was obtained from a wild strain E . coli 79-1454 . The recombinant plasmid was digested by EcoRI and generated three segments, medium segment (3.2Md) was removed, the largest and the smallest segment was ligased, then the mixture was transformed into E . coli RRI, screening Ap(r) Tc(s) clones, one of recombinants was named E . coli RR1(pNZ8802) . The recombinant plasmid molecular weight is smaller, but expression of K88ac antigen is higher than first cloning . Subcloning can adhere to mucosae of piglet's intesting . Therefore, the recombinant can be use for oval living vaccine. Bioorg Khim, 1989 Apr, 15(4), 562 - 5 {Synthesis of oligoribonucleotides with the use of RNA polymerases of E . coli and immobilized synthetic DNA-templates}; Denisova LIa et al.; A new approach to synthesis of oligoribonucleotides is suggested, based on transcription by E . coli RNA polymerase of synthetic immobilized DNA-templates with AUG as primer . The approach has been experimentally verified by synthesis of two oligonucleotides, viz., a RNA fragment of the fr phage (16 nucleotides long) and a RNA fragment of the tickborne encephalitis virus (18 nucleotides long) . Fraction of the synthesized RNA fragments in the whole nucleotide material is about 20% . The templates can be used repeatedly . Sequences of the oligoribonucleotides were confirmed . Advantages of this approach and its usefulness for SP6 DNA-dependent RNA polymerase are discussed. EMBO J, 1989 Apr, 8(4), 1271 - 7 Autoregulation and multiple DNA interactions by a transcriptional regulatory protein in E . coli pili biogenesis; Forsman K et al.; An operon mediating biogenesis of digalactoside-binding pilus-adhesin of serotype F13 in uropathogenic Escherichia coli includes the regulatory gene papB . The papB gene product was found to act as transcriptional activator of an operon which includes the papB gene and several pap cistrons encoding the proteins of the pilus polymer . Studies of how pap gene expression was affected by increasing amounts of PapB protein in the cells showed that high levels did not stimulate transcription but caused repression . Results from in vitro studies demonstrated that the PapB protein was a sequence-specific DNA-binding protein . Binding studies using gel mobility shift assays and DNase I protection (footprinting) showed that PapB protein binds to three separate sites . A sequence greater than 200 bp upstream of the promoter, and directly adjacent to a binding site for the cAMP receptor protein-cAMP complex, appeared as a preferential PapB binding site . A second site was localized to sequences overlapping the -10 region of the promoter and a third binding site was found within the coding sequence of the papB gene itself . The data suggest that the PapB protein has a dual function as activator/repressor of pilus-adhesin transcription and that its autoregulatory mode of action involves differential binding to separate sites. Int J Radiat Biol, 1989 Apr, 55(4), 593 - 604 Radiosensitization of E . coli B/r by arylhydrazonopropanedinitriles; Crump PW et al.; Several arylhydrazonopropanedinitriles and an arylhydrazonopropane-diethyl ester (derivatives of well-known uncouplers of oxidative phosphorylation) have been studied with respect to their ability to radiosensitize E . coli B/r under oxic and hypoxic conditions . Of the compounds studied, 2-carboxyphenylhydrazono-propanedinitrile and 2-carboxyphenylhydrazonopropanediethylester were found to be the most efficient radiosensitizers under hypoxia, whilst the former compound was also found to provide radiosensitization under oxic conditions . Increased radiosensitization by 4-carboxyphenylhydrazonopropanedinitrile was observed on decreasing the pH of the irradiation incubation medium . The results are discussed with respect to the physicochemical properties of these compounds and their reactivity with thiols, for which data are presented. Biochimie, 1989 Apr, 71(4), 411 - 6 Structure of cDNA of cytosolic aspartate aminotransferase of chicken and its expression in E . coli; Mattes U et al.; The structure of the mRNA of chicken cytosolic aspartate aminotransferase has been determined by analysis of cDNA and genomic clones . Two transcripts of different length were found that appear to arise from the alternate use of 2 polyadenylation signals in the 3' untranslated region . The expression product of the full-length construct in E . coli proved to be catalytically active and possessed the expected molecular weight. Bioorg Khim, 1989 Apr, 15(4), 492 - 8 {Expression of synthetic human angiogenin gene in E . coli in cleaved beta-galactosidase-angiogenin protein}; Kovalenko SP et al.; A synthetic gene coding for human angiogenin was cloned in pUR290 plasmid in frame with beta-galactosidase, both parts of the resultant fused protein being joined through an Asp-Pro sequence . The fused protein, synthesised in E . coli cells upon IPTG induction and isolated as inclusion bodies, possessed angiogenic activity on the chick chorioallantois membrane and was cleaved upon acid treatment to yield free angiogenin. Biochim Biophys Acta, 1989 Mar 16, 995(1), 54 - 8 Kinetic consequences of site-specific mutation of Glu-239----Gln in E . coli aspartate transcarbamylase: comparison with catalytic subunits and Phe-240 mutant enzyme; Hsuanyu Y et al.; The kinetic characteristics of E . coli aspartate transcarbamylase, altered by site-specific mutagenesis of Glu-239----Gln, have been determined by equilibrium isotope-exchange kinetics and compared to the wild-type system . In wild-type enzyme, residue Glu-239 helps to stabilize the T-state structure by multiple bonding interactions with Tyr-165 and Lys-164 across the c1-c4 subunit interface; upon conversion to the R-state, these bonds are re-formed within c-chains . Catalysis of both the {14C}Asp in equilibrium C-Asp and {32P}ATP in equilibrium Pi exchanges by mutant enzyme occurs at rates comparable to those for wild-type enzyme . Saturation with different reactant/product pairs produced kinetic patterns consistent with strongly preferred order binding of carbamyl-P prior to Asp and carbamyl-Asp release before Pi . The kinetics for the Gln-239 mutant enzyme resemble those observed for catalytic subunits (c3), namely a R-state enzyme (Hill coefficient nH = 1.0) and Km (Asp) approximately equal to 6 mM . The Glu-239----Gln mutation appears to destablize both the T- and R-states, whereas the Tyr-240----Phe mutation destablizes only the T-state. Cell, 1989 Mar 10, 56(5), 891 - 904 Reverse transcriptase-dependent synthesis of a covalently linked, branched DNA-RNA compound in E . coli B; Lim D et al.; We have found a branched DNA-RNA compound in E . coli B, that is similar in its secondary structure, but not its nucleotide sequence, to the previously described branched DNA-RNA compounds in myxobacteria . This compound is not produced in E . coli K12 . We have cloned a 3.5 kb chromosomal segment of E . coli B, which, when transferred into E . coli K12, leads to the production of the DNA-RNA compound . We describe the isolation of the DNA-RNA compound, the determination of its nucleotide sequence, and the nucleotide sequence of the genes required for its formation . The sequence contains the coding regions for the DNA component, the RNA component, and an open reading frame encoding a reverse transcriptase . This reverse transcriptase is shown to be required for the formation of the DNA-RNA compound in vivo and in vitro. Electrophoresis, 1989 Mar, 10(3), 165 - 72 Free flow electrophoresis as a method for the purification of enzymes from E . coli cell extract; Kuhn R et al.; The application of the four techniques of free flow electrophoresis (zone electrophoresis, isotachophoresis, isoelectric focusing and field step electrophoresis) for the purification of proteins from a complex protein mixture was investigated . For this purpose alpha-amylase (EC 3.2.1.1) from Aspergillus oryzae was added and reisolated from E . coli cell extract . The chosen enzyme and the biological extract are models for many industrial separation problems . In optimized experiments purity, purification factor, yield, throughput and efficiency were calculated . The best results were obtained with field step electrophoresis in combination with zone electrophoresis . High purity (0.82 mg enzyme/mg total protein) and high throughput (111 mL sample/h) were achieved using this technique . Field step electrophoresis gave the best throughput (330 mL sample/h), but low purity (0.63 mg enzyme/mg total protein) . This technique can also be used for a simple concentration of the sample . With zone electrophoresis a purity of more than 0.95 mg enzyme/mg total protein was obtained, which was the best of all techniques . However, the enzyme concentration was decreased due to dilution with buffer solution after the separation . Isotachophoresis was the most difficult technique, combined with a relatively low recovery of 31% of the enzyme activity . In a purification scheme, free flow electrophoresis is able to substitute one or even several chromatography steps with a negligible loss of biological activity. Immunology, 1989 Mar, 66(3), 394 - 7 Comparison of concentration and avidity of specific antibodies to E . coli in breast milk and serum; Sennhauser FH et al.; To investigate the relationship between mucosal and systemic immunity we analysed the specific anti-Escherichia coli antibody concentration and avidity of IgA in colostrum and IgG in paired blood samples from 47 mothers giving birth to premature neonates . The avidity of each sample, expressed as an avidity index, was determined using a novel enzyme immunoassay (EIA)-based procedure, while specific antibody determinations were performed by means of conventional sandwich EIA techniques . All subjects had detectable antibody to E . coli in serum and breast milk . The median avidity index for specific IgA antibody in breast milk (3.53 M NH4SCN, range 2.77-4.90) was significantly higher (P less than 0.0001) than that for specific IgG antibody in serum (median 2.03 M NH4SCN, range 1.15-3.65) . Using Spearman correlation analysis, a weak but significant association was found between the avidity of colostral IgA antibody and the avidity of systemic IgG antibody to pooled E . coli polysaccharides (rs = 0.29, P = 0.02) . There was also a weak correlation between the concentrations of specific serum IgG antibody and of specific colostral IgA antibody (rs = 0.36, P = 0.006) . There was no correlation between the concentration of IgA anti-E . coli antibody in colostrum and the avidity of colostral IgA antibody (rs = 0.14, P less than 0.05) . Similarly, there was no correlation between the concentration and the avidity of serum IgG anti-E . coli antibody (rs = 0.23, P less than 0.05) . The findings of this study suggest independent regulation of concentration and avidity of specific IgA antibody in preterm breast