Mol Gen Mikrobiol Virusol, 1985 Oct, (10), 8 - 19 {Effect of mutation changes in RNA-polymerase and transcription termination factor rho on expression of various operons in E . coli}; Iarulin VR et al.; Six mutations, impairing DNA polymerase of E . coli in combination with the wild type gene for rho factor or ts-mutation rho 15 have been studied in relation to the expression of seven operons having different types of regulation . The expression of genes for glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase is shown to be constitutive and resistant to mutationally altered RNA polymerase and rho factor . The expression of genes for adenine phosphoribosyltransferase and of deo operon is regulated by rho dependent attenuators with attenuation being lifted incomplete medium.Mutation rho 15 decreases the level of enzymes of thr and lac operons independent of mRNA levels of these operons . Mutation rho 15 effect on posttranscriptional level is modified by mutations damaging RNA polymerase . The data obtained suppose RNA polymerase to affect all stages of realization of genetic information, beginning with promoter recognition and RNA synthesis and including the protein synthesis on mRNA. Cell, 1985 Oct, 42(3), 941 - 9 Overproduction of FtsZ induces minicell formation in E . coli; Ward JE Jr et al.; The ftsZ gene in E . coli K-12 is an essential cell division gene . We report that a two to sevenfold increase in the level of the FtsZ protein resulted in induction of the minicell phenotype . An increase in the level of FtsZ beyond this range resulted in an inhibition of all cell division . Unlike the classical minicell mutant, the formation of minicells induced by increased levels of FtsZ did not occur at the expense of normal divisions, indicating that increasing FtsZ resulted in additional division events per cell cycle . In addition, increased FtsZ caused cell division to be initiated earlier in the cell cycle . These results are consistent with the level or activity of FtsZ controlling the frequency of cell division in E . coli. Biull Eksp Biol Med, 1985 Oct, 100(10), 470 - 2 {Genetic regulation of the function of E . coli plasmid pAP42 transfer genes}; Maksimenko LV et al.; Sensitivity of the genetic transfer system of F-like plasmid pAP42 marked with the transposons Tn1 and TN9 to fertility inhibitors of six reference Fin-groups was studied . It was shown that transfer function and donor-specific piliformation of the plasmid under study were inhibited by reference plasmids of FinU and FinV groups, surface exclusion by plasmids of FinU and FinQ groups . The different influence of the FinOP group plasmid on transfer functions of the marked plasmids pAP42::Tn1 and pAP42::Tn9 that is likely to be connected with the effect of incorporated transposons was determined. J Biomol Struct Dyn, 1985 Oct, 3(2), 269 - 80 Hydrogen exchange of individual amide protons in the E . coli lac repressor DNA-binding domain: a nuclear magnetic resonance study; Boelens R et al.; Proton exchange in lac repressor headpiece was studied by COSY and 2D NOE spectroscopy . The exchange rates of amide protons, stabilized by the hydrogen bonds of the three alpha-helices of the headpiece, could be determined quantitatively . The exchange rates in these helices showed repetitive patterns of about three to four residues . A correlation with the position of the amide proton in the interior or the exterior of the alpha-helix of the protein was found . The exchange data strongly support the validity of the three-dimensional structure, as determined recently (Kaptein, R . et al., J . Mol . Biol . 182, 179-182 (1985)). EMBO J, 1985 Oct, 4(10), 2687 - 93 Promotion of RNA transcription on the insertion element IS30 of E . coli K12; Dalrymple B et al.; Two promoters of RNA transcription have been identified on IS30 by an in vivo assay, in which various DNA fragments with IS30 sequences were inserted in front of the promoterless galK gene of plasmid pFD51 . Both promoters have a similar activity of approximately 10% of the activity of the lacUV5 promoter . Promoter P30A precedes the long open reading frame (ORFA), and its proposed -35 region lies within the left-hand terminal inverted repeat of IS30 . However, the apparent activity of promoter P30A is significantly reduced when measured in the 3' region of ORFA . Thus, either the activity of promoter P30A is controlled by an IS30-encoded product from the same element, or some termination of transcription from