Gene, 1989 Nov 15, 83(1), 117 - 24 Overexpression of native human beta 2-microglobulin in Escherichia coli and its purification; Parker KC et al.; beta 2-Microglobulin (beta 2M), the small subunit of human leukocyte antigen (HLA) class-I proteins, has been synthesized in Escherichia coli and purified in mg amounts . A beta 2m cDNA clone was fused in-frame behind DNA encoding the signal sequence for the outer membrane protein, OmpA . Three different constructions were made, whose products differed by the insertion of either an extra Ala residue, the hexapeptide AEFLEA {single-letter amino acid (aa) code}, or no aa between the OmpA signal sequence and beta 2M-coding sequence . All three protein products were correctly processed by bacterial signal peptidase, as determined by N-terminal sequencing, and all three were secreted as soluble proteins into the periplasmic space . However, the signal sequence of the preprotein with the inserted hexapeptide, AEFLEA, was cleaved to a much greater degree than the other two preproteins . When there was no insertion, the mature protein was identical to human beta 2M, as analyzed by sodium dodecyl sulfate-polyacrylamide-gel electrophoresis, circular dichroism, and native isoelectric focusing . This 'bacterial beta 2M', radiolabeled with Bolton-Hunter reagent, was able to exchange into papain-solubilized HLA-B7, as determined by Sephadex G-75 chromatography and immune precipitation, indicating that bacterial beta 2M could complex with the heavy chain of HLA-B7. Biochem Biophys Res Commun, 1989 Nov 15, 164(3), 955 - 60 Affinity purification of the HIV-1 protease; Heimbach JC et al.; An inhibitor of the HIV-1 protease has been employed in the generation of a resin which allows the rapid purification of this enzyme . A peptide substrate analogue, H2N-Ser-Gln-Asn-(Phe-psi{CH2N}-Pro)-Ile-Val-Gln-OH, was coupled to agarose resin . The HIV-1 protease was expressed in E . coli and the supernatant from lysed cells was passed through the affinity resin . Active HIV-1 protease was then eluted with a buffer change to pH 10 and 2 M NaCl . Final purification to a homogeneous preparation, capable of crystallization, was achieved with hydrophobic interaction chromatography . Solutions containing HIV-1 protease bound to competitive inhibitors do not bind to the column. Biochem Biophys Res Commun, 1989 Nov 15, 164(3), 1437 - 45 Studies on site directed mutant pig citrate synthases; Evans CT et al.; The DNAs encoding the non-mutant and mutant forms of pig citrate synthase (PCS) were subcloned into an expression system to determine their synthesis and stability in E . coli gltA- cells that are defective in bacterial citrate synthase . GltA- cells that expressed the non-mutant PCS DNA grew on defined minimal acetate media and produced a constant level of PCS (0.43 U/mg protein) . In contrast, when the gltA- cells were transformed with the DNA encoding PCS mutations in His274 or Asp375 the cells did not grow on minimal acetate media . The presence of the mutant PCS proteins in E . coli was confirmed by protein blot and immunoisolation analyses using an antibody specific for porcine heart citrate synthase . The activities of the mutant PCS enzymes were two orders of magnitude less than the non-mutant enzyme in the total cell lysates . The data indicate that the active site amino acids, His274 and Asp375, are essential for the catalysis activity of citrate synthase. Biochem Biophys Res Commun, 1989 Nov 15, 164(3), 1339 - 47 Production in Escherichia coli of a biologically active subfragment of von Willebrand factor corresponding to the platelet glycoprotein Ib, collagen and heparin binding domains; Pietu G et al.; A full-length cDNA for vWF has been cloned from a human lung cDNA library and a fragment of this cDNA has been modified to allow its expression in E . coli . This fragment, which corresponds to Val 449-Asn 730 of vWF and includes the GPIb-binding domain and binding sites for collagen and heparin, was subcloned into an expression vector containing an inducible lambda PL promoter . On induction, the expressed recombinant vWF subfragment migrated with a mol wt of around 38,000 after SDS-PAGE . It was identified as a vWF fragment by Western blotting using either a polyclonal or a monoclonal antibody which inhibits the binding of vWF to GPIb . Following solubilization in urea, the bacterial extract inhibited ristocetin-induced platelet aggregation and bound to ristocetin-treated platelets, to collagen and to heparin. Biochem Biophys Res Commun, 1989 Nov 15, 164(3), 1253 - 61 Separation of yeast proteasome subunits . Immunoreactivity with antibodies against ATP-dependent protease Ti from Escherichia coli; Tanaka K et al.; On two-dimensional gel electrophoresis, proteasomes (multicatalytic proteinase complexes) from the yeast Saccharomyces cerevisiae were separated into a characteristic set of approximately 20 components with molecular weights of 21,000 to 31,000 and isoelectric points of 3.5 to 7.5 . The main components were isolated by reverse-phase high performance liquid chromatography on a TSK gel phenyl-5PW RP column and named YC1 to YC11, in order of their elution . Immuno-blot analysis showed that two components (YC1-alpha and YC1-beta) with molecular weights of 30,800 and 28,300 strongly cross-reacted with antibody against the P-component of ATP-dependent protease Ti from Escherichia coli, but no components were found to react with antibodies against the A-component of protease Ti or another ATP-dependent protease La (the Ion gene product) of Escherichia coli . These results indicate a structural relationship between eukaryotic proteasomes and bacterial ATP-dependent protease Ti. Biochem Biophys Res Commun, 1989 Nov 15, 164(3), 1149 - 56 Sequence specificity of pausing by DNA polymerases; Weisman-Shomer P et al.; We have constructed recombinant M13 DNA templates containing stretches of oligo (purines) and oligo (pyrimidines) . Each of these inserts hinders the advancement of the large fragment of E . coli Pol I during DNA synthesis . The pattern of blockage is independent of changes in KCl or Mg2+ concentrations and pausing is moderately alleviated at lower pH . Blockage is not affected by either the concentration of template or by the position of the DNA primer . The pattern of pause sites is similar for calf thymus DNA polymerase-alpha, implying that replicative barriers are determined by the structure of the DNA at its growing point . There is a lack of correlation between the position of pause sites with different inserts and known alternate DNA structures . Thus, the homo-oligomeric inserts may possess a different structure when complexed with DNA polymerase . This concept accounts for the appearance of unique new upstream and downstream pause sites that result from the insertion of each oligonucleotide. Biochem Biophys Res Commun, 1989 Nov 15, 164(3), 1040 - 4 A versatile non-radioactive technique for restriction analysis of recombinant DNA; Knochenhauer S et al.; Restriction analysis of recombinant DNA is most frequently performed according to Smith and Birnstiel by labeling 5'-termini with 32P, followed by partial digestion, separation, and autoradiographic detection of labeled fragments . We describe a rapid, non-radioactive technique for restriction analysis of recombinant DNA which combines Southern blotting of partial restriction digests and hybridization with a vector-specific probe labeled with the steroid-hapten Digoxigenin for immunological detection . This technique has several advantages compared to conventional methods . Labeling with 32P is not necessary and as the labeled DNA-fragment used as probe is vector-specific, it can be applied for numerous constructs using the particular cloning vector (e.g.pBR322) . Furthermore, the probe can be stored for several months and can be reused many times. Arch Biochem Biophys, 1989 Nov 15, 275(1), 11 - 5 On the inhibition of deoxycytidylate hydroxymethylase by 5-fluoro-2'-deoxycytidine 5'-monophosphate; Subramaniam R et al.; Studies were performed to determine whether 5-fluoro-2'-deoxycytidine 5'-monophosphate (FdCMP) is an inhibitor of deoxycytidylate hydroxymethylase and whether it could form an isolable covalent complex with the enzyme and the cofactor, 5,10-methyl-enetetrahydrofolate . The results showed that although FdCMP is a competitive inhibitor of dCMP hydroxymethylase, it does not cause time-dependent inhibition of the enzyme in the presence of cofactor . Further, although uv difference spectral evidence was found for FdCMP-cofactor-enzyme complex, the complex was not sufficiently stable to isolate on nitrocellulose filters . We conclude that FdCMP is not a mechanism-based inhibitor of dCMP hydroxymethylase. J Biol Chem, 1989 Nov 15, 264(32), 19132 - 7 Catalytic role of histidine 147 in Escherichia coli thymidylate synthase; Dev IK et al.; Nine mutant thymidylate synthases were isolated that only differed in sequence at position 147 . The wild-type enzyme (which had a histidine residue at 147) and mutant enzymes were purified to near homogeneity and their kinetic properties were compared . Although the kcat values for the mutant enzymes were 10-10,000-fold lower than for the wild-type enzyme, the Km values for both 2'-deoxyuridylate and 5,10-methylenetetrahydrofolate were nearly identical for all the enzymes indicating that His-147 is not significantly involved in initial substrate binding . By comparing the wild-type (His-147) to the glycine (Gly-147) enzyme, the side chain of His-147 was estimated to lower the activation energy of the catalytic step by 1.6-2.9 kcal mol-1 . In contrast to the wild-type enzyme, the activity of the Gly-147 enzyme decreased when the pH was raised above 7.5 . The activity loss coincided with the deprotonation of a residue that had a pKa of 9.46 +/- 0.2 and an enthalpy of ionization (delta Hion) of 12.1 +/- 0.9 . These values are consistent with the involvement of a lysine or an arginine residue in the catalytic process . An inspection of the rates of ternary complex formation among enzyme, 5-fluoro-2'-deoxyuridylate, and 5,10-methylenetetrahydrofolate for the mutant enzymes indicated that His-147 is not needed for the proton removal from C-5 of 2'-deoxyuridylate but rather participates in an initial catalytic step and alters the pKa value of a catalytically important lysine or arginine residue. J Biol Chem, 1989 Nov 15, 264(32), 18907 - 11 Determination of the secondary structure of interleukin-8 by nuclear magnetic resonance spectroscopy; Clore GM et al.; The solution conformation of interleukin-8 (IL-8), a small protein of 72 residues with a wide range of proinflammatory activities, has been investigated by two-dimensional NMR spectroscopy . The 1H-NMR spectrum of IL-8 is assigned in a sequential manner and regular elements of secondary structure are identified on the basis of a qualitative interpretation of the nuclear Overhauser, coupling constant and amide exchange data . The IL-8 monomer contains a triple stranded anti-parallel beta-sheet arranged in a Greek key and a long C-terminal helix (residues 57-72) . It is shown that IL-8 is a dimer in solution in which the interface is principally formed by six backbone hydrogen bonds between residues 25, 27, and 29 of one monomer and residues 29, 27, and 25, respectively, of the other . As a result, the two units of the dimer form a contiguous six-stranded anti-parallel beta-sheet . The secondary structure of IL-8 is similar to that found in the crystal structure of the sequence related protein platelet factor 4. Gene, 1989 Nov 15, 83(1), 147 - 52 Recombinant selection by microinjection: a simple cDNA cloning procedure for production of exclusively sense RNA transcripts; Digweed M et al.; A new strategy for cDNA cloning is presented, designed particularly for identification of recombinants by functional analysis, after microinjection into somatic cells . First-strand synthesis is primed by the oligodeoxyribonucleotide: (formula; see text) After second-strand synthesis and blunting, double-stranded cDNA is formed, which carries restriction sites for NotI and ApaI downstream from the coding sequence . The cDNA is ligated into a plasmid, between two promoters for phage T7 and T3 RNA polymerases . Following transfection and amplification in Escherichia coli, plasmids are extracted from the library or sublibraries . Linearisation with NotI, prior to in vitro transcription, cleaves the plasmids between the 3'-end of the coding sequence and the adjacent promoter and thus ensures that only sense RNAs, suitable for microinjection, are produced after addition of the RNA polymerases . Use of NotI, a rare cutter in the human genome, should ensure that the cDNA inserts are not damaged during linearisation . In the unlikely event that this does happen, a site for ApaI is also available . The method is demonstrated for the human adenine phosphoribosyltransferase-encoding gene. Anal Biochem, 1989 Nov 15, 183(1), 177 - 89 Enzymatic quantification of sphingosine in the picomole range in cultured cells; Van Veldhoven PP et al.; An enzymatic method to quantify the mass levels of free sphingosine in cellular lipid extracts was developed . The assay is based upon the observation that ceramide is phosphorylated by Escherichia coli diacylglycerol kinase . Although sphingosine is not recognized by the enzyme, it can be converted to a substrate by acylation with hexanoic anhydride . Using a mixed micellar assay, previously reported for the mass quantification of diacylglycerol, the short-chain ceramide (N-C6-sphingosine), generated by acylation, is quantitatively phosphorylated to N-C6-{32P}sphingosine phosphate . This assay allows quantification of sphingosine over a broad range from 25 to 5000 pmol . When this assay was applied to standard compounds, reverse-phase thin-layer chromatography of the reaction products was adequate to separate the phosphorylated derivatives of long-chain ceramide and N-C6-sphingosine . However, the presence of other lipids in extracts from biological samples (mainly monoalkylglycerols