milk . Similar results were seen for specific IgG antibody in serum . The correlations between systemic and mucosal antibody with respect to both concentration and avidity were significant, but are relatively weak and therefore suggest that there also may be independent factors which afford differential regulation of systemic and mucosal antibody responses. Biofizika, 1989 Mar-Apr, 34(2), 322 - 3 {N,N'-dicyclohexylcarbodiimide-sensitive K+-dependent H+ secretion from anaerobically grown E . coli cells}; Trchunian AA et al.; Dependence of N,N'-dicyclohexylcarbodiimide (DCC)-sensitive H+ secretion of K+ activity was discovered . This dependence took place only in anaerobically grown bacteria, and only at the structural intact DCC-sensitive H+-ATPase complex and K+-ionophore Trk. Biochimie, 1989 Mar, 71(3), 307 - 12 Structural analysis of human skeletal beta-tropomyosin produced in E . coli; Ferraz C et al.; We have cloned the cDNA coding the beta-tropomyosin of human muscle in an expression vector whose expression depends upon a promotor that can be induced by isopropyl-beta-thiogalactopyranoside . We show that a new protein was synthesized by bacteria containing the engineered plasmid . This protein was heat stable and reacted with antibodies against tropomyosin . We have purified this protein and further identified it by determining its amino acid composition and sequencing the NH2 terminal . Unlike the native muscle tropomyosin, the NH2 terminal is not acetylated and contains a methionine . The circular dichroism spectrum is compatible with 100% alpha-helices . These results show that the protein synthesized in E . coli possesses a native structure. Mutat Res, 1989 Mar, 217(2), 123 - 34 KIN, a mammalian nuclear protein immunologically related to E . coli RecA protein; Angulo JF et al.; A polypeptide of about 120 kDa, called KIN, has been identified in rat FR 3T3 cells by immunoblotting using affinity-purified antibodies against the RecA protein of Escherichia coli (38 kDa) . The KIN protein as shown by fluorescent light microscopy and electron microscopy is essentially concentrated in the nucleus . Its level is higher in proliferating than in quiescent cells . Cell treatment with mitomycin C increases the level of the KIN protein . We sought similar proteins in other mammalian cells . Proteins with the same electrophoretic mobility were detected in mouse, monkey and human cell lines as well as in rat and mouse embryos. Mol Microbiol, 1989 Mar, 3(3), 295 - 302 Molecular cloning and expression in Escherichia coli K-12 of the O101 rfb region from E . coli B41 (O101:K99/F41) and the genetic relationship to other O101 rfb loci; Heuzenroeder MW et al.; The gene cluster (rfb region) which determines the synthesis of O101 lipopolysaccharide (LPS) O-antigen was cloned from the Escherichia coli O101:K99:F41 reference strain B41 to give plasmid pPM1301 . The smallest subclones represented by pPM1305 and pPM1330 expressed O-antigen in E . coli K-12 similar to (but not identical to) B41, as judged by immunogold electron microscopy and silver staining of LPS separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) . At least six proteins were detected by minicell analysis of proteins encoded by pPM1305, which suggests that O-antigen synthesis is genetically complex . Restriction and deletion analysis demonstrated that a minimum of 8.9 kb and a maximum of 11.8 kb are required for O101 O-antigen biosynthesis in E . coli K-12 . Examination of LPS banding patterns of other O101 isolates by SDS-PAGE suggested heterogeneity of LPS structure . Southern DNA hybridization analysis using radiolabelled subclones of pPM1305 demonstrated that there was close relationship among the O101 ETEC isolates. Acta Chir Scand, 1989 Mar, 155(3), 155 - 7 Factors influencing the outcome of E . coli peritonitis in rats; Andersson R et al.; Substances that may influence the course and outcome of intra-abdominal sepsis were investigated in an experimental model of Escherichia coli peritonitis in rats . All rats received an intraperitoneal injection of E . coli . In the first set of experiments, substances commonly contaminating the abdominal cavity after trauma were intraperitoneally injected, and the following mortality rates were found: saline solution (controls) 27%, hemoglobin solution 80% (p less than 0.01), whole blood 20% (p greater than 0.05), whole blood together with bile 93% (p less than 0.001) and bile 87% (p less than 0.01) . In the second set of experiments, intravenous injection of commonly used solutions gave mortality rates of 20% (controls) for saline solution, 80% for dextran (p less than 0.01) and 47% for Intralipid (p greater than 0.05) . E . coli peritonitis in rats thus was aggravated by intraperitoneal hemoglobin, bile or whole blood plus bile, and also by intravenous dextran. Acta Chir Scand, 1989 Mar, 155(3), 151 - 4 Influence of dextran on blood clearance and organ distribution of radiolabelled E . coli and on survival in experimental E . coli septicemia; Andersson R et al.; Previous reports indicated impaired function of the reticuloendothelial system following administration of dextran . In the present experimental study, intravenous injection of Escherichia coli resulted in four deaths among 15 rats pretreated with i.v . dextran 70, but none in 15 controls (p less than 0.05) . To evaluate the influence of dextran on bacterial clearance from blood and distribution in organs, 125I-labelled, heat-killed E . coli was injected 1 or 24 hours following i.v . injection of 1.0 ml sterile saline solution or 1.0 ml dextran 70 . After both of these intervals, dextran resulted in significant (p less than 0.05) decrease of blood clearance compared with the controls . The organ distribution in counts/minute (cpm)/g tissue was compared in liver, spleen and lungs . Splenic uptake was reduced at both times following dextran administration, while there was increase in pulmonary uptake at 1 hour and in hepatic uptake at 24 hours post-dextran (p less than 0.005 and p less than 0.02, respectively) . Evaluation of cpm/total organ weight showed no change after dextran injection, except for increased (p less than 0.05) pulmonary uptake after 1 hour. Biochim Biophys Acta, 1989 Mar 1, 1007(2), 158 - 66 The pnd gene in E . coli plasmid R16: nucleotide sequence and gene expression leading to cell Mg2+ release and stable RNA degradation; Sakikawa T et al.; The pnd gene promotes the degradation of stable RNA in the presence of rifampicin at 42 degrees C, but is repressed during normal growth (Ohnishi, Y . and Akimoto, S . (1980) J . Bacteriol . 144, 833-835) . We have determined the sequence of a third srnB-pnd-type gene, and have analyzed the effects of its expression from an inducible promoter . The nucleotide sequence of the pnd gene of the R16 plasmid exhibits an open reading frame for a polypeptide with 50 amino-acid residues, with high sequence homology to the pnd gene of a plasmid (R483) of a different incompatibility group . A possible base-paired stem and loop structure, which may participate in the regulation of gene expression, was detected between the promoter and the initiation codon, analogous to that in two comparable genes, srnB in the F and pnd in the R483 plasmid . When bacterial cells containing a lac-pnd fusion plasmid were incubated with a lac inducer at 30 degrees C, magnesium was released from the cells in bulk, and spheroplasts of the cells lysed even in hypertonic solution . Furthermore, when Mg2+ efflux was inhibited in the medium containing 5 mM Mg2+ or in Tris-HCl buffer, the degradation of stable RNA at 42 degrees C was inhibited . These results suggest that expression of the pnd gene effects a release of cellular magnesium by a membrane alterations, resulting in the stable RNA degradation at a higher temperature. Biochem Biophys Res Commun, 1989 Feb 28, 159(1), 87 - 94 Chemical synthesis and expression of the HIV-1 protease gene in E . coli; Louis JM et al.; The 297bp HIV-1 protease gene was constructed from five discrete synthetic fragments and expressed in E . coli . A soluble protein product of 11.5 Kd was detected by immunoblotting using protease specific antisera . A quantitative assay system, utilizing a synthetic nonapeptide spanning the cleavage site between p17-p24 in the gag polyprotein, was used to measure the specific protease activity in crude extracts . The protease hydrolyzed tyrosyl-proline bonds with an approximate specific activity of 43 pmoles/min/micrograms of total protein . The chemical synthesis of the protease gene and it's expression provides a feasible method for rapid mutant analysis, important for structure-function studies and rational design of potential inhibitors. Nucleic Acids Res, 1989 Feb 25, 17(4), 1475 - 91 New RNA-protein crosslinks in domains 1 and 2 of E . coli 30S ribosomal subunits obtained by means of an intrinsic photoaffinity probe; Hajnsdorf E et al.; Functionally active 70S ribosomes containing 4-thiouridine (s4U) in place of uridine were prepared by a formerly described in vivo substitution method . Proteins were crosslinked to RNA by 366 nm photoactivation of s4U . We observe the systematic and characteristic formation of 30S dimers; they were eliminated for analysis of RNA-protein crosslinks . M13 probes containing rDNA inserts complementary to domains 1 and 2 of 16S RNA from the 5'end up to nucleotide 868 were used to select contiguous or overlapping RNA sections . The proteins covalently crosslinked to each RNA section were identified as S3, S4, S5, S7, S9, S18, S20 and S21 . Several crosslinks are compatible with previously published sites for proteins S5, S18, S20 and S21; others for proteins S3, S4, S7, S9, S18 correspond necessarily to new sites. Cell, 1989 Feb 24, 56(4), 641 - 9 A division inhibitor and a topological specificity factor coded for by the minicell locus determine proper placement of the division septum in E . coli; de Boer PA et al.; The E . coli minicell locus (minB) was shown to code for three gene products (MinC, MinD, and MinE) whose coordinate action is required for proper placement of the division spetum . Studies of the phenotypic effects of expression of the three genes, alone and in all possible combinations, indicated the following: cell poles contain potential division sites that will support additional septation events unless specifically inactivated; the minC and minD gene products act in concert to form a nonspecific inhibitor of septation that is capable of blocking cell division at all potential division sites; and the minE gene codes for a topological specificity factor that, in wild-type cells, prevents the division inhibitor from acting at internal division sites while permitting it to block septation at polar sites. Nucleic Acids Res, 1989 Feb 11, 17(3), 1139 - 57 Terminal sequences do not contain the rate-limiting decay determinants of E . coli cat mRNA; DeFranco C et