P30A occurs within the coding region of ORFA . Promoter P30C precedes a short open reading frame (ORFC) in-frame with ORFA, but in the opposite strand . Reading frame ORFC is closely followed by a terminator of RNA transcription, T30C . None of the other potential open reading frames predicted from the DNA sequence, with one possible exception, are preceded by a promoter of RNA transcription active in the assay . No significant transcription was detected out of the left-hand end of the complete element . However, a small amount, probably due to read-through from promoter P30A, was detected out of the right-hand end of a complete copy of IS30 . In addition the right-hand end of IS30 has been shown to have the potential to create promoters by insertion. Nucleic Acids Res, 1985 Sep 25, 13(18), 6767 - 86 Evidence that E . coli ribosomal protein S13 has two separable functional domains involved in 16S RNA recognition and protein S19 binding; Schwarzbauer J et al.; We have found that E . coli ribosomal protein S13 recognizes multiple sites on 16S RNA . However, when protein S19 is included with a mixture of proteins S4, S7, S8, S16/S17 and S20, the S13 binds to the complex with measurably greater strength and with a stoichiometry of 1.5 copies per particle . This suggests that the protein may have two functional domains . We have tested this idea by cleaving the protein into two polypeptides . It was found that one of the fragments, composed of amino acid residues 84-117, retained the capacity to bind 16S RNA at multiple sites . Protein S19 had no affect on the strength or stoichiometry of the binding of this fragment . These data suggest that S13 has a C-terminal domain primarily responsible for RNA recognition and possibly that the N-terminal region is important for association with protein S19. FEBS Lett, 1985 Sep 23, 189(2), 318 - 24 Rare codons in E . coli and S . typhimurium signal sequences; Burns DM et al.; Codon usage has been examined in the signal sequences of 27 genes encoding proteins which possess leader peptides, and are inner-membrane located or exported . The results have been compared with codon usage in the corresponding coding sequences of most of the mature proteins . A bias is observed in the usage of rare codons for two of the three hydrophobic amino acids for which there are rare codons . Since hydrophobic residues are predominant in leader peptides, we suggest that a resulting concentration of rare codons in the signal sequence may play a role (or have played a role in the evolutionary past) in the secretion process by delaying translation. FEBS Lett, 1985 Sep 23, 189(2), 315 - 7 E . coli Ada regulatory protein repairs the SP diastereoisomer of alkylated DNA; Hamblin MR et al.; Using HPLC and 31P NMR spectroscopy on a chemically synthesized asymmetric mixture of the diastereoisomers of thymidyl(3'----5')thymidyl-O-methyl phosphate absolute configuration has been correlated with chromatographic mobility . The methyl phosphotriester system in alkylated DNA which is repaired by the Ada regulatory protein of E . coli has consequently been established to possess the Sp configuration. Biochem Biophys Res Commun, 1985 Sep 16, 131(2), 856 - 62 Involvement of calcium in the transient depolarization of E.coli cytoplasmic membrane induced by phage adsorption: a study with the fluorescent calcium indicator QUIN2; Boulanger P et al.; Adsorption of phage T4 to its outer membrane receptor (the lipopolysaccharide) triggers a transient depolarization of the cytoplasmic membrane which is prevented upon addition of EGTA, suggesting that calcium is necessary for the transmission of the signal between the two membranes . Using the fluorescent indicator QUIN2, we show that T4 adsorption triggers the release of envelope-bound calcium, the amount of which increases with the number of infecting phages . Since this amount was the same whether the cells were pretreated or not with EDTA-Tris, this suggests that this calcium originated from the high affinity sites of the lipopolysaccharide. Nucleic Acids Res, 1985 Sep 11, 13(17), 6343 - 60 Specific ribosomal RNA recognition by a fragment of E . coli ribosomal protein S4 missing the C-terminal 36 amino acid residues; Changchien LM et al.; We have previously investigated the role of the N-terminal region of ribosomal protein S4 to participate in 30S ribosome assembly and function (1-3) . In this report we extend these studies to the two fragments produced by the chemical cleavage of