which are also a substrate for the diacylglycerol kinase) interfered and necessitated an additional purification step . The most efficient purification step devised was a combination of anion- and cation-exchange chromatography . The mass levels of free sphingoid bases in different cultured cells were quantified using this assay . Levels varied between 8 to 20 pmol/10(6) cells . When normalized to phospholipids, sphingosine levels varied between 0.01 and 0.04 mol% . The lowest levels were found in L929 cells, while Schwann cells derived from Twitcher mice contained the highest levels . These levels were significantly higher than those of Schwann cells derived from normal mice. Biochem J, 1989 Nov 15, 264(1), 151 - 60 Cloning and expression in Escherichia coli of a rat hepatoma cell cDNA coding for 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase; Crepin KM et al.; In liver, the 470-residue bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2/FBPase-2) catalyses the synthesis and degradation of fructose 2,6-bisphosphate, a potent stimulator of glycolysis . In rat hepatoma (HTC) cells, this enzyme has kinetic, antigenic, and regulatory properties, such as insensitivity to cyclic AMP-dependent protein kinase and lack of associated FBPase-2 activity, that differ from those in liver . To compare the sequence of the HTC enzyme with that of the liver enzyme, we have cloned the corresponding fully-coding cDNA from HTC cells . This cDNA predicts a protein of 448 residues in which the first 32 residues of liver PFK-2/FBPase-2 including the cyclic AMP target sequence have been replaced by a unique N-terminal decapeptide . The rest of the protein is identical with the liver enzyme . An N-terminally truncated recombinant peptide of 380 residues containing the PFK-2 and FBPase-2 domains was expressed in Escherichia coli as a beta-galactosidase fusion protein . It was recognized by anti-PFK-2 antibodies but its enzymic activities were barely detectable . In contrast, a cDNA fully-coding for the HTC enzyme could be expressed in E . coli as a beta-galactosidase-free peptide that exhibited both PFK-2 and FBPase-2 activities . This peptide had those PFK-2 kinetic properties of the HTC enzyme that differ from the liver enzyme . These data, together with immunoblot experiments, suggest that the lack of associated FBPase-2 activity in HTC cells results from a post-translational modification of the enzyme rather than from the difference in amino acid sequence . As well as this peculiar type of PFK-2/FBPase-2 mRNA, HTC cells also contained low concentrations of the liver-type mRNA . Unlike in liver, neither mRNA was induced by dexamethasone in these cells. Gene, 1989 Nov 15, 83(1), 125 - 35 Human ornithine decarboxylase(ODC)-encoding gene: cloning and expression in ODC-deficient CHO cells; Holtta E et al.; We have cloned a full-length human ornithine decarboxylase (ODC)-encoding gene from a genomic library of human myeloma cells which overproduce ODC due to a selective gene amplification . Correct expression of the cloned gene was assessed by transfecting it into a Chinese hamster ovary (CHO) cell mutant devoid of ODC activity . Transfection with a 10-kb BamHI DNA fragment of the genomic clone, conferred ODC activity to the recipient cells and relieved them of dependence on exogenous polyamines for growth . A set of 40 transformants was isolated, eight of which were further characterized . The transfected ODC gene appeared to be hypomethylated at the cytosine residues in the sequence CpG . The transfectants were all responsive to serum stimulation, but showed different levels of ODC expression depending on both copy number and integration site of the transfected ODC gene . ODC serum induction in the transfectants was sensitive to cycloheximide and polyamine additions, and the half-life of the enzyme was very short, like that in normal CHO cells . These results suggest that the human ODC gene we transfected contains all the elements needed for normal control of ODC expression. Biochem Biophys Res Commun, 1989 Nov 15, 164(3), 1402 - 10 Purification and DNA-binding properties of human papillomavirus type 16 E6 protein expressed in Escherichia coli; Imai Y et al.; Unfused human papillomavirus type 16 (HPV 16) E6 protein was expressed in Escherichia coli using a lambda PL promoter system . The protein was isolated from the cells as inclusion bodies, extracted by 6 M guanidine-HCl, and purified by chromatography . The purified protein had high affinity to DNA and was demonstrated for the first time to bind to a specific sequence within the long control region of HPV 16. Biochem Biophys Res Commun, 1989 Nov 15, 164(3), 1053 - 9 Localization of c-ERB A proteins in rat liver using monoclonal antibodies; Schmidt ED et al.; Monoclonal antibodies were raised against the nuclear thyroid hormone receptors encoded by c-ERB A genes and against a purified nuclear receptor fraction . These antibodies recognize the c-ERB A protein in nuclear extracts from rat liver and are able to compete with thyroid hormone in Scatchard analyses . In sections of rat liver they react with all the hepatocyte nuclei as well as with the cells of the hepatic bile ducts . Comparison with another putative T3 receptor antibody, described previously, showed that distinct 57 kD proteins with a different cellular distribution were recognized. J Natl Cancer Inst, 1989 Nov 15, 81(22), 1698 - 704 Antibodies against the human papillomavirus type 16 early proteins in human sera: correlation of anti-E7 reactivity with cervical cancer; Jochmus-Kudielka I et al.; By Western blot technique, 519 samples of human sera were tested for the presence of antibodies to the human papillomavirus (HPV) type 16 proteins E4 and E7 that had been expressed in Escherichia coli as fusion proteins . Sera were obtained from patients attending the University hospitals for reasons unrelated to HPV infections (controls), from patients with HPV-associated lesions, as well as from patients suffering from cervical cancer . Within the control population, 18.1% of them had antibodies that reacted with the E4 protein, and 3.9% of them had antibodies that reacted with the E7 protein . No sex-specific difference in the antibody prevalence was observed . The highest proportion of anti-E4 antibody-positive individuals (40.7%) was observed in the age group between 11 and 20 years . The frequency of anti-E4-positive sera was threefold higher in patients with HPV-associated genital lesions than that in age-matched controls . Antibodies against the HPV16 E7 protein were found 14 times more frequently in patients with cervical cancer, compared with age- and sex-matched controls (P less than .00001) . From these data, we concluded that anti-E4 antibodies may be correlated with virus replication and that anti-E7 antibodies may represent a marker for cervical cancer development. J Biol Chem, 1989 Nov 15, 264(32), 18966 - 72 Human bisphosphoglycerate mutase . Expression in Escherichia coli and use of site-directed mutagenesis in the evaluation of the role of the carboxyl-terminal region in the enzymatic mechanism; Garel MC et al.; Bisphosphoglycerate mutase is an erythrocyte-specific enzyme whose main function is to synthesize 2,3-diphosphoglycerate, the allosteric effector of hemoglobin . In addition to its main 2,3-diphosphoglycerate synthase activity, the enzyme displays phosphatase and mutase activities both involving 2,3-diphosphoglycerate in their reaction . The three activities have been demonstrated to be catalysed at a unique active site . To study the structure of such an active site we have developed a recombinant system producing mutants of human bisphosphoglycerate mutase in Escherichia coli, by site-directed mutagenesis . For this purpose the human bisphosphoglycerate mutase cDNA that we had previously cloned has been used to construct a procaryotic high level expression vector bearing the "tac" promoter . Human bisphosphoglycerate mutase produced in E . coli, a species which does not normally synthesize this enzyme, represented 8% of the total soluble bacterial protein and displayed the three catalytic activities (synthase, mutase, and phosphatase) characteristic of the enzyme . Since it has been suggested that the carboxyl-terminal region may be implicated in the catalytic activity of the enzyme, three variants deleted in this part of the protein were produced . Our results indicate that a minimal deletion of 7 amino acid residues in the carboxyl-terminal portion of the human bisphosphoglycerate mutase completely abolished the three catalytic activities of the enzyme . In contrast, the effects of the deletion of the last two lysine residues were limited to a 38% reduction in the synthase activity . These results show that the carboxyl-terminal amino acid residues are either directly or indirectly implicated in the three catalytic functions of the human bisphosphoglycerate mutase, and that the two terminal lysine residues are not essential for the major part of the enzymatic mechanism of the enzyme. Cancer Res, 1989 Nov 15, 49(22), 6318 - 23 Expression of human DNA topoisomerase I in yeast cells lacking yeast DNA topoisomerase I: restoration of sensitivity of the cells to the antitumor drug camptothecin; Bjornsti MA et al.; Yeast Saccharomyces cerevisiae strains that are permeable to the antitumor alkaloid camptothecin are killed by the drug if they express DNA topoisomerase I, the cellular target of the drug (J . Nitiss and J.C . Wang, Proc . Natl . Acad . Sci . USA, 85: 7501-7505, 1988) . We show that in a yeast strain permeable to camptothecin but lacking DNA topoisomerase I, sensitivity to the drug was restored upon expression of human DNA topoisomerase I from a plasmid-borne human complementary DNA clone . When the human enzyme was expressed from a galactose-inducible, glucose-repressible yeast promoter, PGAL1, sensitivity to camptothecin was observed in the presence of galactose but not in the presence of glucose . Expression of human DNA topoisomerase I in Escherichia coli was also demonstrated by the complementation of a conditional lethal E . coli DNA topoisomerase I mutant . These systems can be used in the study of human DNA topoisomerase I-camptothecin interactions and in the screening of additional therapeutics targeting the enzyme. J Biol Chem, 1989 Nov 15, 264(32), 19192 - 9 Cloning and characterization of cDNA for mitochondrial GTP:AMP phosphotransferase of bovine liver; Yamada M et al.; Three different types of cDNA clones for mitochondrial GTP:AMP phosphotransferase (AK3) were isolated from a cDNA library of bovine liver poly(A)+ RNA . Nucleotide sequencing revealed that each of these clones consisted of a common 5'-untranslated region, a common AK3-coding sequence and a 3'-untranslated region with different sizes . By Northern blot analysis, three species of AK3 mRNA apparently corresponding to the isolated cDNA clones were detected, which would be a result of varying terminations and polyadenylations of the primary transcript . From comparison of the size of the product synthesized in vitro from the message directed by the isolated cDNA with that of the purified AK3 protein, AK3 appeared to have no cleavable NH2-terminal sequence as found in other mitochondrial proteins . The AK3 cDNA was expressed in Escherichia coli, which resulted in complementation of an adenylate kinase mutation of E . coli . The AK3 product was exported to the periplasmic space through the bacterial inner membrane . The possible involvement of the NH2-terminal sequence of the protein in targeting to the mitochondrial matrix was discussed. Biochemistry, 1989 Nov 14, 28(23), 9028 - 43 Mechanism of adenylate kinase . Is there a relationship between local substrate dynamics, local binding energy, and the catalytic mechanism? Sanders CR 2nd, Tian GC, Tsai MD. Adenylyl (beta,gamma-methylene)diphosphonic acid (AMPPCP) labeled with deuterium at the adenine ring ({8-2H}AMPPCP) and at the beta,gamma-methylene group (AMPPCD2P), as well as adenosine 5'-monophosphate labeled at the adenine ring ({8-2H}AMP), was synthesized and used for deuterium nuclear magnetic resonance (NMR) determination of effective correlation times (tau c) of the free nucleotide and the complexes with adenylate kinase (AK) . Extensive and rigorous control experiments and theoretical analysis were performed to justify the validity of the experimental approaches, particularly the fast exchange condition, and the reliability of the tau c values obtained . For the free nucleotide, the results suggest that the phosphonate group of free AMPPCP possesses appreciable local mobility relative to the adenine ring and that complexation with Mg2+ greatly reduced such a local mobility . For the complexes with AK, effective tau c values of 7, 15, 28, 28, and 27 ns were obtained for AMPPCD2P, MgAMPPCD2P, {8-2H}AMPPCP, Mg{8-2H}AMPPCP, and {8-2H}AMP, respectively . These results suggest that the adenine ring of substrates is rigidly bound in all cases, that the phosphonate chain of AMPPCP possesses considerable local mobility, and that Mg2+ reduces such local mobility but does not totally immobilize it . The local dynamics of the analogues bound to AK was correlated with local binding energies for the binding of MgAMPPCP and MgATP to AK estimated from the binding studies by proton NMR and other techniques, in conjunction with the binding theory of Jencks {Jencks, W . P . (1981) Proc . Natl . Acad . Sci . U.S.A . 78, 4046-4050} . The results suggest that no general correlation exists between the local rigidity of portions of a bound substrate and the corresponding (ground state) local binding energy contributed by these portions . In particular, the adenosine moiety contributes little to the binding energy despite the fact that the adenine ring is rigidly bound; the triphosphate (PPPi) moiety behaves oppositely; Mg2+ immobilizes the triphosphate chain but does not enhance binding . Finally, isomers of the substitution-inert beta,gamma-bidentate Cr(III) complexes of adenosine 5'-triphosphate (CrATP) were used to probe two unresolved catalytic problems implicitly related to the local mobility of the phosphonate chain of AMPPCP in the AK-MgAMPPCP complex . The first problem concerns the result of electron paramagnetic resonance (EPR) studies that (Rp)- but not (Sp)-{beta-17O}ATP caused a line broadening in the Mn(II) EPR spectrum of the AK-MnATP complex {Kalbitzer, H . R., Marquetant, R., Connolly, B . A., & Goody, R . S . (1983) Eur . J . Biochem . 