al.; The mechanism of E . coli chloramphenicol acetyltransferase (cat) mRNA decay was investigated . Alteration of the 5' untranslated terminus does not appear to have an effect on the turnover rate of the mRNA . Similarly, changes at the 3' terminus of the message, including the addition of a stable stem and loop structure, do not affect the half-life of the message . The data suggest that 5' and 3' terminal untranslated sequences do not contain the rate-limiting determinants for cat message decay . Decay rates for various segments of the cat mRNA were measured and indicate that all regions of the message have similar stabilities . The current model of cat mRNA degradation involves a rate-limiting endonucleolytic decay event that occurs internal to the message followed by degradation of the cleavage products. Sci China B, 1989 Feb, 32(2), 186 - 92 The expression of enterotoxin A-B+ gene of V . cholerae in E . coli; Ma QJ et al.; The cloning in E . coli of a cholerae toxin gene that is A-B+ has been successfully constructed by using DNA recombinant techniques . E . coli cells carrying the recombinant plasmid pMM-CTB have been shown to produce a large amount of CTB subunits which are secreted as extracellular proteins. EMBO J, 1989 Feb, 8(2), 569 - 76 Isolation of point mutations that affect the folding of the H chain of human ferritin in E.coli; Luzzago A et al.; We have approached the problem of folding and assembly of the heavy (H) chain of human ferritin by isolating point mutations that affect this process . Apoferritin is an ideal model system to approach the problem of protein folding and assembly into multimeric structures . We have developed a recombinant hybrid molecule that allows us to select for ferritin mutants in which the folding-assembly process is altered or completely impaired . The selection procedure is based on a recombinant protein which consists of a fusion between the H chain of human ferritin and the alpha-peptide of beta-galactosidase . In the wild type situation, the alpha-peptide domain is segregated inside the apoferritin shell upon assembly and is unable to interact with the substrate and perform its enzymic function . We show that by selecting for mutations that restore beta-galactosidase activity we are able to identify ferritin mutations that affect the folding-assembly process . The selective procedure was applied to the analysis of the amino acid side chains that are important for the attainment of the correct conformation of the carboxy-terminal E helix in the 4-fold axis. Klin Wochenschr, 1989 Feb 1, 67(3), 113 - 8 A rapid phospholipase A2 bioassay using 14C-oleate-labelled E . coli bacterias; Meyer T et al.; Two methods of phospholipase A2 determination using 14C-labelled E . coli bacterias as substrate were compared . One method works with a filter membrane for separation of cleaved 14C-oleate from remaining phospholipids, the other uses the well-known thin-layer chromatography for lipid analysis . Some features of human serum phospholipase A2 regarding pH and Ca2+ dependency were investigated . Possible sources of errors were discussed . It was shown that either method can differentiate between normal and pathologically elevated phospholipase A2 levels, but that the filter method is superior in terms of sensitivity and workload. Eur J Biochem, 1989 Feb 1, 179(2), 399 - 404 Modulation of the affinity of the single-stranded DNA-binding protein of Escherichia coli (E . coli SSB) to poly(dT) by site-directed mutagenesis; Bayer I et al.; A vector for site-directed mutagenesis and overproduction of the Escherichia coli single-stranded-DNA-binding protein (E . coli SSB) was constructed . An E . coli strain carrying this vector produces up to 400 mg pure protein from 25 g wet cells . The vector was used to mutate specifically the Phe60 residue of E . coli SSB . Phe60 had been proposed to be located near the single-stranded-DNA-binding site . Substitution of the Phe60 residue by Val, Ser, Leu, His, Tyr and Trp gave proteins with no or only minor conformational changes, as detected by NMR spectroscopy . The affinity of the mutant E . coli SSB proteins for single-stranded DNA decreased in the order Trp greater than Phe (wild-type) greater than Tyr greater than Leu greater than His greater than Val greater than Ser, leading to the conclusion that position 60 is a site of hydrophobic interaction of the protein with DNA. FEBS Lett, 1989 Jan 30, 243(2), 299 - 302 Nucleotide residues of tRNA, directly interacting with proteins within the complex of the 30 S subunit of E . coli ribosome with poly(U) and NAcPhe-tRNA(Phe); Abdurashidova GG et al.; With the aid of photoinduced tRNA-protein cross-linking, nucleotide residues A21, U45 and U60 were shown to interact directly with proteins S5, S7 and S9 respectively, in the complex of the 30 S subunit of E . coli ribosome with poly(U) and NAcPhe-tRNA(Phe) . These nucleotide residues are located in the central part of the L-form in the tertiary structure of RNA. FEBS Lett, 1989 Jan 30, 243(2), 293 - 8 Identification of the magnesium, europium and lead binding sites in E . coli and lupine tRNAPhe by specific metal ion-induced cleavages; Marciniec T et al.; The Pb, Eu and Mg-induced cleavages in E . coli and lupine tRNAPhe have been characterized and compared with those found in yeast tRNAPhe . The pattern of lupine tRNAPhe hydrolysis closely resembles that of yeast tRNAPhe, while several major differences occur in the specificity and efficiency of the E . coli tRNAPhe hydrolysis . The latter tRNA is cleaved with much lower yield in the D-loop, and interestingly, cleavage is also detected in the variable region, that is highly resistant to hydrolysis in eukaryotic tRNAs . The possible location of tight Pb, Eu and Mg binding sites in E . coli tRNAPhe is discussed on the basis of the specific hydrolysis data. FEBS Lett, 1989 Jan 30, 243(2), 186 - 92 Synthesis of a (desSer1 Ile29 Leu89) chicken cystatin gene, expression in E . coli as fusion protein and its isolation; Auerswald EA et al.; A synthetic gene coding for the cysteine proteinase inhibitor (desSer1 Ile29 Leu89) chicken cystatin was cloned and expressed in E . coli . The gene was assembled from 12 oligonucleotides and inserted into vector pUC 8 . Expression as fusion protein was performed in a temperature-inducible E . coli system . The expression product was synthesized as 20% of total E . coli protein . The fusion protein was purified, the chicken cystatin homologue was split off with CNBr and the N-terminal sequence confirmed up to position 37 . The properties of the purified material correspond to those of natural chicken cystatin . The recombinant cystatin variant binds anti-chicken cystatin IgG, is inhibitorily active and displays Ki values with papain and with cathepsin B similar to those determined for natural chicken cystatin. FEMS Microbiol Lett, 1989 Jan 15, 48(2), 129 - 32 Detection of a homologue to an E . coli glutamate synthase gene in a cyanobacterium; Lightfoot DA et al.; The gltBY locus of E . coli, which codes for the two subunits of pyridine nucleotide dependent glutamate synthase, was used as a probe to detect homologues in genomic DNA of Synechococcus PCC6301, a unicellular cyanobacterium . Non-overlapping probes from gltB detected a single homologue with extensive homology, however gltY probes do not detect a strong homologue . The possibility that the gltB homologue encodes a ferredoxin-dependent glutamate synthase of the type found in cyanobacteria, algae and plants is discussed. Nucleic Acids Res, 1989 Jan 11, 17(1), 317 - 34 The interaction of E . coli integration host factor and lambda cos DNA: multiple complex formation and protein-induced bending; Kosturko LD et al.; The interaction of E . coli's integration Host Factor (IHF) with fragments of lambda DNA containing the cos site has been studied by gel-mobility retardation and electron microscopy . The cos fragment used in the mobility assays is 398 bp and spans a region from 48,298 to 194 on the lambda chromosome . Several different complexes of IHF with this fragment can be distinguished by their differential mobility on polyacrylamide gels . Relative band intensities indicate that the formation of a complex between IHF and this DNA fragment has an equilibrium binding constant of the same magnitude as DNA fragments containing lambda's attP site . Gel-mobility retardation and electron microscopy have been employed to show that IHF sharply bends DNA near cos and to map the bending site . The protein-induced bend is near an intrinsic bend due to DNA sequence . The position of the bend suggests that IHF's role in lambda DNA packaging may be the enhancement of terminase binding/cos cutting by manipulating DNA structure. Free Radic Res Commun, 1989, 8(1), 1 - 9 The molecular genetics of superoxide dismutase in E . coli . An approach to understanding the biological role and regulation of SODS in relation to other elements of the defence system against oxygen toxicity; Touati D; Superoxide-mediated oxidative stress initiates a chain of events resulting in numerous cellular injuries . We have used genetics and E . coli to investigate the role and regulation of superoxide dismutase (SOD) and its relationship with the other constituents of the oxygen toxicity defence system . Structural SOD genes have been cloned and sequenced, permitting us to refine structural analysis and to isolate SOD-deficient mutants . The conditional oxygen sensitivity of these mutants, together with their increased mutation rate, demonstrated the essential biological role of SOD . Furthermore the complementation of SOD-lacking E . coli deficiencies by introducing a plasmid containing the gene encoding the human SOD supported the proposal that superoxide dismutation is the physiological function of SOD . Regulation of the MnSOD, through which the global SOD level in E . coli is modulated, has been studied using operon and protein fusions with the lactose operon, and led to the conclusion of a multicontrol of MnSOD . Isolation and characterization of regulation mutants are in progress, with the aim of identifying effectors and targets involved in the response to superoxide-mediated oxidative stress. Oncogene Res, 1989, 5(1), 61 - 6 Expression of human and viral ets genes in E . coli production of human ets-2-specific monoclonal antibodies; Seth A et al.; Full-length and deletion variants of the ets proto-oncogene and v-ets oncogene of E26 virus were expressed in E . coli using the previously described expression vectors pANH1, pJL6 and pJLA16 . The recombinant proteins were expressed at greater than 5% of the total cellular proteins and were characterized by Western blot analysis using ets-specific antipeptide antibodies . A number of monoclonal antibodies were raised against p35, the expressed product of the partial human ets-2 proto-oncogene construct . These monoclonal antibodies are