protein S4 at the tryptophan residue 167 . We find that the carboxyl terminal fragment (168-203) does not bind 16S RNA nor does it participate in assembly with the other 20 proteins from the 30S ribosome . In contrast, the larger fragment (1-167), does bind 16S RNA specifically . If the S4-fragment (1-167) is used to replace protein S4 in the complete 30S assembly reaction, all 20 of the other 30S proteins are incorporated . We conclude that the carboxyl terminal section of the protein S4 is not directly involved in binding 16S RNA or in the assembly of any of the other 30S proteins. Nucleic Acids Res, 1985 Sep 11, 13(17), 6283 - 98 Interaction of unfolded tRNA with the 3'-terminal region of E . coli 16S ribosomal RNA; Helk B et al.; Fragments of tRNA possessing a free TpsiC-loop or a free D-loop form stable complexes with the colicin fragment (1494-1542) of 16S ribosomal RNA from E . coli . The colicin fragment does not bind to tRNA in which the T-loop and the D-loop are involved in tertiary interactions . Colicin cleavage of the 16S rRNA from E . coli is inhibited by aminoacyl-tRNA or tRNA fragments, indicating that a similar interaction may take place on the intact 70S ribosomes . The oligonucleotide d(G-T-T-C-G-A)homologous to the conserved sequence G-T-psi-C-Pu-(m1)A in the TpsiC-region of many elongator tRNAs binds to the conserved sequence U-C-G-mU-A-A-C (1495-1501) of the 16S rRNA . It is suggested that the 3'-end of the 16S rRNA may provide the part of the binding site for the elongator tRNAs on bacterial ribosomes. FEBS Lett, 1985 Sep 9, 189(1), 145 - 9 Expression in E . coli of finger-domain lacking tissue-type plasminogen activator with high fibrin affinity; Kagitani H et al.; Tissue-type plasminogen activator (t-PA) has a high affinity for fibrin and induces lysis of fibrin (fibrinolysis) on the surface of fibrin without degrading circulating fibrinogen . cDNA for t-PA which lacks the 'finger-domain' (the site for fibrin affinity) was isolated from Detroit 562 cells . Analysis of the nucleotide sequence revealed a lack of the sequences which code for the finger-domain . A plasmid (pDPAT 1) containing the Escherichia coli tac promoter/operator and the cDNA sequence coding for 'finger-domain lacking t-PA' was constructed for expression in E . Coli . The polypeptide so produced was a new type of t-PA lacking finger-domain, but revealed plasminogen activator activity with the function of fibrin affinity. Prikl Biokhim Mikrobiol, 1985 Sep-Oct, 21(5), 611 - 6 {Amination in E . coli strains effectively producing threonine}; Astaurova OB et al.; The effect of the mutation of threonine and homoserine resistance (thrr) on the activity of the enzymes catalysing the biosynthesis of glutamic acid, glutamate synthase (EC 1.4.1.13) and glutamate dehydrogenase (EC 1.4.1.4), and on the productivity of a threonine-producing E . coli strain obtained by gene engineering was being studied . The resistance to threonine was found to correlate well with the increasing activities of the abovementioned enzymes and with a higher productivity of the E . coli strain. EMBO J, 1985 Sep, 4(9), 2287 - 93 Sequence of gene malG in E . coli K12: homologies between integral membrane components from binding protein-dependent transport systems; Dassa E et al.; The MalG protein is needed for the transport of maltose in Escherichia coli K12 . We present the sequence of gene malG . The deduced amino acid sequence corresponds to a protein of 296 amino acid residues (mol . wt . = 32 188 daltons) . This protein is largely hydrophobic (hydrophobic index = 0.83) and is thus presumably an integral inner membrane protein which could span the membrane through six hydrophobic segments . We provide direct evidence from fusion proteins for the translation frame and we also identified the in vitro made MalG protein . We have found a sequence which is highly conserved between MalG and MalF, the other integral inner membrane protein of the maltose transport system . This conserved sequence is also present in all known integral membrane proteins of binding protein-dependent transport systems, always at the same distance (approximately 90 residues) from their COOH terminus . We discuss briefly this finding. Biochem Int, 1985 Sep, 11(3), 281 - 90 Poly(dG-dC) in the Z-form inhibits E . coli DNA polymerase I and AMV DNA polymerase activity; Brahmachari SK et al.; The effect of Z-conformation of DNA on its template activity in DNA synthesis reactions in vitro has been