133, 221-227}.(ABSTRACT TRUNCATED AT 400 WORDS) Biochemistry, 1989 Nov 14, 28(23), 9088 - 94 Analysis of the ribonuclease H activity of HIV-1 reverse transcriptase using RNA.DNA hybrid substrates derived from the gag region of HIV-1; Mizrahi V; The RNase H activity associated with recombinant p66/p51 HIV-1 reverse transcriptase (RT) has been analyzed in the absence of DNA synthesis by using homogeneous RNA.DNA substrates . The substrates consisted of SP6 runoff transcripts from a portion of the gag region of the HIV-1 genome hybridized to complementary single-stranded DNA from either an M13 subclone or a phagemid transcription vector subclone . The corresponding hybrids either carried a 5'-mismatch of seven nucleotides or were fully base-paired . Analysis of recombinant HIV-1 p66/p51 RT by an activated gel assay employing these substrates suggested that the RNase H activity was exclusively associated with the p66 polypeptide . Denaturing gel electrophoresis was used to analyze the oligonucleotide products generated by hydrolysis of the hybrids by HIV-1 RT, M-MuLV RT, and Escherichia coli RNase H . The significant difference in the time-dependent distribution of products of HIV-1 RT vs E . coli RNase H catalyzed cleavage of 5'-mismatched hybrids indicated that the preparation of recombinant HIV-1 RT was free of contaminating bacterial RNase H . Although the HIV-1 RT associated RNase H activity shares many of the general mechanistic features of other retroviral enzymes {Gerard, G . F . (1981) Biochemistry 20, 256-265}, the appearance of unique intermediates and end products in the course of hydrolysis of 5'-mismatched and fully base-paired hybrids indicated a significant difference in the sequence dependence of the kinetics of RNase H cleavage by HIV-1 RT and M-MuLV RT. Nucleic Acids Res, 1989 Nov 11, 17(21), 8755 - 65 Promoter selectivity of Escherichia coli RNA polymerase: omega factor is responsible for the ppGpp sensitivity; Igarashi K et al.; Transcription in vitro of stringently controlled Escherichia coli genes by purified RNA polymerase holoenzyme is inhibited by guanosine tetraphosphate (ppGpp) . In order to examine possible role of omega factor in this ppGpp sensitivity, RNA polymerases with or without the omega factor were reconstituted and tested for their ppGpp sensitivity using an in vitro mixed transcription system . RNA polymerase lacking the omega factor was found virtually insensitive to ppGpp but the addition of a purified omega factor restored the ppGpp sensitivity of this omega-free RNA polymerase . These results raise a possibility that the omega factor is a regulatory protein of RNA polymerase and is involved in the ppGpp-mediated alteration of the promoter selectivity. Nucleic Acids Res, 1989 Nov 11, 17(21), 8741 - 53 Overproduction and crystallization of FokI restriction endonuclease; Kita K et al.; To overproduce FokI endonuclease (R.FokI) in an Escherichia coli system, the coding region of R.FokI predicted from the nucleotide sequence was generated from the FokI operon and joined to the tac promoter of an expression vector, pKK223-3 . By introduction of the plasmid into E . coli UT481 cells expressing the FokI methylase gene, the R.FokI activity was overproduced about 30-fold, from which R.FokI was purified in amounts sufficient for crystallization . The removal of a stem-loop structure immediately upstream of the R.FokI coding region was essential for overproduction. Nucleic Acids Res, 1989 Nov 11, 17(21), 8485 - 93 High level gene expression in mammalian cells by a nuclear T7-phase RNA polymerase; Lieber A et al.; Here we describe a novel expression system for mammalian cells which is based on transcription of hybrid genes containing T7 phage promoters by a T7 phage RNA polymerase targeted to the nucleus of the host cells . The RNA polymerase gene of T7 phage has been modified by substituting a sequence encoding the nuclear location signal of SV40 large T antigen for the N-terminal part of the polymerase gene . Expression of the modified gene is driven by the mouse metallothionein promoter in transfected mouse Ltk- cells resulting in high concentration of the polymerase in the nucleus . Nuclear T7 RNA polymerase directs efficient transcription of the cat gene under control of a T7 promoter . T7 constructs are expressed at a level at least 6 fold higher than the prototype pRSVcat . The unique properties of this heterologeous expression system are discussed. Nucleic Acids Res, 1989 Nov 11, 17(21), 8645 - 56 Physical studies of 5S RNA variants at position 66; Zhang P et al.; Two variants of the 5S RNA of E . coli have been examined by imino proton NMR spectroscopy, one of them a deletion of A66 (Christiansen, J., Douthwaite, S.R., Christensen, A . and Garrett, R.A . (1985) EMBO J . 4, 1019-1024) and the other a replacement of A66 with a C (Goringer, H.U . and Wagner, R . (1986) Biol . Chem . Hoppe-Seyler 367, 769-780) . Both are of interest because the role the bulged A in helix II of 5S RNA is supposed to play in interactions with ribosomal protein L18 . The data show that the structural perturbations that result from these mutations are minimal, and assign the resonances of some of the imino protons around position 66 . Some mutations at or near position 66 greatly reduce the L18-dependent increase in the circular dichroism of 5S RNA at 267 nm first observed by Bear and coworkers (Bear, D.G., Schleich, T., Noller, H.F . and Garrett, R.A . (1977) Nucl . Acids Res . 4, 2511-2526). Nature, 1989 Nov 9, 342(6246), 204 - 6 Images of single-stranded nucleic acids by scanning tunnelling microscopy; Dunlap DD et al.; The scanning tunnelling microscope has the potential to resolve the structure of biological molecules with atomic detail . Progress has been made in the imaging of dried, unshadowed double helices of DNA4-7 and in recording images of DNA under water . Also, images of unshadowed complexes of DNA with the RecA protein from Escherichia coli indicate that this technique may not be restricted to thin biological samples . Here we present images of polydeoxyadenylate molecules aligned in parallel, with their bases lying flat on a surface of highly oriented pyrolytic graphite and with their charged phosphodiester backbones protruding upwards . Based on these images, a molecular model has been built which suggests the presence of a hydrogen bond that could stabilize the parallel alignment . Our micrographs demonstrate the potential application of scanning tunnelling microscopy in structural studies of nucleic acids and provide evidence that it could be used to sequence DNA. Nature, 1989 Nov 9, 342(6246), 142 - 8 Intermediate states in the movement of transfer RNA in the ribosome; Moazed D et al.; Direct chemical 'footprinting' shows that translocation of transfer RNA occurs in two discrete steps . During the first step, which occurs spontaneously after the formation of the peptide bond, the acceptor end of tRNA moves relative to the large ribosomal subunit resulting in 'hybrid states' of binding . During the second step, which is promoted by elongation factor EF-G, the anticodon end of tRNA, along with the messenger RNA, moves relative to the small ribosomal subunit. FEBS Lett, 1989 Nov 6, 257(2), 447 - 50 Colicin N and its thermolytic fragment induce phospholipid vesicle fusion; Massotte D et al.; Colicin N, a bacteriocin encoded on a plasmid belonging to the pore-forming class of colicins, induces phospholipid vesicle fusion at acidic pH as demonstrated by fluorescence resonance energy transfer . Its C-terminal thermolytic fragment has properties very similar to the native molecule . The fusion is protein concentration-dependent and is regulated by (a) group(s) with a pK of approximately 4.6 . The physiological relevance of this characteristic common to all colicins tested so far is discussed. FEBS Lett, 1989 Nov 6, 257(2), 269 - 73 Proteolytic activity of the plum pox potyvirus NIa-protein on excess of natural and artificial substrates in Escherichia coli; Garcia JA et al.; The plum pox potyvirus (PPV) NIa protease expressed from a medium copy number plasmid was able to process an excess of substrate expressed from a high copy number plasmid, in a binary Escherichia coli expression system . The delta B7 NIa protease mutant only partially processed the NIb-CP junction but its efficiency was independent of the amount of substrate . The delta B7 mutant essentially did not recognize an artificial cleavage site which was quite efficiently recognized by the wild-type protease . No competitive inhibition of the proteolytic activity by the presence of excess of different protease mutants was observed. Eur J Biochem, 1989 Nov 6, 185(2), 341 - 6 Substitution of Val20 by Gly in elongation factor Tu . Effects on the interaction with elongation factors Ts, aminoacyl-tRNA and ribosomes; Jacquet E et al.; Substitution of V20 by G in the consensus element G18HVDHGK24 of EF-Tu (referred to as EF-TuG20) strongly influences the interaction with GDP as well as the GTPase activity {Jacquet, E . & Parmeggiani, A . (1988) EMBO J . 7, 2861-2867} . In an extension of this work we describe additional properties of the mutated factor, paying particular attention to the interaction with the macromolecular ligands . Our results show that the conformational transitions induced by the mutation strongly favor the regeneration of the active complex EF-TuG20.GTP, almost as effectively as with wild-type EF-Tu in the presence of elongation factor Ts . Addition of elongation factor Ts further enhances the rate of the GDP to GTP exchange of the mutated factor . Remarkably, EF-TuG20.GDP can support the enzymatic binding of aminoacyl-tRNA to ribosome.mRNA at low MgCl2 concentration, an effect that with wild-type EF-Tu can only occur in the presence of kirromycin . Our results show that EF-TuG20.GDP shares common features with the GTP-like conformation induced by kirromycin on wild-type EF-Tu . The ability of the ribosome to activate the EF-TuG20 center for GTP hydrolysis is strongly decreased, while the stimulation by aminoacyl-tRNA is conserved . The ribosomal activity is partially restored by addition of aminoacyl-tRNA plus poly(U), showing that codon/anticodon interaction contribute to correct the anomalous interaction between ternary complex and ribosomes . The impaired activity of EF-TuG20 in poly(Phe) synthesis is related to the degree of defective GTP hydrolysis and, most interestingly, it is characterized by a striking increase of the fidelity of translation at high MgCl2 concentration . This effect probably depends on a more selective recognition of the ternary complex by ribosome.mRNA, as a consequence of a longer pausing of EF-TuG20 on the ribosome . In conclusion, position 20 in EF-Tu is important for coordinating the allosteric mechanisms controlling the action of EF-Tu and its ligands. FEBS Lett, 1989 Nov 6, 257(2), 257 - 9 Translational active mRNPs from rabbit reticulocytes are qualitatively different from free mRNA in their translatability in cell-free system; Minich WB et al.; The translatability of polyribosomal and free mRNPs from rabbit reticulocytes and their mRNA was compared . Both classes of mRNPs turned out to be active in rabbit reticulocyte lysates . Considerable differences between mRNPs and mRNA have been revealed . The most striking feature of mRNPs was that high concentrations of mRNPs do not inhibit protein biosynthesis, whereas high concentrations of mRNA strongly inhibit this process . This inhibition is specific for mRNA and does not occur at the addition of the same amount of rRNA from E . coli . The features of mRNP translation are not the result of addition of the supplementary translation factors within particles . The specific function of mRNP proteins in the process of translation is under discussion. Eur J Biochem, 1989 Nov 6, 185(2), 365 - 70 Role of the cell surface-exposed regions of outer membrane protein PhoE of Escherichia coli K12 in the biogenesis of the protein; Agterberg M et al.; To investigate the role of the cell surface-exposed regions of outer membrane protein PhoE of Escherichia coli K12 in the biogenesis of the protein, deletions were generated in two presumed cell surface-exposed regions of the protein . Intact cells expressing these mutant proteins were recognized by PhoE-specific monoclonal antibodies, which recognize conformational epitopes on the cell surface-exposed parts of the protein and/or were sensitive to a PhoE-specific phage . This shows that the polypeptides were normally incorporated into the outer membrane . When the deletions extended four amino acid residues into the seventh presumed membrane-spanning segment, the polypeptides accumulated in the periplasm . In conclusion, exposed regions of PhoE protein apparently do not play an essential role in outer membrane localization, which is consistent with the observation that these regions are hypervariable when PhoE is compared to the related proteins OmpF and OmpC . In contrast, the membrane-spanning segments are essential for the assembly process. FEBS Lett, 1989 Nov 6, 257(2), 465 - 7 Uni-site catalysis by Escherichia coli F1-ATPase with different numbers of bound nucleotides; Hanada H et al.; We prepared two types of E . coli F1 by slightly different gel filtration procedures of the purified F1: F1(II) contained about 2 mol, and F1(V) about 5 mol of bound adenine nucleotides per mol of the enzyme . Thus F1(II) had more than 2, possibly 3, vacant catalytic sites, while F1(V) had less than one vacant catalytic site . The rate of ATP hydrolysis in uni-site catalysis (in the presence of inorganic phosphate) was about 3-fold