highly specific and recognize the p56 ets-2 product from several human cell lines. Ann Ist Super Sanita, 1989, 25(1), 163 - 9 Molecular analysis of mutagenesis in E . coli; Fuchs RP; In this paper we present a general strategy that is suitable for the analysis of the forward mutation spectrum induced by any physical or chemical mutagen or carcinogen . The assay is based upon the inactivation of the tetracycline resistance gene located on plasmid pBR322 . Plasmid DNA is treated in vitro with the mutagen and transformed into the host bacteria of choice . Mutant clones are selected and analysed by sequencing . We present also two techniques that allow the determination of the DNA modification spectrum . The comparison for a given mutagen of the modification spectrum and the induced mutation spectrum permits the identification of hot spot sequences . Using different chemical mutagens (derivatives of the carcinogenic aromatic amide N-2-acetylaminofluorene, cis-platinum, etc.) this assay was found to be able to detect the different classes of mutagenic events: base substitutions, frameshifts, insertions and deletions . The advantages and limitations of this assay are discussed. Bioorg Khim, 1989 Jan, 15(1), 78 - 89 {Klenow fragment of DNA-polymerase I from E . coli . III . The role of internucleotide phosphate groups of the matrix in its binding with the enzyme}; Volchkova VA et al.; The modification of Klenow fragment of DNA polymerase I E . coli was investigated by the affinity reagents d(Tp)2C{Pt2+(NH3)2OH}(pT)7 and d(pT)2pC{Pt2+(NH3)2OH}(pT)7 . The template binding site of the enzyme was modified by these reagents in the presence of NaF (5 mM), which inhibits selectively the 3'----5'-exonuclease activity of the enzyme and therefore prevents the reagent from degradation . NaCN destroyed covalent bonds between reagents and enzyme, restoring activity of the Klenow fragment . The affinity of different ligands (inorganic phosphate, nucleoside monophosphates, oligonucleotides) to the template binding site of Klenow fragment was estimated . Minimal ligands capable to bind with the template site were shown to be triethylphosphate (Kd 290 microM) and phosphate (Kd 26 microM) . Ligand affinity increases by the factor 1.76 per an added (monomer unit from phosphate to d(pT) and then for oligonucleotides d(Tp)nT (n 1 to 19-20) . At n greater than 19-20, the ligand affinity remained constant . The complete ethylation of phosphodiester groups lowers affinity of the oligothymidylates to the enzyme by approximately 10 times, and comparable decrease of Pt2+-oligonucleotide affinity to polymerase is caused by the absence of Mn2+-ions . The data obtained led to suggestion that one Me2+-dependent electrostatic contact of the template phosphodiester group with the enzyme takes place (delta G = -1.45...-1.75 kcal/mole) . Formation of a hydrogen bond with the oxygen atom of P = O group of the same template phosphate is also assumed (delta G = -4.8...-4.9 kcal/mole) . Other template internucleotide phosphates do not interact with the enzyme but the bases of oligonucleotides take part in hydrophobic interactions with the template binding site . Gibbs energy changes by -0.34 kcal/mole when the template is lengthened by one unit. Folia Biol (Praha), 1989, 35(2), 65 - 77 Cloning of Japanese quail ovalbumin cDNA in E . coli; Klaudiny J et al.; Japanese quail oviduct total cDNA was synthesized from total mRNA by the classical method, using AMV reverse transcriptase, the Klenow fragment of DNA polymerase I and S1 nuclease . The results of the synthesis of total ss-cDNA partially differed from those published for the synthesis of chicken total ss-cDNA . The presumed causes of the differences in the complete reverse transcription of mRNAcon and incomplete reverse transcription of mRNAlys and a large part of mRNAov in our case are discussed . An atypical strategy was used for cloning full-length cDNAov . The total cDNA was dC-tailed, then fractionated by analytical agarose electrophoresis and 4 cDNA fractions of different lengths were isolated from the gel using DEAE cellulose membranes . A cDNA fraction about 1500-2500 bp long containing full-length cDNAov was annealed with dG-tailed PstI-linearized plasmid pBR322 and cloned into competent E . coli DHl cells . Seventy-two clones were screened for the presence of full-length cDNAov, initially by insert size and then by means of hybrid-arrested translation . Four clones containing 1900-1980 bp cDNAov were obtained . The cDNA ends in one of these clones were sequenced . Comparison of these sequences with those of chicken mRNAov indicated that almost full-length cDNAov's had been cloned . They lacked a small number of nucleotides at their 5' ends, which had probably been split off during the degradation of the hairpin loop by S1 nuclease . A sequence of 134 bases from the 5' end of mRNAov is presented and compared with the known sequence of chicken mRNAov . The advantages of the cloning strategy employed, in particular, its cloning efficiency and the possibility of simultaneously identifying clones of also other oviduct cDNA species (in this work: cDNAY and, tentatively, cDNAcon), are discussed. Arch Esp Urol, 1989 Jan-Feb, 42(1), 11 - 7 {Morphopathologic study of renal glomeruli in Wistar rats in shock experimentally induced with E . coli}; Mendez Sanchez A et al.; The present study analyzed the structural and ultrastructural changes observed in the kidneys of Wistar rats inoculated with E . coli bacteria suspended in saline solution . For the study, we divided 64 Wistar rats into 2 groups . Two ml . of the suspension containing 9.5 X 10(10) E . coli 0 group 26 CECT, no . 351 were given to the rats in group A via the oral route . Rats in group B were inoculated with 1 ml . of the same suspension . Structurally, we observed an increased glomerular area caused by the increased number and activation of mesangial cells . These showed developed organoids, podocytes with dilated organoids of the cytoplasmic vacuolar system, and very fine, disorganized pedicels . The parietal cells revealed vacuolized cytoplasm, and the basement membranes of the glomerular capillaries were thickened and arranged in layers . In the lumen of the glomerular, vessels, we observed histiocytic elements on endothelial walls, with large amounts of lysosomal elements and residual bodies . Inoculation of the E . coli suspension causes renal shock, which is more intense when innoculation is via the intraperitoneal route, causing severe changes in organ function. Radiobiologiia, 1989 Jan-Feb, 29(1), 23 - 9 {The mutagenic effect of accelerated heavy ions on E . coli cells}; Amirtaev KG et al.; In studying E . coli mutation rate as a function of dose of different types of ionizing radiation it was found that mutagenic efficiency of helium ions (LET-22, 54 and 72 keV/microns) was higher than that of gamma-rays . As LET increased the mutagenic efficiency decreased . The mutation rate for all types of radiation under study was both a power function and a linear-quadratic function of dose. J Steroid Biochem, 1989 Jan, 32(1A), 5 - 11 DNA binding of glucocorticoid receptor protein A fusion proteins expressed in E . coli; Bonifer C et al.; In an effort to obtain large quantities of glucocorticoid receptor (GR) protein for functional and structural studies, several truncated versions of the human glucocorticoid receptor (hGR) have been expressed in E . coli as C-terminal fusion proteins with protein A . The amount of expressed protein was between 5 and 25 mg/l in the culture . South-Western blotting was initially used to demonstrate the DNA binding capacity of fusion proteins containing the DNA binding domain of GR . The hybrid proteins were highly enriched in the insoluble fraction after cell lysis . For further purification and characterization the fusion proteins were solubilized in 8 M urea . The concentration of denaturing agent was reduced by dilution and the fusion proteins were allowed to refold . The renatured GR protein A fusion proteins bound to DNA in a nitrocellulose filter binding assay . We also show that it is possible to purify the renatured fusion protein to apparent homogeneity using a single chromatographic step on DNA-cellulose. Mutat Res, 1989 Jan, 210(1), 93 - 102 On the possible role of cytosine deamination in delayed photoreversal mutagenesis targeted at thymine-cytosine dimers in E . coli; Ruiz-Rubio M et al.; While delayed photoreversal (PR) mutagenesis has been interpreted as a measure of misincorporation step in targeted mutagenesis, the specificity to produce glutamine tRNA suppressor mutations (C to T transitions) at sites in DNA where a thymine-cytosine dimer (T = C) may target mutation suggests a deamination model: deamination T = C to T = U and trans-U DNA replication after PR . We describe here two enquires that did not support the latter model: (a) Uracil DNA glycosylase activity as estimated from the restricted plating efficiency of phage T5 containing uracil-substituted DNA showed no variation that might allow an exceptional opportunity for mutation at U in DNA, and (b) . The kinetics of delayed PR mutagenesis were unaltered if UV-irradiated cells were held in buffer suspension for 2 h at 41 degrees C (a procedure known to allow deamination T = C to T = U) and then assayed . Other results with cells containing both umuC and ung (uracil DNA glycosylase) defects showed the magnitude of T = C deamination sufficient to provide T = U at the critical site of mutation to an extent greater than the mutation frequencies produced by delayed PR mutagenesis, and considerations of the kinetics led to the suggestion that the deamination model could apply if there were an optimum period 30-130 min post-UV for efficient recovery of DNA replication after PR . The results underscored the feasibility of delayed PR mutagenesis by deamination and trans-U replication, but a selection between the two models could not be determined. Free Radic Res Commun, 1989, 8(1), 47 - 53 Effects of E . coli 0111.B4 lipopolysaccharide on spin-labelled murine macrophage and hepatocyte membranes; Jackson SK et al.; Macrophages and hepatocytes from normal and BCG-primed mice have been spin-labelled in their membranes with 5- and 16-doxyl stearic acid . Incubation of spin-labelled cells from BCG-primed animals with lipopolysaccharide from E . coli 0111.B4 produced a detectable and transient disturbance in the cell membranes as reflected by an increase in the order parameter measured from the electron spin resonance spectra of 5-doxyl-stearate . This membrane disturbance was maximal at 3-4 hours of incubation and was only detected with cells from mice primed with BCG . Spectra obtained from the 16-doxyl-stearate-labelled cells showed no change in order parameter on incubation with lipopolysaccharide. Mol Gen Mikrobiol Virusol, 1989 Jan, (1), 19 - 24 {Transposition of insertional sequence