studied . Normal poly(dG-dC) in the B-form, brominated and unbrominated in the Z-form have been compared for their template activity in DNA synthesis reactions mediated by AMV DNA polymerase and E . coli DNA polymerase I . The results indicate that poly(dG-dC) in the Z-form is totally inactive as a template for DNA synthesis and further that it is a strong competitive inhibitor of copying of the B-form DNA. Prikl Biokhim Mikrobiol, 1985 Sep-Oct, 21(5), 691 - 7 {Adsorption of spin-labeled flocculants on E . coli cells}; Kuptsova NI et al.; The interaction of intact E . coli cells with polymeric flocculants polyethylenimine and dextran was being studied by spin-labelling . The nitroxyl groups of spin-labelled polymers are reduced during formation of the adsorption layer on the cell surface . Some peculiarities of redox reactions between polymer macroradicals and units of the bacterial electron-transport chain were studied depending of temperature and flocculation regime . The course of the reduction of spin-labelled polymer radicals and characteristics of EPR spectra of the flocculant give evidence on the formation of a loose polymeric layer on the surface of E . coli cells. Mol Cell Biochem, 1985 Sep, 68(1), 59 - 66 Influence of E . coli lipopolysaccharide binding to rat alveolar type II cells on their functional properties; Aracil FM et al.; The interaction between lipopolysaccharide from E . coli 0111:B4 and rat alveolar type II pneumocytes and its influence on the functional properties of the cells and their membranes were studied . Type II cells were isolated by a novel procedure involving digestion of the lung connective tissue with elastase and Percoll-gradient centrifugation . Binding of (14C)lipopolysaccharide to type II cells resulted in a partially reversible; non-specific, high affinity process . (14C)Choline incorporation into phosphatidylcholine by type II cells was stimulated by lipopolysaccharide, the maximum effect being observed at 10-20 micrograms/ml . 45Ca2+ uptake by type II cells was also increased by lipopolysaccharide . Using plasma membranes from lung homogenates an increase of membrane microviscosity versus the amount of lipopolysaccharide was shown . These results indicate that E . coli lipopolysaccharide interacts with alveolar type II cells by binding reversibly to particular ingredients of the membrane bilayer and induces a modification of ion permeability and fluidity of the membrane. Nucleic Acids Res, 1985 Aug 26, 13(16), 5995 - 6013 The pathway of E . coli RNA polymerase-promoter complex formation as visualized by footprinting; Hofer B et al.; The pathway of E . coli RNA polymerase-promoter complex formation was probed by characterization of low temperature intermediates at the major coat protein promoter of phage fd DMA . Three different complexes could be distinguished . One of them represents the active 'open' complex, the other two have to be regarded as 'closed' . The promoter contacts of the lower temperature complexes were totally comprised within the contacts of the higher temperature complexes . Increase in temperature led to extension of contacts into the downstream direction, while the upstream border of the complexes remained virtually unchanged . Only in the 'open' complex contacts were extended beyond the start site of transcription. Biochem Biophys Res Commun, 1985 Aug 15, 130(3), 1177 - 84 Evaluation of recombinant DNA-directed E.coli produced alpha 1-antitrypsin as an anti-neutrophil elastase for potential use as replacement therapy of alpha 1-antitrypsin deficiency; Straus SD et al.; alpha 1-antitrypsin (alpha 1AT) deficiency is an inherited disorder almost always associated with the development of panacinar emphysema in the fourth to fifth decades . One source of alpha 1AT for chronic replacement therapy of such individuals is that produced by E.coli directed by a cDNA coding for the human alpha 1AT molecule . Using TG1(E.coli), an alpha 1AT molecule produced by E.coli transformed with the plasmid-expressing vector pTG922, the present study shows that recombinant DNA-directed E.coli-produced alpha 1AT is as an effective inhibitor of neutrophil elastase as alpha 1AT purified from plasma . Importantly, TG1(E.coli) inhibited human neutrophil elastase with an association rate constant of 1.3 +/- 0.4X10(7) M-1 sec-1, similar to that of normal plasma alpha 1AT (1.1 +/- 0.1, p greater than 0.2) . Furthermore, when TG1(E.coli) was added to alpha 1AT-deficient plasma obtained