higher with F1(II) than with F1(V), suggesting that ADP and inorganic phosphate bound at the catalytic sites of F1(V) changed the kinetics of uni-site catalysis significantly. FEBS Lett, 1989 Nov 6, 257(2), 311 - 4 Point mutations in conserved amino acid residues within the C-terminal domain of HIV-1 reverse transcriptase specifically repress RNase H function; Schatz O et al.; Two single site substitutions (E478----Q and H539----F) were introduced into the C-terminal RNase H domain of HIV-1 reverse transcriptase . These mutant proteins were expressed in Escherichia coli and purified by Ni2+-nitrilotriacetic acid affinity chromatography . Both enzymes are clearly defective in RNase H function, but exhibit wild type reverse transcriptase activity. J Mol Biol, 1989 Nov 5, 210(1), 79 - 89 Evidence for partial export of Vitreoscilla hemoglobin into the periplasmic space in Escherichia coli . Implications for protein function; Khosla C et al.; The Vitreoscilla hemoglobin protein has been implicated in earlier studies to serve a globin-like function under oxygen-limited growth conditions . Evidence is presented using fractionation as well as proteinase K accessibility techniques to prove that a considerable amount of this protein is localized in the periplasmic space of the cell . Genetic evidence points towards the existence of information within the N-terminal domain of the protein that plays a role in the process of protein export . However, this sequence is not cleaved in the process of translocation . Analysis of the primary structure of this region reveals several unusual features . Instead of positively charged residues at its amino terminus, it has a negative charge . The overall hydrophobicity of the central region of this sequence is significantly lower than in typical leader peptides due to the presence of a charged residue . In keeping with the likelihood that such an export signal may not be very efficient, a substantial fraction of the total cellular hemoglobin can also be detected in the cytoplasm . Heme is incorporated in both cytoplasmic and periplasmic globin as indicated by the ability of protein from both fractions to bind carbon monoxide . The secretion of this protein into the periplasm raises questions concerning the physiological significance of its localization . Dimensional analysis of a model based on the facilitated diffusion hypothesis, which was initially proposed to account for the effects of eukaryotic globins on oxygen transport, suggests that periplasmic globin can support an additional oxygen flux to the respiratory apparatus that may be physiologically significant. J Biol Chem, 1989 Nov 5, 264(31), 18733 - 41 Mannose permease of Escherichia coli . Domain structure and function of the phosphorylating subunit; Erni B et al.; The mannose permease of Escherichia coli is a component of the phosphotransferase system . It transports mannose and related hexoses by a mechanism that couples sugar transport with sugar phosphorylation . It is a complex consisting of two transmembrane subunits (II-PMan and II-MMan) and a hydrophilic subunit (IIIMan) . IIIMan also exists in a soluble form as dimer in the cytoplasm . Each monomer of IIIMan consists of two structurally and functionally distinct domains which are linked by a flexible hinge of the sequence KAAPAPAAAAPKAAPTPAKP . Both domains are transiently phosphorylated . The NH2-terminal domain (P13) is phosphorylated at N-3 of His-10 by the cytoplasmic phosphorylcarrier protein phospho-HPr . The COOH-terminal domain (P20) is phosphorylated by P13 at N-1 of His-175 . Phosphoryltransfer occurs not only between P13 and P20 on the same IIIMan subunit but also between isolated domains and between domains on different subunits of the dimer . In the presence of the IIMan subunits, the phosphoryl group is directly transferred from His-175 of P20 to the sugar substrates of the permease . The P13 domain contains the contact sites for dimerization of IIIMan . The P20 domain contains the contact sites for interaction with the IIMan subunits . By reconstructing the ptsL gene, the two domains were expressed as individual polypeptides and the length of the hinge between P13 and P20 was changed . The in vivo and in vitro activities of mutant IIIMan were little affected by these modifications . The hinge is highly sensitive to proteolytic cleavage in vitro and its specificity for proteases can be modified by introducing the appropriate specificity determinants. J Biol Chem, 1989 Nov 5, 264(31), 18645 - 6 Crystallization of the complex of a monoclonal Fab fragment with the histidine-containing protein of the phosphoenolpyruvate: sugar phosphotransferase system of Escherichia coli; Delbaere LT et al.; Single crystals of the complex of a monoclonal Fab fragment with the histidine-containing protein of the phosphoenolpyruvate:sugar phosphotransferase system of Escherichia coli have been grown . This represents one of the first Fab-protein antigen complexes in which the same Fab fragment has previously been crystallized in the uncomplexed state and the structure solved (Prasad, L., Vandonselaar, M., Lee, J . S., and Delbaere, L . T . J . (1988) J . Biol . Chem . 263, 2571-2574) . Single crystals up to 0.25 x 0.50 x 0.05 mm in size were grown by the technique of washing and reseeding . The space group is C2, with unit cell dimensions a = 130.0, b = 68.1, and c = 77.6 A; beta = 97.3 degrees; and Z = 4 . There is one Fab-histidine-containing protein complex/asymmetric unit, and the solvent content is estimated to be 57%. J Biol Chem, 1989 Nov 5, 264(31), 18627 - 31 A novel flagellar Ca2+-binding protein in trypanosomes; Engman DM et al.; A 24-kDa protein of Trypanosoma cruzi, the protozoan parasite that causes Chagas' disease, is recognized by antisera from both humans and experimental animals infected with this organism . Near its C terminus are two regions that have sequence similarity with several Ca2+-binding proteins and that conform to the "E-F hand" Ca2+-binding structure . We expressed a cDNA encoding this protein in Escherichia coli and showed that both the recombinant protein and the 24-kDa native trypanosome protein do indeed bind Ca2+ . The protein's low Ca2+-binding capacity (less than 2 mol of Ca2+/mol of protein) and high Ca2+-binding affinity (apparent Kd less than 50 microM Ca2+) are consistent with binding of Ca2+ via the E-F hand structures . Immunofluorescence assays using a mouse antiserum directed against the fusion protein localized the native protein to the trypanosome's flagellum . The protein's abundance, Ca2+-binding property, and flagellar localization suggest that it participates in molecular processes associated with the high motility of the parasite. J Biol Chem, 1989 Nov 5, 264(31), 18340 - 8 Formation of heteroduplex DNA promoted by the combined activities of Escherichia coli recA and recBCD proteins; Roman LJ et al.; We have established an in vitro reaction in which heteroduplex DNA formation is dependent on the concerted actions of recA and recBCD proteins, the major components of the recBCD pathway of genetic recombination in vivo . We find that heteroduplex DNA formation requires three distinct enzymatic functions: first, the helicase activity of recBCD enzyme initiates heteroduplex DNA formation by unwinding the linear double-stranded DNA molecule to transiently form single-stranded DNA (ssDNA); second, recA protein traps this ssDNA before it reanneals; third, recA protein catalyzes the pairing of this ssDNA molecule with another homologous ssDNA molecule, followed by the renaturation of these molecules to form heteroduplex DNA . The first two functions should be important to all in vitro reactions involving recA and recBCD proteins, whereas the third will be specific to the DNA substrates used . The rate-limiting step of heteroduplex DNA formation is the trapping by recA protein of the ssDNA produced by recBCD enzyme . A model for this reaction is described. J Biol Chem, 1989 Nov 5, 264(31), 18296 - 301 Inactivation of Escherichia coli L-threonine dehydrogenase by 2,3-butanedione . Evidence for a catalytically essential arginine residue; Epperly BR et al.; Incubation of homogeneous preparations of L-threonine dehydrogenase from Escherichia coli with 2,3-butanedione, 2,3-pentanedione, phenylglyoxal, or 1,2-cyclohexanedione causes a time- and concentration-dependent loss of enzymatic activity; plots of log percent activity remaining versus time are linear to greater than 90% inactivation, indicative of pseudo-first order inactivation kinetics . The reaction order with respect to the concentration of modifying reagent is approximately 1.0 in each case suggesting that the loss of catalytic activity is due to one molecule of modifier reacting with each active unit of enzyme . Controls establish that this inactivation is not due to modifier-induced dissociation or photoinduced nonspecific alteration of the dehydrogenase . Essentially the same Km but decreased Vmax values are obtained when partially inactivated enzyme is compared with native . NADH (25 mM) and NAD+ (70 mM) give full protection against inactivation whereas much higher concentrations (i.e . 150 mM) of L-threonine or L-threonine amide provide a maximum of 80-85% protection . Amino acid analyses coupled with quantitative sulfhydryl group determinations show that enzyme inactivated 95% by 2,3-butanedione loses 7.5 arginine residues (out of 16 total)/enzyme subunit with no significant change in other amino acid residues . In contrast, only 2.4 arginine residues/subunit are modified in the presence of 80 mM NAD+ . Analysis of the course of modification and inactivation by the statistical method of Tsou (Tsou, C.-L . (1962) Sci . Sin . 11, 1535-1558) demonstrates that inactivation of threonine dehydrogenase correlates with the loss of 1 "essential" arginine residue/subunit which quite likely is located in the NAD+/NADH binding site. J Biol Chem, 1989 Nov 5, 264(31), 18751 - 60 Site-specific substitution of glutamate for aspartate at position 59 of rat oncomodulin; Hapak RC et al.; Replacement of the aspartate residue at position 59 of rat oncomodulin by glutamate by oligonucleotide-directed mutagenesis has afforded a protein which more closely resembles rat parvalbumin, at least judged by its interaction with the luminescent lanthanide ion Eu3+ . The single-peak 7F0----5D0 spectrum observed at pH 5.0 with the fully bound wild-type protein is replaced by one which clearly shows two features at 5791 and 5796 A, arising from Eu3+ ions bound at the CD and EF sites, respectively . Furthermore, the pH dependence of the spectrum is substantially altered; the pKa observed for the CD domain, in which aspartate 59 residues, is shifted upward from pH 6.0 for the wild-type recombinant protein to pH 6.8 in the D59E mutant . Moreover, the maximum in the high-pH spectrum is shifted from 5781 to 5784 A . All three changes are indicative of a CD binding domain having increased parvalbumin-like character . Interestingly, however, the D59E substitution has only a modest effect on the Ca2+- and Mg2+-binding properties of the CD domain . For the wild-type protein, KCa = 7.8 x 10(-7) M and KMg = 3 x 10(-3) M . These affinities are more than an order of magnitude weaker than those seen for various parvalbumins and substantiate previous claims for calcium specificity made for the oncomodulin CD domain . Replacement of aspartate 59 by glutamate resulted in minor increases in affinity of the CD domain for Ca2+ (KCa = 5.5 x 10(-7) M) and Mg2+ (KMg = 1 x 10(-3) M) . These findings strongly suggest that residues in oncomodulin besides aspartate 59 are important determinants of the observed calcium specificity of the CD calcium-binding domain . The consequences of the substitution at residue 59 appear to be confined to the CD domain . For the EF site in wild-type recombinant oncomodulin, KCa = 4.2 x 10(-8) M and KMg = 1.6 x 10(-4) M . The corresponding values for the D59E site-specific variant are identical within experimental error (KCa = 4.2 x 10(-8) M and KMg = 1.8 x 10(-4) M). J Biol Chem, 1989 Nov 5, 264(31), 18693 - 700 Characterization of OmpR binding sequences in the upstream region of the ompF promoter essential for transcriptional activation; Rampersaud A et al.; In Escherichia coli the expression of the outer membrane porin gene ompF requires the transcriptional activator protein OmpR . Previous DNase I footprinting experiments with purified OmpR localized the OmpR binding site from positions -105 to -60 (relative to the transcriptional start site) in the ompF promoter, and three tandem 10-base pair sequences elements (Fa, Fb, and Fc) within this region were proposed to be important for OmpR recognition . In order to elucidate the roles of the F boxes for transcriptional activation of ompF, various F box deletions and point mutations were constructed and analyzed for their effects on ompF-lacZ expression and OmpR binding . Removal of 102 nucleotides, which included a portion of the OmpR binding region (the Fa box), evidenced the largest decrease in transcriptional activation and significantly reduced OmpR binding . Additional deletion of four more base pairs in this target site (representing half of the Fb box) further reduced ompF expression . OmpR interactions with DNA sequences representing the OmpR binding region were analyzed by DNA mobility shift experiments . A 43-base pair ompF oligonucleotide containing the Fa, Fb, and Fc regions was sufficient for OmpR-dependent DNA binding using either purified OmpR or cell supernatants . The central C residue in each F box was changed to a T and unique patterns of protein-DNA complexes were observed that were different from that of the wild type binding site . The most dramatic effect on OmpR binding was observed when the C to T transversion occurred in the Fb box, and this mutation also reduced the level of ompF-lacZ expression . Our results indicate that the F boxes play important roles in the activation of ompF expression, and we suggest that OmpR may interact cooperatively with these boxes. J Biol Chem, 1989 Nov 5, 264(31), 18582 - 8 In vitro analysis of the process of translocation of OmpA across the Escherichia coli cytoplasmic membrane . A translocation intermediate accumulates transiently in the absence of the proton motive force; Tani K et al.; The proton motive force (delta mu H+) plays an important role, although it is not absolutely essential, in the in vitro translocation of secretory proteins, such as OmpA, across the cytoplasmic membrane of Escherichia coli (Yamada, H., Tokuda, H., and Mizushima, S . (1989) J . Biol . Chem . 