IS21 in E . coli cells activated by an exogenous promotor}; Dubeikovskii AN; Transpositional activity of the IS21-element was found to be activated during its cloning in the pUC18 vector plasmid . The activated IS-element restores ability to transposition of the deficient IS21-element present in the same cell . Simultaneously with the activated transposition the increase in frequencies of precise exclusion of IS-element is registered . The activation of IS-element is concluded to be directed by exogenous promoter. C R Acad Sci III, 1989, 308(14), 401 - 6 {Neutralising properties for HIV virus of hybrid protein MalE-CD4 expressed in E . coli and purified in 1 step}; Clement JM et al.; Genetic fusions allowing the expression in E . coli of hybrid proteins between a bacterial periplasmic maltose binding protein (MalE) and the CD4 molecule (the receptor of the HIV virus) have been constructed . One of them has kept most of the properties of each constituent: it is exported, can be purified in one step on an affinity column, interacts with anti-MalE and anti-CD4 antibodies, binds HIV gp 120 protein and inactivates HIV virus in an in vitro test. Folia Biol (Praha), 1989, 35(1), 35 - 41 Expression of a bovine leukaemia virus envelope fusion protein in E . coli; Zajac V et al.; An expression plasmid containing 1.224 bp of the bovine leukaemia virus (BLV) env gene was constructed . The polypeptides encoded by six recombinant plasmids were analysed by electrophoretic transfer blot analysis . Two new proteins of 150-160 kDa and 60 kDa, respectively, were found in whole cellular extracts using sera of naturally infected cattle and/or by use of mouse monoclonal antibodies against gp51. EMBO J, 1989 Jan, 8(1), 83 - 90 Expression of active hormone and DNA-binding domains of the chicken progesterone receptor in E . coli; Eul J et al.; Bacterially-expressed fusion proteins containing the DNA-(region C) or hormone-binding (region E) domains of the chicken progesterone receptor (cPR) fused to the C terminus of Escherichia coli beta-galactosidase were analysed for the specificity of interaction with natural and synthetic hormone-responsive elements (HREs) and progestins, respectively . The purified fusion protein containing the progestin-binding domain bound progesterone with an apparent Kd of 1.0-1.5 nM and was specifically photocross-linked with the synthetic progestin R5020 in crude bacterial lysates . Labelling of intact bacterial cells with {3H}R5020 revealed that the majority, if not all, of the bacterially produced hormone-binding domain was active . No differences in the binding to a synthetic palindromic glucocorticoid/progestin-responsive element (GRE/PRE) were found when the bacterially produced cPR DNA-binding domain was compared in methylation interference assays with the full-length chicken progesterone receptor form A expressed in eukaryotic cells . The study of dissociation kinetics, however, revealed differences in the half-life of the complexes formed between the palindromic GRE/PRE and either the receptor form A or the fusion protein containing the cPR DNA-binding domain . DNase I protection experiments demonstrated that the bacterially produced region C of the cPR generated specific 'footprints' on the mouse mammary tumour virus long terminal repeat (MMTV-LTR) which were nearly identical to those previously reported for the rat glucocorticoid receptor. Radiobiologiia, 1989 Jan-Feb, 29(1), 30 - 5 {The effect of genotype on the induction of prophage lambda in E . coli cells by ionizing radiation of various LET}; Bonev MN et al.; A study was made of the influence of the repair genotype on lambda prophage induction by ionizing radiation of different LET in lysogenic E . coli cells . Bacterial strains W3110, P3478, GC244, and 30SO were exposed to gamma-rays and helium ions of 22 keV/microns . Induction of the prophage in GC244 and 30SO strains deficient by lexA and recA genes was either inhibited (GC244) or lacking (30SO) . Inducibility of P3478 carrying polA mutation was 12 and 5 times as high as that of the wild type strain after exposure to gamma-radiation and helium ions, respectively. Acta Leprol, 1989, 7 Suppl 1, 212 - 6 Transformation of Mycobacterium smegmatis with E . coli plasmids; Zainuddin ZF et al.; One limiting factor in the studies of tuberculosis and leprosy is the lack of a versatile system for genetic analysis and manipulation of mycobacteria . One strategy used in constructing a plasmid vector for transforming Mycobacterium smegmatis was to insert fragments of a mycobacterial plasmid into an Escherichia coli plasmid . We found that the parental E . coli plasmid is capable of self-replication in M . smegmatis yielding chloramphenicol-resistant colonies . Plasmids from different passages of one M . smegmatis transformant were recovered and characterised by restriction digest analysis . The plasmid from the earlier passage was found to be indistinguishable from the original plasmid by restriction analysis . Plasmids from later preparations, however, were found to have undergone modifications in the M . smegmatis host resulting in an apparent increase in transformation efficiency for M . smegmatis . These plasmids can be used as a shuttle vector for the genetic manipulation of mycobacterial species. Prikl Biokhim Mikrobiol, 1989 Jan-Feb, 25(1), 3 - 14 {Beta-galactosidase from E . coli as a marker in immunoenzyme analysis}; Markarian AN et al.; In the present review the authors analyze factors influencing sensitivity of the enzyme immunoassay (EIA) . beta-Galactosidase from E . coli was chosen as a marker enzyme . Physico-chemical properties of the enzyme, detection methods and various ways of obtaining chemical conjugates with antigens and antibodies are discussed . Some examples of using beta-galactosidase as a label in homogeneous and heterogeneous EIA are given which enables different compounds to be analyzed with a high sensitivity . New approaches employing gene engineering for constructing "fusions" between beta-galactosidase and antigens (alternative to chemical conjugates) are discussed that can be used in the near future. Acta Biochim Pol, 1989, 36(3-4), 195 - 203 Crystallographic studies on E . coli Trp aporepressor; Joachimiak A et al.; Two crystal forms of Trp aporepressor, an inactive, unliganded form of Trp repressor have been obtained which are suitable for high resolution X-ray diffraction analysis . Trp aporepressor crystallizes in two forms: orthorhombic, P 2(1)2(1)2 and tetragonal P 4(1) (or P 4(3} which diffract to 1.8 A and 2.4 A, respectively . The orthorhombic crystals contain one monomer in the asymmetric unit, therefore the twofold axis relates two subunits of the dimer as in the case of the previously described Trp repressor (R . Schevitz et al., 1985, Nature, 317, 782-786) and Trp pseudorepressor (C . Lawson & P . B . Sigler, 1988, Nature, 333, 869-871) . The tetragonal crystals have two dimers in the asymmetric unit and are nearly isomorphous with the tetragonal crystals of Trp repressor and Trp pseudorepressor grown under similar conditions but in the presence of an activator or inhibitor, respectively. Biochimie, 1989 Jan, 71(1), 167 - 74 Involvement of ion channels in the transport of phage DNA through the cytoplasmic membrane of E . coli; Letellier L et al.; Upon infection, phage DNA is transported through the bacterial cytoplasmic membrane . This crossing is accompanied by a transient increase in the permeability of the cytoplasmic membrane toward ions and small solutes . This has led several authors to propose that DNA might cross the cytoplasmic membrane through channels . In the first part of the review we present data that we obtained with phage T4 and that strongly support this proposal . We then present the structural and ionic characteristics of these channels . In the second part, we summarize data obtained by several authors concerning the permeability changes induced by different phages and show that these results are compatible with a model of phage DNA transfer through channels . Finally, we discuss the possible origin of these channels. Acta Chir Scand, 1989, 155(1), 7 - 13 Influence of aprotinin, a protease inhibitor, on porcine E . coli shock . Studies on coagulation, fibrinolytic and hemodynamic response; Svartholm E et al.; The effect of a protease inhibitor, aprotinin, on hemostasis (a2M, a2AP, AT III, prothrombin-convertin activity, fibrinogen, fibrinogen monomers and fibrinolytic activity on fibrin plates) was investigated in pigs with septic shock . Anesthetized pigs were given live Escherichia coli i.v . (n = 7), or aprotinin (1,000,000 KIE i.v.) 15 min before live E . coli (n = 6), or Ringer's solution only (sham controls, n = 8) . Septic shock developed in all E . coli-infused pigs . Pulmonary vascular resistance increased, platelet and leukocyte counts fell and signs of systemic activation of the coagulation and fibrinolytic systems appeared in the E . coli groups, but aprotinin attenuated the effects on these systems and also on the pulmonary circulation . Five of the six aprotinin-pretreated pigs survived, but none of the seven with septic shock and no pretreatment . Thus the shock induced by infusion of live E . coli and the resultant changes in the coagulation and fibrinolytic systems and in cardiopulmonary hemodynamics were diminished by aprotinin pretreatment. Biochim Biophys Acta, 1988 Dec 20, 951(2-3), 255 - 60 Nucleotide excision by E . coli DNA polymerase I in proofreading and non-proofreading modes; Lecomte PJ et al.; Escherichia coli DNA polymerase I exists in at least two distinct kinetic forms . When it binds to a template, the proofreading activity is usually switched off . As the enzyme progresses along the template, it becomes more and more competent for excision . This phenomenon introduces a link between fidelity and processivity . Processivity is best studied when the chain-length distributions of synthesized polymers are stationary . Even then, however, one cannot avoid multiple initiations on a given template by the same molecule of the enzyme . When synthesis is initiated with primers of lengths 15 or 20, a strange phenomenon is observed . It seems that the polymerase starts by hydrolyzing the primer down to a length of 7-10 nucleotides and only then starts to add nucleotides . It does so in a low-accuracy mode, suggesting that, while the exonuclease is clearly active, it does not contribute to proofreading . The warm-up of the proofreading function is therefore reinterpreted as a switch between two modes of behaviour: a mode 1 of low accuracy in which the 3'----5' exonuclease, while active, is uncoupled from the polymerase and does not contribute to proofreading, and a mode 2 of high accuracy in which the exonuclease is kinetically linked to the polymerase activity. Science, 1988 Dec 9, 242(4884), 1415 - 8 Human insulin-degrading enzyme shares structural