from homozygous alpha 1AT type Z individuals, the TG1(E.coli) remained functional and augmented the anti-neutrophil elastase activity of the serum proportional to the amount of TG1(E.coli) added . These observations suggest that if sufficient amounts of recombinant DNA methodology-produced alpha 1AT molecules could be safely delivered to the alveolar structures of alpha 1AT-deficient individuals, they would function to protect the alveolar walls from elastolytic attack. Nucleic Acids Res, 1985 Aug 12, 13(15), 5515 - 25 The sequence of the distal end of the E . coli ribosomal RNA rrnE operon indicates conserved features are shared by rrn operons; Liebke H et al.; The 1440 nucleotides of the distal region of the E . coli ribosomal RNA operon found on the lambda aroE transducing phage has been sequenced . We show that the lambda aroE hybrid rrn operon ends after a solitary 5S RNA gene with rrnE distal sequence . A single terminator structure of dyad symmetry followed by a run of six T's have been identified and compared to other sequenced rrn terminator hairpins . Immediately adjacent to the hairpin is a region of interrupted but conserved sequence that is shared by rrnE, rrnB and rrnD . An open reading frame of 127 amino acids abuts the terminator structure . Another open reading frame of 147 amino acids is found on the opposite strand several hundred nucleotides downstream. Biochim Biophys Acta, 1985 Aug 8, 830(2), 206 - 12 Phenylalanyl-tRNA synthetase from E . coli MRE-600: analysis of the active site distribution on the enzyme subunits by affinity labelling; Khodyreva SN et al.; Affinity labelling has been employed to localize the substrate-binding sites on the enzyme subunits of phenylalanyl-tRNA synthetase (L-phenylalanine:tRNAPhe-ligase, EC 6.1.1.20) of Escherichia coli MRE-600 (alpha 2 beta 2-type) . N-Chlorambucilylphenylalanyl-tRNA, N-bromoacetylphenylalanyl-tRNA, tRNAPhe containing an azido group at the eighth position of the molecule (S4U), tRNAPhe containing azido groups at different points of the molecule, p-azidoanilidate of phenylalanine, adenosine 5'-trimethaphosphate and N-bromoacetyl-L-phenylalaninyladenylate were used in experiments . It has been shown that tRNA-binding sites are formed on heavy beta-subunits of the enzyme . Phenylalanyl-tRNA is also localized on beta-subunits, while the aminoacyl moiety of aminoacyl-tRNA is localized near the contact region of subunits . The phenylalanine-binding site is located on light alpha-subunits of the enzyme . Adenosine 5'-trimethaphosphate and the analogue of phenylalanyladenylate modify both types of enzyme subunits . In our opinion, the catalytic center of tRNA aminoacylation is formed in the contact region of subunits, and the aminoacyl moiety is transferred into tRNA (from the alpha- into beta-subunit in the region of their contact). FEBS Lett, 1985 Aug 5, 187(2), 339 - 44 Characterisation of HlyC and mechanism of activation and secretion of haemolysin from E . coli 2001; Nicaud JM et al.; In this paper the DNA sequence of the cloned hlyC gene from E . coli 2001 is presented . The gene encodes a protein of 20 kDa which is able to activate the 107 kDa polypeptide encoded by hlyA . This gives rise to a haemolytically active protein which differs from the inactive form in stability and by its migration when analysed by polyacrylamide gel electrophoresis under non-denaturing conditions . We also show that the inactive form is secreted in the presence of the transport functions hlyB and hlyD . This result rules out any role for the hlyC gene product in the transport of HlyA across the inner membrane. Mol Gen Mikrobiol Virusol, 1985 Aug, (8), 15 - 9 {Effect of dioxidine, antineoplastic agents and other mutagens on the precise excision of Tn1 and Tn10 transposons in E . coli K12}; Bakai TS et al.; Induction of precise excision of transposons Tn1 and Tn10 from the genes met::Tn1 and cys::Tn10 by chemical agents, having mutagenic and DNA damaging activities, has been studied . The drugs dioxydin, NMU, photrin, phopurine, thiophosphamid, rongeron as well as sodium azide, 2-NP, DDDTDP are shown to differ in their ability to stimulate the precise excision of transposons of different classes and in the efficiency of stimulated process . Results of the present paper are in proof of the possible using of experimental model, based on registering the precise excision of transposons, for screening the mutagenic and cancerogenic activities of chemical agents from the