264, 1723-1728) . The transient accumulation in membrane vesicles of a possible translocation intermediate of OmpA was observed in the absence of delta mu H+ . The intermediate was detected on a polyacrylamide gel as a proteinase K-resistant band corresponding to a molecular weight of 26,000 . The intermediate did not possess the signal peptide . The appearance of this band was inhibited in the absence of ATP or the presence of adenosine 5'-(beta,gamma-imino)triphosphate (AMP-PNP) and enhanced upon the addition of SecA . Upon the addition of NADH that energizes the membrane, the intermediate was converted to the translocated form of OmpA, even in the presence of AMP-PNP . These results suggest different requirements of ATP and delta mu H+ for the early and late stages of the translocation reaction . The SecA requirement for the early stage of the translocation has also been suggested . In addition to this band, two other bands were observed at higher positions on the gel, when the translocation reaction was performed in the absence of delta mu H+ . Although these two bands also represented the mature form of OmpA, which was partly protected from the proteinase K treatment by the membrane vesicles, the accumulation was not transient . These bands did not appear when the translocation reaction was performed in the presence of dithiothreitol . Together with other evidence, the above observations suggest that OmpA, which has an intramolecular disulfide bridge, cannot undergo the translocation unless delta mu H+ is imposed. J Biol Chem, 1989 Nov 5, 264(31), 18577 - 81 A high concentration of SecA allows proton motive force-independent translocation of a model secretory protein into Escherichia coli membrane vesicles; Yamada H et al.; The in vitro translocation of OmpF-Lpp, a model secretory protein, into inverted membrane vesicles of Escherichia coli obligatorily requires the proton motive force (delta mu H+) in the conventional assay system (Yamada, H., Tokuda, H., and Mizushima, S . (1989) J . Biol . Chem . 264, 1723-1728) . The translocation, however, took place efficiently, even in the absence of delta mu H+, when the system was supplemented with additional SecA . With the stripped membrane vesicles, which are permeable to protons, or in the absence of NADH, the supplementation of SecA remarkably stimulated the translocation activity . The further addition of NADH did not significantly enhance the translocation activity under the SecA-enriched conditions . OmpF-Lpp thus translocated could be recovered from the vesicular lumen by sonication, indicating that complete translocation occurred in the absence of delta mu H+ . It is suggested that delta mu H+ is required for high affinity interaction of SecA with the presumed secretory machinery in the cytoplasmic membrane and that a high concentration of SecA modulates the delta mu H+ requirement. J Biol Chem, 1989 Nov 5, 264(31), 18527 - 30 Protein synthesis initiation factor eIF-4D . Functional comparison of native and unhypusinated forms of the protein; Smit-McBride Z et al.; Protein synthesis initiation factor eIF-4D is a relatively abundant protein in mammalian cells and possesses a unique amino acid residue, hypusine . The role of the hypusine modification in eIF-4D function was addressed by studying the function of eIF-4D variants lacking hypusine . The cloned human cDNA encoding eIF-4D was overexpressed in Escherichia coli and a precursor form lacking hypusine was purified . This protein fails to stimulate methionyl-puromycin synthesis in vitro, nor does it significantly inhibit the action of native eIF-4D . Mammalian expression vectors were constructed with the wild-type cDNA and a mutant form in which the codon for lysine-50 (the residue hypusinated) was altered by site-directed mutagenesis to that for arginine . Transient co-transfection of COS-1 cells with the eIF-4D vector and a vector expressing dihydrofolate reductase led to strong synthesis of both eIF-4D and dihydrofolate reductase . This indicates that normal cellular levels of eIF-4D are saturating in these cells and that excess levels of eIF-4D are not detrimental . Cotransfection with the eIF-4D arginine variant caused no effect on dihydrofolate reductase synthesis, in agreement with the in vitro experiments . The inability of the unhypusinated eIF-4D variants to stimulate methionyl-puromycin synthesis in vitro and to affect protein synthesis in vivo strongly suggests that the hypusine modification is required for eIF-4D activity and for its interaction with the 80 S initiation complex in protein synthesis. J Biol Chem, 1989 Nov 5, 264(31), 18314 - 9 Serine to cysteine mutations in trp repressor protein alter tryptophan and operator binding; Chou WY et al.; The tryptophan repressor regulates expression of the aroH, trpEDCBA, and trpR operons in Escherichia coli . The protein contains no cysteine residues, and the presence of this reactive side chain would allow introduction of spectral probes to monitor binding reactions . Three mutant trp aporepressors, each with a point mutation from serine to cysteine, were produced at positions 67, 86, and 88 by oligonucleotide-directed site-specific mutagenesis . This single conservative substitution affected both tryptophan and operator DNA affinities in all three purified proteins . Cysteine substitution for serine at position 67 decreased tryptophan binding by approximately 6-fold and the operator DNA affinity by approximately 50-fold . The proximity of this amino acid to Gln-68 which is involved in binding to operator DNA (Otwinowski, Z., Schevitz, R . W., Zhang, R.-G., Lawson, C . L., Joachimiak, A., Marmorstein, R . Q., Luisi, B . F., and Sigler, P . B . (1988) Nature 335, 321-329) may account for this effect . Substitution at position 86 diminished tryptophan binding by approximately 4-fold and operator DNA binding by approximately 130-fold . The participation of Ser-86 in the hydrogen bond network required for operator binding (Otwinowski, Z., Schevitz, R . W., Zhang, R.-G., Lawson, C . L., Joachimiak, A., Marmorstein, R . Q., Luisi, B . F., and Sigler, P . B . (1988) Nature 335, 321-329) presumably accounts for the DNA binding effects . The diminished corepressor activity in these two mutants may derive from distortions of the binding region, as the tryptophan and DNA binding sites are intimately related . The mutation at position 88 altered tryptophan binding the most of the three mutants (approximately 18-fold) and operator binding least (approximately 12-fold) . Ser-88 forms a hydrogen bond with the amino group of bound tryptophan (Schevitz, R . W., Otwinowski, Z., Joachimiak, A., Lawson, C . L., and Sigler, P . B . (1985) Nature 317, 782-786), and alteration of the geometry of the side chain would be anticipated to perturb the topology of the binding site . The diminished operator affinity may derive from improper alignment of the tryptophan ligand, crucial for high affinity operator binding (Otwinowski, Z., Schevitz, R . W., Zhang, R.-G., Lawson, C . L., Joachimiak, A., Marmorstein, R . Q., Luisi, B . F., and Sigler, P . B . (1988) Nature 335, 321-329).(ABSTRACT TRUNCATED AT 400 WORDS) J Mol Biol, 1989 Nov 5, 210(1), 39 - 49 Mutant rho factors with increased transcription termination activities . II . Identification and functional dissection of amino acid changes; Mori H et al.; We have determined the nucleotide sequences of three mutant rho genes encoding hyperfunctional rho proteins (rho S) together with their parent allele, rho-ts702 . These mutant rho factors contain the following amino acid changes as deduced from their sequences: (1) the thermo-labile mutant, rho-ts702, has Thr304 substituting for Ala; (2) rho S-77 and rho S-81, which are selectively altered in the primary polynucleotide binding site, share an identical mutation, Leu3----Phe; (3) rho S-82, which is altered in both the primary and secondary polynucleotide binding sites, carries three amino acid substitutions together, Leu3----Phe, Asp156----Asn and Thr323----Ile . Dissection and functional characterization of each mutation in rho S-82 have revealed that Ile323 alone is responsible for alterations in both the secondary RNA interaction and the terminator selectivity observed with the original mutant, rho S-82 . Taken together, these results not only confirm our proposal in the accompanying paper that the primary and secondary RNA binding sites differently contribute in determining the overall efficiency and site-specificity of termination, respectively, but also support the possibility that these binding sites exist as structurally distinct domains in rho protein . In contrast, Asn156 was shown to cause decreased termination efficiency, though it had no influence on RNA interactions . Thus, this amino acid residue appears to be associated with still another rate-determining step of termination, for instance, interactions between rho and RNA polymerase . On the basis of Chou-Fasman secondary structure predictions as well as amino acid sequence comparison with F1-ATPase, we discuss how the proposed domains are structurally and functionally related to the putative ATPase reactive center of rho protein. J Mol Biol, 1989 Nov 5, 210(1), 23 - 37 Mutant rho factors with increased transcription termination activities . I . Functional correlations of the primary and secondary polynucleotide binding sites with the efficiency and site-selectivity of rho-dependent termination; Tsurushita N et al.; We have characterized rho proteins from mutants of Escherichia coli, rho s-81 and rho s-82, which are hyperactive in termination . The two mutant rho proteins are differentially altered both in termination activities and in RNA interactions . rho s-81 generally elicits enhanced termination on various templates such as phage T7 DNA and a DNA restriction fragment containing the trpE intracistronic rho-dependent terminators, either measured as a whole or examined for individual sites . On the other hand, rho s-82 has strikingly different preferences toward individual termination sites, exhibiting overall termination activities higher or lower than normal, depending on templates . From measurements of the rho ATPase activity with T7 RNA and various homoribopolymers as cofactors, both mutant rho proteins are shown to have broadened RNA base specificities in contrast to the stringent requirement for cytosine observed with the wild-type rho . Functional tests on the two kinds of polynucleotide binding sites known for rho have indicated that rho s-81 is mainly altered in the primary site, whereas rho s-82 is simultaneously affected in the secondary binding site as well as the primary site . Thus, we conclude that the primary and secondary sites contribute distinctly in determining the overall efficiency and site-specificity of termination, respectively . Further analysis of detailed termination points at the trpE and lambda tR1 terminators has revealed that major RNA transcripts generated by the wild-type rho and rho s-81 are notably rich in adenine and poor in cytosine for the 3'-terminal five to ten nucleotides, whereas those preferentially terminated by rho s-82 are conversely richer in cytosine than adenine . This finding suggests that rho may recognize the RNA-DNA hybrid region at the 3' end of a nascent transcript in its secondary binding reaction. J Biol Chem, 1989 Nov 5, 264(31), 18808 - 17 Intrinsic properties of reverse transcriptase in reverse transcription . Associated RNase H is essentially regarded as an endonuclease; Oyama F et al.; The intrinsic properties of reverse transcriptase in reverse transcription were studied using a synthetic, partial ovalbumin mRNA with a synthetic DNA oligonucleotide annealed to the 3'-end of the RNA as a model substrate . With or without concomitant cDNA synthesis, the RNase H activity of avian myeloblastosis virus (AMV)-reverse transcriptase cleaved the substrate at a site which would leave a hybrid of between 7 and 14 base pairs between the 3' termini of the RNA and DNA oligonucleotide . Variability in the exact size of the hybrid probably reflects some weak base preference for cleavage by the enzyme . These short hybrids can be recognized as substrates by Escherichia coli RNase H and can be utilized by reverse transcriptase as sites for continuation of cDNA synthesis . Substrates with 5'-triphosphorylated termini, 3'-OH, 3'-phosphate, 3'-end hairpin structures and 20 base pair hybrids on the middle region of long RNA more than 300 bases or on circular RNA were all cleaved by AMV-reverse transcriptase-associated RNase H, indicating that the RNase H activity is essentially regarded as an endonuclease degrading RNA moiety in RNA-DNA hybrid . The modes of action of reverse transcriptase from murine leukemia virus and Rous-associated virus 2 were the same as that of AMV-reverse transcriptase, except that the size of the remaining hybrid and the specificity for cleavage depended on the reverse transcriptase . We propose a possible model to explain the mode of action of RNase H and RNA-dependent DNA polymerase activities in reverse transcription. J Biol Chem, 1989 Nov 5, 264(31), 18457 - 62 Interaction of RNA polymerase II with structurally altered nucleosomal particles . Transcription is facilitated by loss of one H2A.H2B dimer; Gonzalez PJ et al.; The loss of one H2A.H2B dimer from the nucleosomal core increases its affinity for RNA polymerase II and its efficiency as a transcription template, allowing transcription of the entire DNA present in the particle . In contrast, the