and functional homologies with E . coli protease III; Affholter JA et al.; A proteinase with high affinity for insulin has been proposed to play a role in the cellular processing of this hormone . A complementary DNA (cDNA) coding for this enzyme has been isolated and sequenced . The deduced amino acid sequence of the enzyme contained the sequences of 13 peptides derived from the isolated protein . The cDNA could be transcribed in vitro to yield a synthetic RNA that in cell-free translations produced a protein that coelectrophoresed with the native proteinase and could be immunoprecipitated with monoclonal antibodies to this enzyme . The deduced sequence of this proteinase did not contain the consensus sequences for any of the known classes of proteinases (that is, metallo, cysteine, aspartic, or serine), but it did show homology to an Escherichia coli proteinase (called protease III), which also cleaves insulin and is present in the periplasmic space . Thus, these two proteins may be members of a family of proteases that are involved in intercellular peptide signaling. Biochim Biophys Acta, 1988 Dec 2, 957(3), 455 - 8 Kinetic mechanism of catalytic subunits (c3) of E . coli aspartate transcarbamylase at pH 7.0; Hsuanyu Y et al.; In contrast to holo-enzyme (c6r6), catalytic subunits (c3) of Escherichia coli aspartate transcarbamylase (carbamoyl-phosphate:L-aspartate carbamoyltransferase, EC 2.1.3.2) do not exhibit allosteric interactions or inhibition effects that complicate kinetic investigations of substrate binding order . Equilibrium isotope-exchange kinetic probes of c3 at pH 7.0 and 30 degrees C produced kinetic saturation patterns consistent with a strongly preferred order random kinetic mechanism, in which carbamoyl phosphate binds prior to aspartate and carbamoyl aspartate is released before Pi . Weak substrate inhibition effects observed with c6r6 did not occur with c3, possibly due to decreased affinity for ligands at the dianion inhibition site. Cell, 1988 Dec 2, 55(5), 817 - 26 Transmembrane signaling by bacterial chemoreceptors: E . coli transducers with locked signal output; Ames P et al.; Methyl-accepting chemotaxis proteins (MCPs) function as transmembrane signalers in bacteria . We isolated and characterized mutants of the E . coli Tsr protein that produce output signals in the absence of overt stimuli and that are refractory to sensory adaptation . The properties of these "locked" transducers indicate that MCP molecules are capable of generating signals that actively augment clockwise and counter-clockwise rotation of the flagellar motors . Transitions between MCP signaling states can be influenced by amino acid replacements in many parts of the molecule, including the methylation sites, at least one of the two membrane-spanning segments, and a linker region connecting the receptor and signaling domains . These findings suggest that transmembrane signaling may involve direct propagation of conformational changes between the periplasmic and cytoplasmic portions of the MCP molecule. Zentralbl Bakteriol Mikrobiol Hyg {B}, 1988 Dec, 187(2), 107 - 11 {The glutamic acid decarboxylase disc test . An approach for the simplification and speeding up of E . coli detection}; Schubert R et al.; Although E . coli is certainly one of the best known and most widely examined bacterial species only few of their characters and properties were found suitable for the distinction of this species from other species especially from the coliform organisms . One of these rare characters is l-glutamic acid decarboxylase . The demonstration of the action of this enzyme, however, has hitherto been difficult and time consuming . A micro-method is described which permits the demonstration of l-glutamic acid decarboxylase--the glutamic acid decarboxylase disc test (gd-disc-test) . The conditions and procedures required are described in detail . The validation of this test was done demonstrating in presence of gamma-aminobutyric acid by paper chromatography. J Bacteriol, 1988 Dec, 170(12), 5529 - 38 Molecular cloning, expression, and regulation in Escherichia coli K-12 of a chromosome-mediated aerobactin iron transport system from a human invasive isolate of E . coli K1; Valvano MA et al.; We have cloned chromosomal genes determining the aerobactin iron transport system from the Escherichia coli K1 strain VW187 . Mapping and hybridization experiments showed that the VW187 aerobactin region was identical to that of the plasmid ColV-K30 . However, in the E . coli K-12 background, the biosynthesis of both siderophore and ferric aerobactin receptor encoded by the VW187-derived recombinant plasmids was not repressed by iron to the same extent found when a recombinant plasmid derived from pColV-K30 was used . RNA-DNA dot-blot hybridization experiments demonstrated that the aerobactin-specific mRNA synthesized by the VW187-derived clones was not iron regulated in E . coli K-12 . In contrast, the synthesis of aerobactin and its receptor in strain VW187 was completely repressed by iron regardless of whether the recombinant plasmids originated from VW187 or pColV-K30 . Similar results were obtained with gene fusions in which a promoterless lac operon was placed under the control of aerobactin promoter regions of either chromosome- or plasmid-mediated aerobactin systems . DNA sequencing of the chromosomal aerobactin promoter region showed changes in bases located immediately upstream to the -35 region compared with the corresponding region in pColV-K30, which is known to be part of the binding site for the Fur repressor protein. J Biomol Struct Dyn, 1988 Dec, 6(3), 587 - 92 Prediction of the recognition sites on 16S and 23S rRNAs from E . coli for the formation of 16S-23S rRNA complex; Thanaraj TA et al.; Interactions between RNA molecules have been postulated to play an important role in the assembly of ribosomes . Using the sequence analysis and the search of continuous complementary regions on 16S rRNA and 23S rRNA, the recognition sites involved in the formation of ribosome of E . coli are postulated . The number of postulated sites was narrowed down by taking available experimental data . The suggestive evidence for correct postulation is obtained from sequence comparison studies of 16S and 23S rRNAs from various species . The sites 891-899 and 1195-1203 on 16S rRNA along with the corresponding complementary sites 1904-1912 and 760-768 on 23S rRNA are predicted to be the most probable candidates for the sites of recognition between 16S and 23S rRNAs . The possibility of the involvement of the additional site 630-638 on 16S rRNA with its complementary site 2031-2039 on 23S rRNA cannot be ruled out. Arzneimittelforschung, 1988 Dec, 38(12), 1856 - 8 Detection of antibodies against pre-S1 proteins in sera of patients with hepatitis B virus infection by ELISA using a pre-S fusion protein expressed in E . coli; Theilmann L et al.; To evaluate the importance of antibodies directed against pre-S1 proteins (anti-pre-S1) of hepatitis B virus (HBV) in human sera, an enzyme-linked immunosorbent assay (ELISA) for their detection in human sera was established, using a bacterially synthesized pre-S1 fusion protein . Using this ELISA, it was found that anti-pre-S1 was present in sera from 10 of 11 patients with acute HBV infection who recovered completely, but only present in 1 of 8 patients where the infection was prolonged . In chronic HBV carriers anti-pre-S1 was present in only 3 out of 8 patients and titers were low . In addition, in 5 out of 17 individuals who had recovered from previous HBV infection, anti-pre-S1 was also detected . Individuals immunized with recombinant HBsAg-vaccine and healthy controls were all negative for anti-pre-S1 . It is suggested that presence of anti-pre-S1 in sera of patients with acute HBV infection correlates with rapid recovery. Biosci Rep, 1988 Dec, 8(6), 619 - 32 Translational control in E . coli: the case of threonyl-tRNA synthetase; Springer M et al.; Genetic studies have shown that expression of the E . coli threonyl-tRNA synthetase (thrS) gene is negatively auto-regulated at the translational level . A region called the operator, located 110 nucleotides downstream of the 5' end of the mRNA and between 10 and 50 bp upstream of the translational initiation codon in the thrS gene, is directly involved in that control . The conformation of an in vitro RNA fragment extending over the thrS regulatory region has been investigated with chemical and enzymatic probes . The operator locus displays structural similarities to the anti-codon arm of threonyl tRNA . The conformation of 3 constituent mutants containing single base changes in the operator region shows that replacement of a base in the anti-codon-like loop does not induce any conformational change, suggesting that the residue concerned is directly involved in regulation . However mutation in or close to the anti-codon-like stem results in a partial or complete rearrangement of the structure of the operator region . Further experiments indicate that there is a clear correlation between the way the synthetase recognises each operator, causing translational repression, and threonyl-tRNA. APMIS, 1988 Dec, 96(12), 1061 - 5 Binding of E . coli to human blood leukocytes; Guan YZ et al.; The spontaneous binding of both piliated and non-piliated E . coli to human lymphocytes, monocytes, and polymorphonuclear leukocytes (PMN) was investigated . It was found that the piliated E . coli showed a higher binding percentage to lymphocytes than the nonpiliated bacteria . A specific antiserum raised in rabbit against E . coli strongly inhibited the binding of the homologous E . coli strain to lymphocytes and monocytes . On the other hand there was no difference in the binding of piliated and non-piliated E . coli to PMNs, and the specific E . coli antiserum did not inhibit the binding . The binding of E . coli to human lymphocytes obtained from healthy donors was investigated in a 3-week follow-up experiment . It was shown that there were significant differences between the binding of cells from different individuals, whereas there were no differences in the binding ability of the cells from the same individual in the 3-week follow up. Mutat Res, 1988 Dec, 203(6), 415 - 26 The E . coli multitest: a set of strains to characterize diverse genotoxic effects; Tenenbaum L et al.; A set of E . coli strains was developed by Toman et al . (1985) to study the effects of chemical and physical agents on forward mutation, homologous recombination and induction of the SOS system . New tester strains have been constructed to improve this test system in order to explore quantitative genotoxicity spectra . Through the use of these strains: (i) SOS induction can be specifically detected without interference from mutagenesis; (ii) SOS-dependent and SOS-independent mutational events can be distinguished; (iii) the sensitivity