environment. FEBS Lett, 1985 Jul 22, 187(1), 38 - 42 Implication of proteases in the respiration dependent inactivation of the lactose permease of E . coli; Therisod H et al.; The lactose permease of E . coli becomes irreversibly inactivated during lactose transport under conditions of high respiratory activity . This inactivation is characterized by a decrease in the steady state of lactose accumulation, a decrease in the influx rate of lactose, and a decrease in the transmembrane electrical potential . We report here that inhibitors of serine proteases (phenylmethylsulfonyl fluoride and N-alpha-P-tosyl-L-lysine chloromethyl ketone) prevent this inactivation, thus implicating proteases in this process. Nucleic Acids Res, 1985 Jul 11, 13(13), 4719 - 38 Long range RNA-RNA interactions in the 30 S ribosomal subunit of E . coli; Spitnik-Elson P et al.; We have attempted to identify long-range interactions in the tertiary structure of RNA in the E . coli 30 S ribosome . Native subunits were cleaved with ribonuclease and separated into nucleoprotein fragments which were deproteinized and fractionated into multi-oligonucleotide complexes under conditions intended to preserve RNA-RNA interactions . The final products were denatured by urea and heat and their constituent oligonucleotides resolved and sequenced . Many complexes contained complementary sequences known to be bound together in the RNA secondary structure, attesting to the validity of the technique . Other co-migrating oligonucleotides, not joined in the secondary structure, contained mutually complementary sequences in locations that allow base-pairing interaction without disrupting pre-existing secondary structure . In seven instances the complementary relationship was found to have been preserved during phylogenetic diversification. G Batteriol Virol Immunol, 1985 Jul-Dec, 78(7-12), 134 - 43 Interaction of human T lymphocytes with E . coli lipopolysaccharide (LPS); Altavilla D et al.; We have examined the interaction of fluorescein isothiocyanate (FITC) labeled E . coli lipopolysaccharide (LPS) with human T lymphocytes by means of cell flow cytometry, in an effort to establish the type and the conditions of this interaction . Our results indicate that the interaction of LPS with human T lymphocytes is optimal at 37 degrees C . This interaction is rapid and reaches the maximum after 5 minutes and is followed by endocytosis . Our results show that sodium azide and trypsin treatment does not affect LPS- human T cell interaction, suggesting a nonspecific lipid-lipid interaction. Carcinogenesis, 1985 Jul, 6(7), 1027 - 31 Repair of O6-propylguanine and O6-butylguanine in DNA by O6-alkylguanine-DNA alkyltransferases from rat liver and E . coli; Morimoto K et al.; DNA substrates containing O6-n-butylguanine, O6-iso-butylguanine, O6-n-propylguanine and O6-iso-propylguanine were prepared by reaction of calf thymus DNA with the appropriate N-alkyl-N-nitrosourea . These substrates were used to test the ability of O6-alkylguanine-DNA alkyltransferases from Escherichia coli and rat liver to remove such alkyl groups from the O6-position of guanine . It was found that all of these adducts were removed by the alkyltransferases, but the branched alkyl chain iso-butyl- and iso-propyl adducts were removed very slowly . Also, when tested with a DNA substrate containing both O6-n-propylguanine and O6-iso-propylguanine, the alkyltransferases removed almost all of the n-propyl-adduct before the iso-propyl-adduct was attacked . Both alkyltransferases showed a decreasing rate of reaction as the size of the alkyl group increased, but there was a significant difference between the rat liver and E . coli alkyltransferase in the relative rates . The rat liver alkyltransferase repaired O6-methylguanine more slowly than the E . coli protein, but was considerably more rapid than the bacterial equivalent when acting on n-propyl- and n-butyl-adducts . The relative rates of repair were methyl much greater than ethyl greater than n-propyl greater than n-butyl greater than iso-propyl, iso-butyl for the E . coli alkyl-transferase and methyl greater than ethyl, n-propyl greater than n-butyl greater than iso-propyl, iso-butyl greater than 2-hydroxyethyl for the rat liver protein . These results indicate that differential rates of repair may contribute to the relative risks of carcinogenesis and mutagenesis by exposure to alkylating agents of different size and that rates of repair may be species specific and