nucleosomal core lacking the amino-terminal ends of histones, which has an affinity for polymerase equal to that of the H2A.H2B-deficient core, shows transcription properties similar to those of the whole nucleosomal core, with synthesis of short RNA chains (40 nucleotides or less) . Similar results were obtained with a bacterial RNA polymerase . The improved efficiency of the H2A.H2B-deficient cores as transcription templates does not appear to be produced by nonspecific loss of protein or structural relaxation of the particle . These results suggest that a particle lacking one H2A.H2B dimer might be a necessary intermediate during in vivo transcription. Biochim Biophys Acta, 1989 Nov 3, 985(3), 313 - 9 Membrane biogenesis in Escherichia coli: effects of a secA mutation; de Cock H et al.; In Escherichia coli K-12, temperature-sensitive mutations in the secA gene have been shown to interfere with protein export . Here we show that the effect of a secA mutation is strongly pleiotropic on membrane biogenesis . Freeze-fracture experiments as well as cryosections of the cells revealed the appearance of intracytoplasmic membranes upon induction of the SecA phenotype . The permeability barrier of the outer membrane to detergents was lost . Two alterations in the outer membrane may be responsible for this effect, namely the reduced amounts of outer membrane proteins, or the reduction of the length of the core oligosaccharide of the lipopolysaccharide, which was observed in phage-sensitivity experiments and by SDS-polyacrylamide gel electrophoresis . Phospholipid analysis of the secA mutant, grown under restrictive conditions, revealed a lower content of the negatively charged phospholipid cardiolipin and of 18:1 fatty acid compared to those of the parental strain grown under identical conditions . These results are in line with the hypothesis that protein export and lipid metabolism are coupled. Biochim Biophys Acta, 1989 Nov 2, 1009(2), 156 - 60 Effects of gene dosage on the expression of human growth hormone cDNA in Escherichia coli; Yamakawa M et al.; Gene dosage effects on the foreign protein production in Escherichia coli have been analyzed with the expression vectors containing one, two and four tandemly arranged human growth hormone cDNAs under various culture conditions . In a limited synthetic culture medium, a proportional relationship was observed between the number of the integrated cDNA and the amount of human growth hormone produced . With a culture medium rich in the nutrients, however, no such relationship was observed . These results suggest that, under the limited culture conditions, high-level expression of a foreign gene can be achieved by tandemly joined multiple copies of cDNA. Am J Clin Pathol, 1989 Nov, 92(5), 682 - 5 Filter-culture technique using amoeba saline transport medium for the noninvasive diagnosis of Acanthamoeba keratitis; Gradus MS et al.; Acanthamoeba, a common free-living amoeba, is increasingly incriminated as a cause of keratitis and corneal ulceration . Between March 1986 and July 1988, specimens from seven patients submitted by ophthalmologists to the City of Milwaukee Health Department's Bureau of Laboratories were culture positive for Acanthamoeba . All patients were contact lens wearers . The specimens were transported at ambient temperature in amoebasaline (5.0 mL) and filtered through 13 mm 0.22 microns cellulose filters . The filters were then plated in cocultivation with Escherichia coli on nonnutrient agar and had positive results for Acanthamoeba in two to five days . Contact lens cases were culture positive for Acanthamoeba in three instances . These results indicate that corneal scraping in amoeba saline transport medium can provide an effective way to diagnose Acanthamoeba keratitis when direct culture of such specimens is not possible. Virology, 1989 Nov, 173(1), 68 - 76 Biological activity of transcripts synthesized in vitro from full-length and mutated DNA copies of tobacco rattle virus RNA 2; Angenent GC et al.; Full-length cDNA clones of RNA 2 of tobacco rattle virus (TRV) strain PLB have been cloned into the transcription vector pPM1 . Products of in vitro transcription by Escherichia coli RNA polymerase, either capped or uncapped, were as infectious as native RNA 2 when coinoculated with RNA 1 of TRV strain TCM . At least 70% of the internal sequence of the cDNA could be deleted without reduction of the replication efficiency of the transcripts . Sequences of 340 nucleotides at the 5' end and 405 nucleotides at the 3' end of PLB RNA 2 were found to be sufficient for replication . The encapsidation of deletion mutants of PLB RNA 2 was investigated after addition of native PLB RNA 1 and RNA 2 . Accumulation of these mutants was distinguished from that of wild-type RNA 2 by insertion of nonviral sequences in the deleted parts . Three mutant forms of RNA 2 with extensive deletions in the coat protein (CP) gene were replicated but failed to encapsidate, while mutants with nonviral sequences inserted downstream from the CP gene showed a large reduction in replication efficiency. Proc Natl Acad Sci U S A, 1989 Nov, 86(21), 8217 - 21 Molecular cloning and sequence analysis of 3-hydroxy-3-methylglutaryl-coenzyme A reductase from the human parasite Schistosoma mansoni; Rajkovic A et al.; cDNA clones encoding the 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase {(S)-mevalonate:NADP+ oxidoreductase (CoA-acylating), EC 1.1.1.34} from the human parasite Schistosoma mansoni have been isolated and characterized . The composite 3459 base pairs of cDNA sequence contains a 2844-base-pair open reading frame corresponding to a protein of 948 amino acids . The predicted S . mansoni HMG-CoA reductase protein contains a hydrophobic amino terminus consisting of seven potential transmembrane domains that are structurally conservative but are not identical in amino acid sequence with HMG-CoA reductases from other species . The hydrophilic carboxyl terminus of the S . mansoni HMG-CoA reductase protein, however, shares 48-52% sequence identity with the carboxyl termini of other HMG-CoA reductases in a region that contains the catalytic domain . When expressed as a fusion protein in Escherichia coli, the carboxyl-terminal domain of the schistosome protein exhibits HMG-CoA reductase enzyme activity. J Pharmacol Exp Ther, 1989 Nov, 251(2), 731 - 4 Kinin antagonist does not protect against the hypotensive response to endotoxin, anaphylaxis or acute pancreatitis; Berg T et al.; The vasodilator bradykinin (Bk) has long been though to participate in shock induced by endotoxemia, anaphylaxis and acute pancreatitis . Recently developed kinin antagonists have made it possible to test this hypothesis . We studied the effect of two of them . DArg0Hyp3-Thi5.8-DPhe7-Bk (45 and 220 micrograms/kg/min) and Lys-Lys-Hyp2-Thi5.8-DPhe7-Bk (100 micrograms/kg/min) on the early hypotensive response to Escherichia coli lipopolysaccharide (LPS) . Rats infused with the antagonist vehicle were used as controls . At 45 micrograms/kg/min, DArg0-Hyp3-Thi5.8-dPhe7-Bk prevented the hypotensive response to high doses of Bk; however, neither antagonist prevented the hypotensive response to LPS . Circulating kinins measured 3 min after injecting LPS or vehicle were similar (16.3 +/- 1.4 vs . 26.0 +/- 7.2 pg/ml; P greater than .23) . In allergically sensitized rats, 500 micrograms/kg/min DArg0-Hyp3-Thi5.8-DPhe-7-Bk did not alter the hypotensive (anaphylactic) response to antigen challenge (P greater than .38) . Similarly, hypotension caused by development of acute pancreatitis in rats was not prevented by infusion of DArg0-Hyp3-Thi5.8-DPhe7-Bk at 200 micrograms/kg/min, 10 min) (P greater than .69) . These results indicate that in the rate formation of kinins is not a major contributor to the hypotensive response observed in early endotoxemia, anaphylaxis and acute pancreatitis. Exp Hematol, 1989 Nov, 17(10), 997 - 1003 A simple three-step purification procedure for interleukin 3 involving absorption to fixed cells; Murthy SC et al.; We have found that treatment of B6SUtA1 cells with 0.01% glutaraldehyde transformed them into mechanically resistant spheres, thereby making it possible to use these high interleukin 3 (IL-3) receptor-bearing cells as a solid phase reagent suitable for the large scale purification of murine IL-3 (mIL-3) . Using this technique, mIL-3 was purified from serum-free pokeweed mitogen-stimulated spleen cell-conditioned medium (PWM-SCCM) approximately 16,000-fold using absorption to B6SUtA1 cells, Sephadex G75 superfine chromatography, and reverse phase high performance liquid chromatography on a C18 column . The overall yield was 16% . The final product consisted of two proteins with molecular weights of 19.5 and 16.5 kd . Both species possessed mIL-3-like activity . N-glycanase treatment of the purified preparation converted all of the 19.5-kd material into the lower molecular weight species, suggesting that the two species represented different glycosylated states of mIL-3 produced by activated T cells . This was confirmed by competition studies that showed that excess pure Escherichia coli-derived recombinant mIL-3, but not granulocyte-macrophage colony-stimulating factor (GM-CSF), could prevent the binding of both species of the PWM-SCCM-derived material to B6SUtA1 cells. Neuron, 1989 Nov, 3(5), 631 - 8 Localization and characterization of the binding site for the regulatory subunit of type II cAMP-dependent protein kinase on MAP2; Rubino HM et al.; Microtubule-associated protein 2 (MAP2) binds, and is a substrate for, type II cAMP-dependent protein kinase . The structural domain in MAP2 that binds the regulatory subunit (RII) of protein kinase II was identified by expressing fragments of a human MAP2 cDNA in E . coli using the pATH11 vector . Fusion proteins were resolved by SDS-PAGE and transferred to nitrocellulose . The filters were probed with purified bovine heart or brain RII, anti-RII monoclonal antibodies, and 125I-labeled protein A . Binding of RII was localized to a 31 amino acid sequence near the N-terminus of the MAP2 molecule . Fusion proteins containing this fragment bound both heart and brain RIIs in a concentration-dependent manner, but bound heart RII with a higher apparent affinity than brain RII . The amino acid sequence of this fragment (DRETAEEVSARIVQVVTAEAVAVLKGEQEKE) is totally conserved between human and mouse MAP2, suggesting an important role for the RII binding site of MAP2 in neuronal function. Int J Food Microbiol, 1989 Nov, 9(3), 215 - 23 The effect of phosphate, sodium chloride, sodium nitrite, storage temperature and pH on the growth of enteropathogenic Escherichia coli in a laboratory medium; Hughes AH et al.; The effect of eight phosphates in combination with sodium chloride/nitrite mixtures on growth of mixed strains of enteropathogenic Escherichia coli over a period of 10 weeks at three pH values (5.6, 6.2, 6.8) and at seven temperatures ranging from 10 degrees C to 35 degrees C is reported . All eight phosphates inhibited growth to varying degrees in at least some of the conditions investigated . Instances of inhibition increased with concentration of sodium chloride/nitrite and were more frequent at lower temperatures and pH values. Mikrobiologiia, 1989 Nov-Dec, 58(6), 969 - 75 {Relation between the damaging effect of surface-active compounds on Escherichia coli cells and the phase of culture growth}; Ivanov AIu et al.; The techniques of cell electrophoresis and electro-orientation spectroscopy were used to study the effect of sodium dodecyl sulfate (SDS) and cetyl trimethyl ammonium bromide (CTAB) on Escherichia coli K-12 cells from the culture at the exponential and stationary growth phases . SDS (2 x 10(-4) M) considerably damaged cells at the exponential phase, particularly at pH less than 6.0, whereas cells at the stationary phase were damaged to a less degree and only at pH less than 5.3 or after their treatment with Trilon B . The damaging effect of SDS decreased in an isotonic medium (0.25 M sucrose) as compared to a hypotonic medium (distilled water) . CTAB also damaged cells at the exponential phase more than those at the stationary phase, and its damaging action decreased with pH . Mg2+, Ca2+, and Sr2+ cations diminished the degree of cell damage with CTAB, but did not exert any noticeable protection in the case of SDS . The different sensitivity of cells at the exponential and stationary growth phases may be associated with changes in their surface electric charge and with the existence of hydrophobic regions on the cell surface . The higher electric charge of cells at the stationary growth phase is presumed to stem from a rise in the amount of surface lipopolysaccharides which bear a negative electric charge. Mikrobiologiia, 1989 Nov-Dec, 58(6), 885 - 91 {Changes in the pool of polyamines {correction of polyvitamins} during transition from anaerobic to aerobic conditions and localization of enzymes for their synthesis in Escherichia coli cells}; Tkachenko AG et al.; The content of intra- and extracellular polyamines and the activity of enzymes mediating their synthesis change depending on the regime of cell aeration . The pool of putrescine rises abruptly upon the transition from anaerobic to aerobic conditions owing to its liberation from the bound state as well as due to an increase in the activity of ornithine decarboxylase; as a result, the structural-functional organisation of membranes is restored . The free pool of cadaverine appears because, presumably, its binding to membranes is upset and the activity of lysine decarboxylase rises . The localisation of the enzymes for polyamine synthesis in the cell seems to be determined by the specific action of their products on particular cellular structures and metabolic processes in Escherichia coli cells. Biochem Int, 1989 Nov, 19(5), 1031 - 8 Studies on the accessibility of nascent non-helical peptide chains on the ribosome; Evers U et al.; The accessibility of nascent polypeptides with special structural elements to the ribosome was