of the recombination system has been considerably increased. Cell, 1988 Nov 18, 55(4), 683 - 92 SecA protein is required for secretory protein translocation into E . coli membrane vesicles; Cabelli RJ et al.; The soluble and membrane components of an E . coli in vitro protein translocation system prepared from a secA amber mutant, secA13{Am}, contain reduced levels of SecA and are markedly defective in both the cotranslational and posttranslational translocation of OmpA and alkaline phosphatase into membrane vesicles . Moreover, the removal of SecA from soluble components prepared from a wild-type strain by passage through an anti-SecA antibody column similarly abolishes protein translocation . Translocation activity is completely restored by addition of submicrogram amounts of purified SecA protein, implying that the observed defects are solely related to loss of SecA function . Interestingly, the translocation defect can be overcome by reconstitution of SecA into SecA-depleted membranes, suggesting that SecA is an essential, membrane-associated translocation factor. J Biol Chem, 1988 Nov 15, 263(32), 17084 - 91 Dihydrofolate reductase (mouse) and beta-galactosidase (Escherichia coli) can be translocated across the plasma membrane of E . coli; Freudl R et al.; Hybrid genes were constructed . One, ompA153-dfr, encoded the precursor of the 325 residue Escherichia coli outer membrane protein OmpA up to residue 153 which was fused to the complete 186-residue dihydrofolate reductase of the mouse . The other, ompA219-lacZ, coded for the same precursor up to residue 219 which was fused to 1017 COOH-terminal residues of the 1023-residue subunit of the beta-galactosidase of E . coli . Full expression of the ompA153-dfr gene caused accumulation of its precursor and of that of the chromosomally encoded OmpA protein . When the amount of product was reduced, no pro-OmpA and very little pro-hybrid protein accumulated . The precursor was processed and the mature protein was fully accessible to trypsin in permeabilized cells . Expression of the ompA219-lacZ gene led to the presence of the hybrid protein at only 20-30% of the amount expected . About 20% of it appeared to be incorporated in the outer membrane . All of the hybrid was quantitatively accessible to trypsin in permeabilized cells . When the hybrid gene was overexpressed, the protein was found associated with the plasma membrane in the cytosol . It is concluded that both beta-galactosidase and dihydrofolate reductase could quantitatively traverse the plasma membrane, provided the amounts synthesized were sufficiently small. Cell, 1988 Nov 4, 55(3), 467 - 75 A consensus sequence of three DNA replication terminus sites on the E . coli chromosome is highly homologous to the terR sites of the R6K plasmid; Hidaka M et al.; Using the "Ter assay" we developed, three separate replication terminus (terC1, terC2, and terC3) sites on the E . coli chromosome were identified . The locations are at 28.3, 35.6, and 33.9 min on the linkage map, respectively . The terC1 site can block the counter-clockwise replication fork only, while the terC2 and terC3 sites inhibit the clockwise fork traveling on the chromosome . DNA sequences of the terC sites required for termination of DNA replication are highly homologous to those of terminus (terR) sites of the R6K plasmid, and the 21 bp consensus DNA sequence of terC is 5'-(A or T) TTAGTTACAACAT (A or C) CT (A or T) (A or T) (A or T) T-3' . In addition, all Ter active pUC-terC plasmids had a low copy number and were unstable in the host cells. Cell, 1988 Nov 4, 55(3), 459 - 66 Identification of the DNA sequence from the E . coli terminus region that halts replication forks; Hill TM et al.; The terminus region of the E . coli chromosome contains two loci, T1 and T2, that inhibit the progress of replication forks and require the trans-acting factor tus . We have identified a 23 bp terminator signal at T1 and T2 that is within 100 bp of the sites of replication arrest . When an oligodeoxyribonucleotide containing the terminator signal was inserted into a plasmid, replication was halted only in a tus+ strain and when the terminator signal was oriented properly . We also found this terminator sequence in the terminus region of the plasmid R6K and in the origin region of RepFIIA class plasmids . In addition, we found striking similarities between the E . coli terminator signal and the terminator sequence of B . subtilis. Biofizika, 1988 Nov-Dec, 33(6), 973 - 7 {A study of the stability of liposomes from the total lipid fraction of E . coli by means of fluorescence spectroscopy and a radioisotope probe}; Cherniavskii VA et al.; Stability of liposomes from total fraction of E . coli lipids was studied by their ability to preserve encapsulated substance under different incubation conditions--+4 degrees C and +37 degrees C for some concentrations of CaCl2 . Two types of vesicules were used: small ones obtained by ultrasonic treatment and larger ones obtained after successive treatment of lipid suspension with Ca2+ ions and EDTA . It has been shown by spectrofluorimetry and radioisotope probe that for both studied types of liposomes their stability decreases with an increase of CaCl2 concentration and rise of temperature . The effect similar to Ca2+ was produced by Mg+2 ions, but not by Na+ ions . Large liposomes are significantly less stable formations than the small ones . Ca+2 ions were shown to induce at first liposome aggregation and then their destruction . Possible action mechanism of metal ions of the second group on liposome stability, the pattern of initiated membrane disturbances and application of these structures as agents during transfer of biologically active compounds in the cells are discussed. Mutat Res, 1988 Nov, 202(1), 19 - 23 Antimutagens against spontaneous and induced reversion of a lacZ frameshift mutation in E . coli K-12 strain ND-160; Clarke CH et al.; Isopropyl-beta-D-thiogalactoside (IPTG), at 0.5 or 1.0 mM, is shown to reduce spontaneous, and to virtually abolish caffeine- or quinacrine-induced reversions of the lacZ frameshift mutation in E . coli ND160 . Guanosine at 200 micrograms/ml and Co2+ at 15 micrograms/ml had an erratic partial antimutagenic effect on spontaneous lac+ reversions . All 3 agents reduce caffeine-induced (500 micrograms/ml) mutagenesis . Spermine (250 micrograms/ml) also reduces quinacrine-induced Lac+ reversion frequencies in this system. Radiobiologiia, 1988 Nov-Dec, 28(6), 767 - 71 {Induction of prophage lambda during the irradiation of E . coli cells exposed to radiations of various LET}; Bonev M et al.; Induction of lambda prophage in lysogenic E . coli cells exposed to ionizing radiation of different LET was studied as a function of dose I(D) . Activities of pleiotropic RecA protein were shown to contribute to the shape of the I(D) curve . The experimental data were fitted by the function I(D) = alpha D(1-exp(-D0-1.D}exp(-beta D) . Inducibility alpha increased with increasing LET which was related to the increased incidence of DNA lesions being a SOS-system call. Mol Gen Mikrobiol Virusol, 1988 Nov, (11), 18 - 20 {Localization of insertion into E . coli chromosome of Tn7-like transposons controlling resistance to trimethoprim and streptothricin}; Tietze E et al.; To localize the insertion sites for Tn7-like transposons Tn1824, Tn1825 and Tn1826 the EcoRI-cleaved fragments of E . coli recA+ and recA- strains chromosome carrying the transposons were hybridized . It was shown that transposition of Tn7-like elements takes place in the strictly defined region of E . coli chromosome, like it had been previously described for transposon Tn7. Mutat Res, 1988 Nov, 194(3), 193 - 205 Repair of the plasmid pBR322 damaged by gamma-irradiation or by restriction endonucleases using different recombination-proficient E . coli strains; Bien M et al.; The plasmid pBR322 was treated with BamHI, PvuII and gamma-irradiation to generate double-strand breaks (dsb) containing differently structured ends . Transformation efficiencies, mutation frequencies and clone analyses of enzymatically damaged DNA are compared with the corresponding results from radiolytically damaged DNA . In E . coli K-12 SFX, the yield of transformants produced by the action of BamHI, PvuII and gamma-irradiation (30 Gy) is 4.3%, 0.14%, and 0.10%, respectively . The survival of open circular DNA (ocDNA) produced by 30 Gy is 1.3% . The transformation efficiencies show only a slight dependence on SOS induction and on the RecA protein . Mutation frequencies to tetracycline sensitivity (tets) per surviving plasmid are 2.6% (BamHI), 11.8% (PvuII) and 0.2% (gamma-irradiated DNA with 30 Gy containing approximately 50% ocDNA and 50% linearized (lin) DNA) . The mutation frequency is low at all radiation doses studied (1-50 Gy) . Only 15% of the DNA of the tets mutants from gamma-irradiated plasmids contained deletions whereas with enzymatically damaged DNA, 30-50% (BamHI) or 90% (PvuII) contained deletions . In all cases, the deletions comprised 500-1700 base pairs (bp) . After SOS induction of the host cells, the mutation frequency of gamma-irradiated plasmids increased by a factor of 4, whereas that of the enzymatically damaged plasmids did not change . For the repair of the enzymatically linearized DNA 2 recombinational pathways are discussed which lead to deletant (pathway I) and non-deletant transformants (pathway II) . In addition, BamHI-linearized plasmids may be repaired by enzyme-induced or spontaneous circular alignment followed by ligation . The high percentage of deletions of the tets mutations for PvuII-linearized DNA with blunt ends is explained by the illegitimate or site-specific recombination pathway I (see text) . The lower percentage of deletions of the tets mutations with BamHI-linearized DNA with short cohesive ends (4 bp) is proposed to be due to a greater contribution of pathway II and/or by circular alignment followed by ligation . The very small yield and the low percentage of deletant mutations of tets mutants from radiolytically damaged DNA is proposed to be due to the large overlapping ends (16-100 bp) of the linDNA which easily leads to circular alignment followed by excision repair . The repair of radiolytically produced ocDNA is predominantly due to excision repair . In agreement with this interpretation is the observation that SOS induction of the host increases the mutation incidence of radiolytically damaged DNA but not of enzymatically damaged DNA. Biochem Biophys Res Commun, 1988 Oct 31, 156(2), 807 - 13 Identification of unusual replacement of methionine by norleucine in recombinant interleukin-2 produced by E . coli; Lu HS et al.; Moderate amounts of norleucine incorporation into recombinant interleukin-2 (IL-2) produced in E . coli have been detected . Incorporation of norleucine occurs both at the amino terminal and internal methionines as confirmed