must be determined from specific measurements rather than extrapolated from data on other organisms. Mutat Res, 1985 Jul, 146(1), 43 - 6 Effect of deficiency in excision repair and umuC function on the mutagenicity with ethylene oxide in the lacI gene of E . coli; Kolman A; The influence of uvrB and umuC genes on the induction of lacI- mutants and nonsense mutants by ethylene oxide (EtO) in the lacI gene of E . coli was studied . The uvrB mutation was characterized by much higher mutation frequencies . In contrast the umuC mutation does not significantly affect the induction kinetics . Thus mutation by EtO is enhanced by the lack of excision repair but not influenced by error-prone repair. Eur J Cancer Clin Oncol, 1985 Jul, 21(7), 859 - 63 Effects of leukocyte interferon (E . coli) on human bone sarcoma growth in vitro and in the nude mouse; Hofmann V et al.; Effects of highly purified human leukocyte interferon (rIFN-alpha 2) on colony formation, DNA synthesis and proliferation in nude mice of tumor cells from eight bone sarcomas have been studied . rIFN-alpha 2 produced a dose-dependent inhibition of {3H}thymidine incorporation by sarcoma cells . Even at high doses (10(4) U/ml), however, {3H}thymidine uptake could not be completely blocked by rIFN-alpha 2 . In a cloning assay three established sarcoma cell lines and five other sarcoma samples obtained after short-term in vitro culture were found to be sensitive to various degrees to rIFN-alpha 2, complete inhibition being seen only at 10(4) U/ml . Three sarcomas were sensitive in the nude mouse model . Scheduling experiments revealed that rIFN-alpha 2 produces a delay in tumor growth only when administered either before or shortly after tumor implantation . Therefore rIFN-alpha 2 appears to be most active when tumor size is small and growth not exponential, indicating that rIFN-alpha 2 may play a role in an adjuvant setting . Growth sarcomas strongly suppressed by rIFN-alpha 2 in the cloning assay was markedly inhibited in the nude mouse . One sarcoma which was only moderately sensitive in the cloning assay was resistant in the animal experiment, confirming the predictive value of the clonogenic assay . Although the present findings demonstrate strong antitumor activity of rIFN-alpha 2 against human bone sarcoma cells they should be interpreted with caution mainly because the high rIFN-alpha 2 levels used in the experiments cannot be maintained in patients over a prolonged period. Biochimie, 1985 Jul-Aug, 67(7-8), 849 - 51 Linker mutagenesis in the gene encoding the periplasmic maltose-binding protein of E . coli; Duplay P et al.; A plasmid carrying the malE gene, coding for the periplasmic maltose-binding protein of E . coli, was submitted to random mutagenesis by the insertion of a BamHI linker . About 25% of the clones recovered had acquired a BamHI site in the gene malE . Most of the linker insertions were accompanied by small deletions with an average size of 30 base pairs . Among 21 mutants synthesizing a stable maltose binding protein, 8 were still able to grow on maltose . A preliminary analysis of these mutants indicates that certain regions of the protein may not be essential for maltose transport. Cell, 1985 Jul, 41(3), 979 - 86 HTLV-III env gene products synthesized in E . coli are recognized by antibodies present in the sera of AIDS patients; Crowl R et al.; The envelope gene of HTLV-III, the retrovirus directly linked to AIDS, encodes a protein of 856 amino acids . Our sequence analysis of the cloned HTLV-III (HXB-3) env gene and its comparison with other isolates reveal significant divergence, especially in the external portion of this protein . A large segment of the env gene (1800 bp) was inserted into the expression vector pEV-vrf3, and a corresponding 68 kd protein, which encompasses both the extracellular and the membrane-associated regions of the native protein, was produced in E . coli . Several smaller polypeptides, which appear to be internal initiation products, were also produced . All 50 AIDS patient sera obtained from different locations in the United States specifically recognized the bacterially synthesized envelope proteins, as judged by Western blots . This suggests that these proteins will be useful for the diagnosis of HTLV-III infection and possibly as a vaccine against AIDS. FEBS Lett, 1985 Jun 17, 185(2), 221 - 5 Direct cross-linking of heptauridilate to E . coli ribosomes by water-soluble carbodiimide in the complex stabilized by