investigated . Poly(C), poly(C, U) and poly(C, A) mRNAs were translated by E . coli ribosomes in vitro . The resulting peptides which were rich in prolines, remained on the ribosomal particles or were released after addition of puromycin . A protease from Aspergillus oryzae hydrolyzed the released peptides rapidly, whereas the degradation of the unreleased ones was only slightly affected . This result shows that the nascent peptides were protected against proteolytic attack by the ribosomal particles . Interestingly, the protease completely degraded the 30S particles whereas the 50S ones remained intact, even after prolonged incubation. Biochem Int, 1989 Nov, 19(5), 1007 - 17 Role of Ca2(+)-calmodulin and protein kinase C in the secretory action of heat-labile enterotoxin of Escherichia coli in mice; Goyal J et al.; The mucosal-to-serosal and serosal-to-mucosal fluxes of Na+ and Cl- were carried out in control and heat-labile enterotoxin treated mice in the presence or absence of Ca2(+)-ionophore A23187, the activator of Ca2(+)-calmodulin or Phorbol-12-myristate-13-acetate (PMA), the activator of Protein kinase C (PKC) or 1-(5-isoquinolinyl sulphonyl)-2-methyl piperazine (H-7), an inhibitor of PKC . There was net secretion of Na+ and Cl- in experimental group in comparison to net absorption in control group . The addition of ionophore or PMA resulted in net secretion of Na+ and Cl- in control group . In experimental group ionophore increased the net secretion of Na+ and Cl- while, PMA could not cause any change in Na+ and Cl- fluxes in experimental group . Calmodulin activity remained unaltered in heat-labile enterotoxin treated mice as compared to control . H-7, reversed the effects of PMA and heat-labile enterotoxin . These studies demonstrate that heat-labile enterotoxin primarily involves PKC in its action. Plasmid, 1989 Nov, 22(3), 244 - 8 CoIV plasmids pCoIV-B188 and pCoIV-K30: genetic maps according to restriction enzyme sites and landmark phenotypic characteristics; Waters VL et al.; The CoIV plasmids are large virulence plasmids of the incompatibility group IncFI . We have obtained the genetic maps of two of the most studied CoIV plasmids, pCoIV-B188 and pCoIV-K30, according to restriction enzyme sites and landmark phenotypic characteristics such as colicin V, the aerobactin iron uptake system, the transfer region, replication regions, and repeated sequences . Although the two plasmids differ in size (pCoIV-B188 is 80 kb and pCoIV-K30 is 144 kb), the maps reveal many regions which are apparently identical or very similar. Mol Biol (Mosk), 1989 Nov-Dec, 23(6), 1596 - 602 {Study of coenzyme reorientation in the active center of tryptophanase by linear dichroism method}; Zakomyrdina LN et al.; Tryptophanase from E.coli was oriented in a compressed slab of polyacrylamide gel and its linear dichroism (LD) and absorption spectra were measured . The free enzyme displays four LD bands at 305, 340, 425 and 490 nm . Two bands at 340 and 425 nm belong to the internal coenzyme-lysine aldimine . The 305 nm band apparently belongs to an aromatic amino acid residue; the sign and form of this band are changed upon the enzyme reaction with substrate analogs . The 490 nm band is present in the LD spectra of holo- and apoenzyme and disappears after treatment with NaBH4 . It is suggested that the 490 nm band belongs to a quinoid enzyme subform . The reaction of tryptophanase with threo-beta-phenyl-DL-serine and L-threonine leads to formation of the external aldimine with a strong absorption band at 420-425 nm . The reduced LD (delta A/A) in this band is one order of magnitude greater than that in the 420 nm of the free enzyme . The complex with D-alanine is characterized by an intermediate LD value in the 425 nm band . In the presence of indole this complex displays the same LD as that observed with beta-phenylserine . The reaction of tryptophanase with L-alanine and oxindolyl-L-alanine leads to formation of the quinoid intermediate with a 500 nm absorption band . The LD value in this band differs from those in the absorption bands of the free enzyme . It is concluded that reorientations of the coenzyme occur in the course of the tryptophanase reaction. Bol Med Hosp Infant Mex, 1989 Nov, 46(11), 736 - 42 {New approaches in the prevention of diarrhea caused by Escherichia coli}; Cravioto A; This article reviews the possibility of preventing diarrhea in young children by knowing how the different enteropathogenic or enterotoxigenic strains of Escherichia coli cause the disease . New approaches on the prevention of diarrheal disease associated to the colonization of these bacteria are discussed based on the response evoked by some of the antigens from maternal milk fed to children with a risk of falling ill at a young age. Anticancer Res, 1989 Nov-Dec, 9(6), 1787 - 91 DNA methylation of the Ha-ras-1 oncogene in neoplastic cells; Barbieri R et al.; The DNA methylation pattern of the human Ha-ras-1 oncogene and the levels of specific mRNA have been analysed in established tumor cell lines and in surgical biopsies . In long-term cultures, the Ha-ras-1 gene is hypomethylated at its 5' end and highly methylated at its 3' portion . The GCGC sites at the 5' end have been shown to become methylated only when E . Coli HhaI methylase is added to isolated DNA, while they remain unmethylated when the enzyme is added to chromatin . These data indicate that the 5' portion of this protooncogene is maintained physiologically unmethylated, possibly because it is necessary for gene transcription . Along the same line of evidence, when levels of mRNA and methylation of the gene were comparatively analysed in breast tumors and autologous normal mammary tissue, no appreciable differences in the degree of methylation were found despite significant differences in mRNA content . These findings show that the levels of Ha-ras- mRNA are independent of the DNA methylation state. Mol Gen Genet, 1989 Nov, 219(3), 489 - 91 Molecular cloning, overexpression and mapping of the slt gene encoding the soluble lytic transglycosylase of Escherichia coli; Betzner AS et al.; The gene of the major autolysin of Escherichia coli, the soluble lytic transglycosylase (Slt), was isolated from an expression gene library . The cloned slt gene was used to determine its chromosomal map position adjacent to trp R at 99.7 min on the E . coli linkage map. Mol Gen Genet, 1989 Nov, 219(3), 359 - 64 Ultraviolet photoproducts at the ochre suppressor mutation site in the glnU gene of Escherichia coli: relevance to "mutation frequency decline"; Garvey N et al.; Ochre suppressor mutations induced by UV in the Escherichia coli glnU tRNA gene are CG to TA transitions at the first letter of the anticodon-encoding triplet, CAA . Premutational UV photoproducts at this site have long been known to exhibit an excision repair anomaly ("mutation frequency decline" or MFD), whereby postirradiation inhibition of protein synthesis enhances their excision and reduces suppressor mutation yields ten-fold . We sought to clarify the basis of this unique repair response by determining the spectrum of UV photoproducts on both strands of a 36 bp region of glnU which includes the anticodon-encoding triplet . We found that four different photolesions are produced within the 3 bp sequence corresponding to the tRNA anticodon: (i) on the transcribed strand, TC (6-4) photoproducts and TC cyclobutane dimers are formed in equal numbers at the site of the C to T transition, indicating that this site is a hotspot for the usually less frequent (6-4) photoproduct; (ii) on the nontranscribed strand, TT dimers are found opposite the second and third letters of the anticodon-encoding triplet, adjacent to the mutation site; and (iii) on the nontranscribed strand, an alkali-sensitive lesion other than a (6-4) photoproduct is formed, apparently at the G in the mutation site . We suggest that mutation frequency decline may reflect excision repair activity at closely spaced UV lesions on opposite strands, resulting in double-strand breaks and the death of potential mutants. Izv Akad Nauk SSSR Biol, 1989 Nov-Dec, (6), 934 - 6 {The effect of CuCl2 on the efflux of inorganic phosphate from Escherichia coli}; Lebedev VS et al.; The influence of CuCl2 on inorganic phosphate efflux from resting E . coli and those treated with glucose has been studied . Maintaining of high phosphate gradient on the membrane is possibly only in case of continuous supply of external metabolic energy . Treatment with CuCl2 does not lead to the increase in permeability of the resting cell membrane for phosphates, but it causes the efflux of phosphates in glucose-treated cells . The above data suggest that the efflux is determined by inhibition of energy influx into cell by CuCl2, not by damaging the cytoplasmic membrane. Biochem Cell Biol, 1989 Nov-Dec, 67(11-12), 812 - 7 Ribosomal protein S1 and initiation factor IF3 do not promote the ribosomal binding of approximately 19-nucleotide-long mDNA and mRNA models; Laughrea M et al.; A number of model mDNAs and mRNAs, about 19 nucleotides in length, were obtained by automated synthesis and T7 RNA polymerase directed transcription from synthetic DNA templates, respectively . These mDNAs and mRNAs had Shine-Dalgarno sequences that were four to eight nucleotides long and their sequence was similar to the R17 coat protein initiation site . The effect of S1 or initiation factors (IFs) on the rate and extent of ribosomal binding of these mDNAs and mRNAs was investigated by nitrocellulose filtration . No effect was detected, suggesting that the main function of S1 or IFs included neither the direct recognition or binding of short messengers to the 30S subunits, nor their rejection as "false" for possessing the wrong sugars or an insufficient length . A pulse-chase experiment with one of the mRNAs also showed that S1 or IF3 did not influence the exchange rate of mRNA bound to the 30S subunit. Arch Exp Veterinarmed, 1989 Nov, 43(6), 811 - 9 {Escherichia coli enterotoxins--their significance for food hygiene and recent findings on the structure and mode of action (review)}; Fehlhaber K et al.; Enterotoxin-forming Escherichia (E.) coli germs may be a cause of food poisoning . Any assessment of E . coli in various types of foodstuff together with an appraisal of its relevance to food hygiene will be of inadequate reliability unless virulence properties of germs are investigated, as well . No comprehensive evaluation can be made of quantitative occurrence of food poisoning caused by enterotoxigenic E . coli strains . Literature data are now being compiled on the occurrence of such strains in food items and on the incidence of intoxications . Reference is made in great detail to recent findings on structure and mode of action of heat-labile and heat-stable enterotoxins of E . coli. Hinyokika Kiyo, 1989 Nov, 35(11), 1911 - 4 {A case of emphysematous pyelonephritis--review of 32 cases in Japanese literature}; Hibi H et al.; A case of emphysematous pyelonephritis is presented . A 66-year-old woman with diabetes mellitus was hospitalized for sudden pyrexia and left abdominal pain on January 13, 1987 . She had shown preshock, pre-disseminated intravascular coagulation, hyperglycemia and renal dysfunction . Plain X-ray films of the abdomen and abdominal computer tomographic scanning showed a gas shadow in the left kidney . Retrograde pyelography demonstrated the left complete ureteral obstruction . A diagnosis was made of emphysematous pyelonephritis associated with diabetes mellitus and ureteral obstruction . Left nephrectomy was performed on January 17, 1987, and the pus obtained from the kidney yielded E . coli . After the operation, she has been doing well with diabetes mellitus under good control without insulin therapy . Thirty two cases of emphysematous pyelonephritis in the Japanese literature including our case are reviewed. Acta Chir Scand, 1989 Nov-Dec, 155(11-12), 561 - 5 Renal glucose and lactate metabolism in endotoxin shock in dogs; Gullichsen E et al.; Renal metabolism of glucose and lactate was studied in ten adult beagle dogs during pentobarbital anesthesia . Six dogs were submitted to hypodynamic shock by means of an intravenous bolus injection of Escherichia coli endotoxin, 0.5 mg/kg over 15 min . Four dogs received only saline solution and served as controls . Sudden cardiac depression, hypotension and moderate renal hypoperfusion were observed in the endotoxin-injected animals . Acidosis and oliguria also occurred during the 5-hour study . Arterial and renal venous glucose concentration increased transiently during the early phase of endotoxin shock . In the control group glucose levels increased slightly by the end of the experiment . Despite marked hyperlactatemia in the endotoxin group, the arteriovenous lactate difference remained almost unchanged . Renal uptake of lactate and output of glucose were not influenced during the moderate renal hypoperfusion caused by endotoxin. Zh Mikrobiol Epidemiol Immunobiol, 1989 Nov, (11), 3 - 8 {The adhesive properties of a nonenteropathogenic strain of Escherichia coli in systems in vitro on isolated rat enterocytes and in vivo}; Gorskaia EM et al.; The adhesive properties and colonizing capacity of E . coli strain O83, isolated from feces of healthy humans and marked according to its resistance to rifampicin and nalidixic acid, were studied . In vivo experiments on germ-free rats revealed that these bacteria were capable of colonizing intestinal mucosa; colonization increased from the small to large intestine and E . coli cells were mainly concentrated in the intestinal lumen and in mucin . In vitro studies showed that this nonenteropathogenic E . coli strain possessed pronounced adhesive properties with respect to the colonic cells of germ-free rats; these properties were considerably less pronounced with respect to the enteric cells of the small intestine . The electron microscopic study of E . coli cells revealed the presence of fimbriae and fibrillae on their surface. Virus Genes, 1989 Nov, 3(2), 153 - 8 Expression