by the isolation of norleucine-containing tryptic peptides which eluted later than the respective methionine-containing peptides by reverse-phase HPLC . The occurrence of norleucine in intact protein and modified peptides was determined by amino acid analysis and amino acid sequencing including Edman degradation and fast atom bombardment mass spectrometry . In the subsequent paper, we determined that norleucine incorporation is caused by the endogenous synthesis of norleucine in E . coli. Nucleic Acids Res, 1988 Oct 25, 16(20), 9545 - 55 A specific and efficient photoreaction between E . coli RNA polymerase and T+1 in the lacUV5 or deoP1 promoter; Jeppesen C et al.; Upon irradiation of the RNA polymerase-lacUV5 or deoP1 promoter complex with short wavelength ultraviolet light (lambda less than or equal to 300 nm) the polymerase is covalently crosslinked at an efficiency of greater than 10% to the first transcribed base of the template DNA strand when this is a thymine . The temperature dependence of this RNA polymerase-T+1 photoreaction strongly indicates a relation to the formation of the open complex . It is suggested that open complex formation is preceded or accompanied by a specific contact between the RNA polymerase and the first transcribed base of the DNA template. FEBS Lett, 1988 Oct 24, 239(1), 41 - 4 Cloning a synthetic gene for human stefin B and its expression in E . coli; Jerala R et al.; A gene coding for human stefin B was synthesized by the solid-phase phosphite method and cloned in the pUC8 cloning vector . The insert with the verified DNA sequence was subcloned into two expression vectors and expressed in E . coli as a fusion protein with beta-galactosidase and as a native protein . The CNBr cleaved fusion protein and the native recombinant stefin B were inhibitory to papain and reacted with antibodies against human stefin B. Cell, 1988 Oct 21, 55(2), 343 - 50 A novel role for cAMP in the control of the activity of the E . coli chromosome replication initiator protein, DnaA; Hughes P et al.; DnaA protein interacts with cAMP with a KD of 1 microM . This interaction stimulates DnaA protein binding to the chromosome replication origin (oriC) and the mioC promoter region, protects DnaA protein from thermal inactivation, releases ADP but not ATP bound to DnaA protein, and restores normal DNA replication activity and ATPase activity in inactive ADP-DnaA protein preparations . A model is proposed in which cellular cAMP levels govern the replication activity of DnaA protein by promoting the recycling of the inactive ADP-DnaA protein form into the active ATP form. Cell, 1988 Oct 7, 55(1), 113 - 23 Transcriptional activation of initiation of replication from the E . coli chromosomal origin: an RNA-DNA hybrid near oriC; Baker TA et al.; Transcription by RNA polymerase preceding the initiation of replication from the E . coli chromosomal origin (oriC) in vitro enables dnaA protein to open the DNA duplex under conditions when its action alone is insufficient . The RNA polymerases of phages T7 and T3 are as effective as the E . coli enzyme in activating initiation . The persistent RNA transcript hybridized to the template creates an R-loop that is responsible for activation . The activating RNA need not cross oriC, but must be less then 500 bp away . Transcripts lacking a 3' OH group are effective, proving that priming of DNA synthesis is not involved in the activation . Thus, transcription activates the origin of an otherwise inert plasmid by altering the local DNA structure, facilitating its opening by dnaA protein during the assembly of replication forks. Bioorg Khim, 1988 Oct, 14(10), 1387 - 92 {Location of the initiating codon AUG in relation to the 5'-end of mRNA mediates the effectiveness of translation in E . coli cells}; Kravchenko VV et al.; A semisynthetic gene for beta-galactosidase (lacZ) and a synthetic DNA fragment containing the "ideal" promoter sequence were used for construction of an artificial operon including translation initiation codon ATG and no SD sequence . Cloning this artificial operon into pBR322 vector resulted in a number of pV plasmids; ATG positions were varied by insertions of synthetic oligonucleotides between lacZ coding sequence and starting point of transcription . It was found that efficiency of beta-galactosidase synthesis in E . coli cells harbouring pV plasmids strongly depended on the relative position of AUG and mRNA 5'-end . High level of the synthesis was provided by translation of mRNA with AUG codon in 5'-terminal position . Amounts of synthesized beta-galactosidase diminished with increase of the distance (2, 4, and 5 nucleotides) between 5'-end of lacZ mRNA and AUG codon. Curr Genet, 1988 Oct, 14(4), 293 - 302 The 5'-upstream region of the yeast 25S rRNA gene contains a promoter element allowing expression in yeast and E . coli; Strobel G et al.; The 25S rRNA gene of Saccharomyces cerevisiae is preceded by a bona fide TATA sequence which allows the initiation of transcription--presumably by polymerase II--from the same strand as the 25S rRNA gene . When the promoter fragment is cloned in front of a lacZ gene equipped with an initiation codon but lacking a promoter, this element permits formation of beta-galactosidase both in yeast and E . coli . Using RNA from yeast transformed with the fusion plasmid, we mapped by primer elongation a single initiation site 63 bp downstream from the presumed TATA sequence, i.e . about 53 bp 5' of the 25S rRNA gene . A similar signal at about the same position was observed when RNA from untransformed wild-type yeast was used as a template for primer elongation . These results suggest that transcription from this polymerase II promoter-like element occurs in vivo . A regulatory function could not be assigned to this transcript . Its initiation is not significantly influenced by heme or carbon source, although two boxes of high homology with upstream activation sequences (UAS) mediating heme dependent expression of the iso-1-cytochrome c gene (CYC1) precede the promoter at the appropriate distance. Biochem Biophys Res Commun, 1988 Sep 30, 155(3), 1261 - 5 Release of 7-alkylguanines from haloethylnitrosourea-treated DNA by E . coli 3-methyladenine-DNA glycosylase II; Carter CA et al.; Previous studies have related DNA modification by the haloethylnitrosoureas to their antitumor activity . Repair of this damage, particularly by O6-alkylguanine-DNA alkyltransferase, has been linked to tumor resistance by several previous investigations . We report here that E . coli 3-methyladenine-DNA glycosylase II can also remove several of the DNA modifications caused by the haloethylnitrosoureas . 7-Chloroethylguanine, 7-hydroxyethylguanine, and diguan-7-ylethane are all released into the supernatant from DNA modified by N-{2-chloroethyl-1,2-14C}-N'-cyclohexyl-N-nitrosourea . Release of diguan-7-ylethane is of particular interest since this entity evidently represents a DNA intrastrand cross-link . If a similar activity is present in mammalian cells, it might be an important source of resistance to the therapeutic action of the haloethylnitrosoureas. FEBS Lett, 1988 Sep 26, 238(1), 116 - 8 Total synthesis of the cystatin alpha gene and its expression in E . coli; Katunuma N et al.; A gene encoding cystatin alpha has been chemically synthesized, cloned and expressed in E . coli . The gene of 318 base pairs was assembled by enzymatic ligation of 19 oligonucleotides and cloned into a pBR322-derived expression plasmid down stream of the tac promoter . The expression product of the synthetic gene has been purified by Sephadex G-50 column chromatography and shown to have the same properties as those of the authentic protein isolated from rat epidermis. Cell, 1988 Sep 23, 54(7), 1003 - 11 ProOmpA is stabilized for membrane translocation by either purified E . coli trigger factor or canine signal recognition particle; Crooke E et al.; We have isolated large amounts of E . coli outer-membrane protein A precursor (proOmpA) . Purified proOmpA is active in membrane assembly, and this assembly is saturable with respect to the precursor protein . A proOmpA-Sepharose matrix allows affinity isolation of trigger factor, a soluble, 63,000 dalton monomeric protein that stabilizes proOmpA in assembly competent form . Comparison of trigger factor's amino-terminal sequence with those in a computer data bank and with those encoded by sec genes, as well as groEL and heat shock gene dnaK, suggests that trigger factor is encoded by a previously undescribed gene . Trigger factor and proOmpA form a 1:1 complex that can be isolated by gel filtration . Purified canine signal recognition particle (SRP) can also stabilize proOmpA for membrane insertion . This postribosomal activity of SRP suggests a unifying theme in protein translocation mechanisms. FEBS Lett, 1988 Sep 12, 237(1-2), 187 - 90 Processed enzymatically active protease (p15gag) of avian retrovirus obtained in an E . coli system expressing a recombinant precursor (Pr25lac-delta gag); Sedlacek J et al.; Processing proteases of avian and mammalian retroviruses cut the polyprotein precursors encoded by the retroviral genes into mature functional proteins . Retroviral processing proteases are still a rather poorly characterized group as to their relation to other proteases, specificity, and mechanism of enzymatic action . In avian retroviruses the generation of the processing protease itself comprises a processing cleavage event - the protease p15gag is cut off the carboxy-terminus of a gag polyprotein precursor, Pr76gag . We report here that direct and efficient production of the avian retrovirus processing protease p15gag (required for structure-function studies and rational design of inhibitors) was obtained in an E . coli system, where massive expression of a size-reduced, recombinant precursor (Pr25lac-delta gag) was accompanied by its structurally accurate processing. Cell, 1988 Sep 9, 54(6), 805 - 12 A novel nucleotide excision repair for the conversion of an A/G mismatch to C/G base pair in E . coli; Lu AL et al.; A protein that binds specifically to A/G mismatches has been detected in E . coli by a gel electrophoresis DNA binding assay . A specific endonuclease is associated with the A/G mismatch-binding protein through two chromatographic steps . The endonuclease is specific for A/G-containing DNA fragments and has no cleavage activity on DNA containing the other seven possible mispairs or homoduplex DNA . The endonuclease simultaneously makes incisions at the first phosphodiester bond 3' to and the second phosphodiester bond 5' to the dA of the A/G mismatch . No incision site was detected on the other strand . These results are consistent with the unidirectional A to C conversion and short repair tract of a novel dam- and mutHLS-independent A/G repair pathway we have recently described . A nucleotide excision repair model is proposed for the