of post-transcriptional regulatory gene of HTLV-I, rex, in Escherichia coli; Zhou ZA et al.; Human T-cell leukemia virus type-I (HTLV-I) has a post-transcriptional regulatory gene termed rex . We have designed the rex gene to express in E . coli . Synthesis of rex protein, p27rex, was examined by immunoblot analysis using anti-p27rex antibody . No difference in electrophoretic mobility in NaDadSO4-PAGE was observed between p27rex expressed in E . coli and in an HTLV-I-infected cell line, MT-2 . Slower migration of p27rex, corresponding to a 27-kD protein, in NaDodSO4-PAGE when compared with the calculated molecular weight from the amino acid sequence (Mr = 20,367) is suggested to be caused not by post-translational modification, but by the intrinsic nature of the protein, which is rich in proline and arginine. Radiat Res, 1989 Nov, 120(2), 294 - 305 Additivity in the sensitizing effects of nitrous oxide and oxygen; Ewing D et al.; In earlier work, we proposed that nitrous oxide (N2O) and low concentrations of oxygen (10(-6) less than {O2} less than 10(-4) mol dm-3) share a common sensitizing mechanism . We also proposed that the basis for sensitization by N2O is different from that by high concentrations of oxygen ({O2} greater than 10(-4) mol dm-3) . We have now tested these proposals with several Escherichia coli strains using mixtures of O2 and N2O . In the strains that are sensitized by N2O, we found that damage from low concentrations of O2 does not add to that from N2O . In contrast, we did find additivity in the sensitizing effects of N2O and high concentrations of O2 . In those E . coli strains that are not sensitized by N2O, the effects of any concentration of O2 are the same in either N2 or N2O . These results are qualitatively the same as those from our previous study with E . coli B/r, and they support our proposals concerning similarities and differences in sensitizing mechanisms of N2O and O2. Mol Biochem Parasitol, 1989 Nov, 37(1), 57 - 64 Cloning and expression of a trypomastigote-specific 85-kilodalton surface antigen gene from Trypanosoma cruzi; Takle GB et al.; An 85-kDa trypomastigote-specific surface antigen gene from Trypanosoma cruzi has been identified by screening a genomic library in lambda gt10 with trypomastigote and epimastigote cDNA . The 1.3-kb genomic clone (pTt34) hybridizes to a single trypomastigote mRNA of 3.7 kb and to multiple bands in genomic Southern blots . Dot-blot experiments show that there are 5-10 copies of this sequence per haploid genome, and these are arranged in a non-tandem manner . pTt34 has been expressed as an anthranilate synthetase fusion protein in Escherichia coli, and inclusion bodies have been used to raise antiserum in rabbits . This antiserum immunoprecipitates a cell surface trypomastigote-specific protein of 85 kDa . The DNA and predicted amino acid sequences of pTt34 are given . Four further clones obtained from a PvuII/HpaI partial genomic library in pUC13 have extended the sequence of the 3' end of pTt34; each of these clones has regions of sequence divergence and each could represent a different member of the gene family. Mol Microbiol, 1989 Nov, 3(11), 1653 - 9 RNA polymerase heterogeneity in Streptomyces coelicolor A3(2); Buttner MJ; Recent genetic and biochemical experiments have revealed the existence of at least seven different sigma factors in Streptomyces coelicolor and demonstrated a role for alternative sigma factors in the control of differentiation and also in the transcription of primary metabolic genes . S . coelicolor has four genes predicted to encode sigma factors that are very closely related to the sigma 70 polypeptide of Escherichia coli. Mol Microbiol, 1989 Nov, 3(11), 1639 - 48 The Escherichia coli regulatory protein MetJ binds to a tandemly repeated 8 bp palindrome; Davidson BE et al.; Site-directed oligonucleotide mutagenesis has been used to isolate thirty four new mutants in the regulatory region of the Escherichia coli K12 gene, metF . The mutants include single base pair (bp) substitutions and insertions, double bp substitutions and one 7bp deletion . The effects of these and another five previously described mutants on the transcriptional regulation of metF have been analysed by using a metF'-lac'Z fusion in a low copy-number plasmid . These data, and those obtained from DNAse protection studies using pure MetJ with wild-type and mutant metF operator DNA, show that the metF operator is comprised of five tandem 8 bp repeat units that overlap the -10 region of the metF promoter . In the presence of the co-repressor S-adenosylmethionine, the DNAse protection studies yielded dissociation constants of 150 nM and 300 nM for the interaction of MetJ with repeat units 1 to 4 and repeat unit 5, respectively . In the absence of co-repressor, the dissociation constants obtained for these interactions are four to five times greater . It is proposed that regulation at the metF operator requires four molecules of MetJ dimer to bind to the five 8 bp repeat units to form a tandem, overlapping array . Interactions between MetJ molecules make an essential contribution to the stability of this protein-DNA complex. Khirurgiia (Mosk), 1989 Nov, (11), 37 - 41 {Peritoneosorption in the treatment of diffuse peritonitis in children}; Sitko LA et al.; The article deals with the results of experimental study on 225 inbred albino rats and experience in the treatment of 143 children with terminal and toxic phases of diffuse peritonitis by correction of the pH of the abdominal cavity medium with standard trisamine solution with one-stage and prolonged peritoneosorption by hemodes under conditions of closed and open management of the abdominal cavity. J Gen Microbiol, 1989 Nov, 135 ( Pt 11), 3057 - 65 The location of a temperature-sensitive trans-dominant mutation and its effect on restriction and modification in Escherichia coli K12; Hubacek J et al.; An Escherichia coli K12 chromosomal EcoRI-BamHI fragment containing a mutant hsdS locus was cloned into plasmid pBR322 . The mcrB gene, closely linked to hsdS, was used for selection of clones with the inserted fragment using T4 alpha gt57 beta gt14 and lambda vir . PvuII phages; the phage DNAs contain methylated cytosines and hence can be used to demonstrate McrB restriction . For the efficient expression of the hsdS gene, a BglII fragment of phage lambda carrying the pR promoter was inserted into the BamHI site of the hybrid plasmid . Under these conditions a trans-dominant effect of the hsdXts+d mutation on restriction and modification was detected . Inactivation of the hsdS gene by the insertion of the lambda phage BglII fragment into the BglII site within this gene resulted in the disappearance of the trans-dominant effect . When the cloned BamHI-EcoRI fragment was shortened by HpaI and EcoRI restriction enzymes, the trans-dominant effect was fully expressed . The results indicate that the Xts+d mutation is located in the hsdS gene . The effect of gene dosage of the HsdS subunit on the expression of Xts+d mutation was studied . The results of complementation experiments, using F'-merodiploids or plasmid pBR322 with an inserted Xts+d mutation, support the idea that the HsdSts+d product competes with the wild-type HsdS product, and has a quantitatively different effect on restriction and modification. J Gen Microbiol, 1989 Nov, 135 ( Pt 11), 2875 - 83 Control of carbon flux to acetate excretion during growth of Escherichia coli in batch and continuous cultures; el-Mansi EM et al.; During growth of Escherichia coli ML308 on pyruvate in a continuous culture (turbidostat) or batch culture, flux of carbon into the cells exceeds the amphibolic capacity of the central pathways . This is balanced by diversion of carbon flux to acetate excretion which in turn diminishes the efficiency of carbon conversion to biomass {g} dry wt (mol substrate)-1} . However, restriction of carbon supply in a chemostat diminishes flux to acetate excretion and at a dilution rate (D = mu) of 0.35 h-1 or less, no flux to acetate excretion was sustained thus permitting perfect balance between carbon input on the one hand, and the output to biosynthesis and energy generation on the other . This, in turn, improves the efficiency of carbon conversion to biomass . Inclusion of 3-bromopyruvate (an inhibitor of pyruvate dehydrogenase) at a concentration which diminishes growth rate (mu) to 0.35 h-1 or less also prevented flux to acetate excretion . Furthermore, in a family of fluoroacetate-resistant strains, excessive flux of pyruvate was balanced by diversion of carbon flux to lactate excretion rather than acetate and a higher growth rate (mu = 0.63 h-1) was sustained. Anal Biochem, 1989 Nov 1, 182(2), 295 - 9 Protein estimation by the product of integrated peak area and flow rate; Buck MA et al.; A convenient method for protein estimation is described, making use of uv detectors and peak integrators that are standard equipment on modern high-performance liquid chromatographs to determine the product of integrated peak area and flow rate of eluting protein at 214 nm (AF214) . We demonstrate that AF214 is proportional to the amount of eluted protein and describe two approaches for calibrating the integrator, by quantitative amino acid analysis and by determining the elution yield of a known amount of applied protein, allowing direct estimation of protein from AF214 . Both approaches yield similar results . The basis for the method is that, for virtually all proteins, absorbance at 214 nm is dominated by the summed contributions from the peptide groups . More accurate estimates can be made when the amino acid composition of the eluting protein is known, since this permits a correction to be made for contributions of amino acid side chains to absorbance at 214 nm . Comparison of AF214 estimates for proteins from the small (30 S) subunit of the Escherichia coli ribosome with those obtained by Bradford analysis shows the latter to give somewhat higher values. Vrach Delo, 1989 Nov, (11), 112 - 6 {The characteristics of acute enterocolitis and Escherichia infections in the Ukrainian SSR}; Kas'ianenko AM et al.; A nosogeographic study in the Ukrainian SSR revealed 3 zones of gastroenterocolitis morbidity: low, average and above average . Also cartographically were determined 3 Escherichia infection morbidity zones in children under 1 year of age . Conformity of pathogenic Escherichia serogroups (025, 026, 055, 0119 and others) among most humans, animals and environment suggest circulation of pathogens by the cycle: man (patient, carrier)--environment--animals (birds) and vice versa. Zentralbl Hyg Umweltmed, 1989 Nov, 189(2), 135 - 46 {The mutagenicity of organic microcontamination in the environment . III . The mutagenicity of selected herbicides and insecticides in the SOS chromotest}; Mersch-Sundermann V et al.; To determine the mutagenicity of selected herbicides and insecticides we examined 26 pure pesticide substances (polychlorinated alicyclic hydrocarbons, phenoxy fatty acids, triazines) with the help of SOS-Chromotest . We used Escherichia coli strain PQ37 in these tests without and with metabolic activation with Aroclor 1254 induced rat liver microsome fraction . None of the substances showed a provable induction of the E . coli-SOS-system, because a great number of the pesticides showed toxic effects against bacteria in high concentrations. Mol Cell Biol, 1989 Nov, 9(11), 5163 - 8 Molecular cloning of mtp70, a mitochondrial member of the hsp70 family; Engman DM et al.; We have isolated a gene from the protozoan parasite Trypanosoma cruzi that encodes a previously unidentified member of the 70-kilodalton heat shock protein (hsp70) family . Among all the eucaryotic hsp70 proteins described to date, this trypanosome protein, mtp70, is uniquely related in sequence and structure to the hsp70 of Escherichia coli, DnaK, which functions in the initiation of DNA replication . This relationship to DnaK is especially relevant in view of the intracellular location of the protein . Within the trypanosome, mtp70 is located in the mitochondrion, where it associates with kinetoplast DNA (kDNA), the unusual mitochondrial DNA that distinguishes this order of protozoa . Moreover, mtp70 is located in the specific region of the kinetoplast in which kDNA replication occurs . In view of the known functions of DnaK, the localization of mtp70 to the site of kDNA replication suggests that mtp70 may participate in eucaryotic mitochondrial DNA replication in a manner analogous to that of DnaK in E . coli. Mol Cell Biol, 1989 Nov, 9(11), 5012 - 21 Role of glutamic acid 177 of the ricin toxin A chain in enzymatic inactivation of ribosomes; Schlossman D et al.; The gene for the A chain of ricin toxin was fused to a beta-galactosidase marker cistron via a DNA sequence encoding a short collagen linker, and the tripartite fusion protein was expressed in Escherichia coli . Site-specific mutagenesis was used to change glutamic acid residue 177 to aspartic acid or alanine . When the mutant proteins were expressed, purified, and tested quantitatively for enzymatic activity, the carboxylate function at position 177 was found not to be absolutely essential for ricin toxin A-chain catalysis. J Infect, 1989 Nov, 19(3), 237 - 49 Adhesion to cells in culture and plasmid profiles of enteropathogenic Escherichia coli isolated from outbreaks and sporadic cases of infant diarrhoea; Scotland SM et al.; Enteropathogenic strains of Escherichia coli (EPEC) that caused 10 outbreaks of infant diarrhoea in the U.K . between 1968 and 1986 were studied . All gave localised adherence (LA) to HEp-2 cells, HeLa cells and Intestine 407 cells in culture . All hybridised with the EPEC adherence factor (EAF) probe . The hybridising sequences were carried on plasmids ranging in size from 26 to 76 MDa . EPEC from sporadic cases of infant diarrhoea occurring between 1979 and 1986 that belonged to the same serotypes as the outbreak strains were also studied . All strains of serotypes O111ab.H2, O114.H2, O119.H6, O127.H6 and O142.H6 gave LA and were