codon-anticodon interaction at both A- and P-sites; Gimautdinova OI et al.; Affinity labelling of E . coli ribosomes is performed by treatment with water-soluble carbodiimide of the complex of ribosomes with (pU)7, tRNAPhe at the P-site and with Phe-tRNAPhe (complex I) and without Phe-tRNAPhe (complex II) at the A-site . The extent of modification is, respectively, 0.06 and 0.026 mol (pU)7 per mol ribosomes . Protein S3 is found as a single labelled protein in complex I, whereas S7, S8, L25 are modified in complex II . Thus, in the absence of a large spacer group within the complex stabilized by codon-anticodon interactions at both A- and P-sites, a highly selective modification occurs. FEBS Lett, 1985 Jun 17, 185(2), 291 - 4 Intersubunit RNA-protein contacts in pre- and post-translocated E . coli ribosome; Abdurashidova GG et al.; Ribosomal proteins participating in intersubunit RNA-protein contacts (directly interacting with RNA of the opposite subunit) were determined by means of ultraviolet-induced cross-links in pre- and post-translocated ribosomal complexes, as well as in the free 70 S ribosome (tight couple) of E . coli . In these 3 complexes at least L1 and L9 proteins interact with 16 S RNA, while S6, S9/11 and S15 react with 23 S RNA . All these proteins ('hinge-joint' proteins) are clustered on the small protuberance of the 50 S subunit and on the platform of the 30 S subunit . Reduction in the number of other (variable) intersubunit RNA-protein contacts in the course of transition from the tight couple to the pre- and, finally, to the post-translocated state, demonstrates gradual loosening of intersubunit interactions in 70 S ribosome . Such a loosening ('opening') of the 70 S ribosome is determined by conformational changes in ribosomal subunits and/or in their relative arrangement, conjugated with alteration of the functional state of the ribosomal complex. Nature, 1985 Jun 13-19, 315(6020), 577 - 8 Inhibition of the Prausnitz-Küstner reaction by an immunoglobulin epsilon-chain fragment synthesized in E . coli; Geha RS et al.; The Prausnitz-Kustner (P-K) reaction is a sensitive test for the presence and activity in the skin of immunoglobulin E, an important class of immunoglobulin mediating allergic reactions . A fragment of the human myeloma ND epsilon-chain gene, encoding the second, third and fourth domains of the IgE constant region (C epsilon 2-4) was assessed here for its ability to inhibit the P-K reaction in vivo . Injection of the fragment in skin sites of healthy human adults prevented subsequent sensitization with serum containing IgE antibody to ragweed antigen . Inhibition of the P-K reaction required a 200-fold molar excess of the C epsilon fragment over the IgE present in the sensitizing serum . The efficacy of the C epsilon fragment in inhibiting the P-K reaction compared favourably with that of natural myeloma IgE (PS) in terms of both blocking concentrations and duration of the blocking effect . The inhibition of the P-K reaction by C epsilon 2-4 fragments was specific and probably caused by the saturation of IgE receptors on mast cells by the recombinant gene product. Nucleic Acids Res, 1985 Jun 11, 13(11), 3953 - 68 Analysis of a sequence region of 5S RNA from E . coli cross-linked in situ to the ribosomal protein L25; Szymkowiak C et al.; 70S ribosomes from E . coli were chemically cross-linked under conditions of in vitro protein biosynthesis . The ribosomal RNAs were extracted from reacted ribosomes and separated on sucrose gradients . The 5S RNA was shown to contain the ribosomal protein L25 covalently bound . After total RNase T1 hydrolysis of the covalent RNA-protein complex several high molecular weight RNA fragments were obtained and identified by sequencing . One fragment, sequence region U103 to U120, was shown to be directly linked to the protein first by protein specific staining of the particular fragment and second by phosphor cellulose chromatography of the covalent RNA-protein complex . The other two fragments, U89 to G106 and A34 to G51, could not be shown to be directly linked to L25 but were only formed under cross-linking conditions . While the fragment U89 to G106 may be protected from RNase T1 digestion because of a strong interaction with the covalent RNA-protein complex, the formation of the fragment A34 to G51 is very likely the result of a double monovalent modification of two neighbouring guanosines in the 5S RNA . The RNA sequences U103 to U120 established to be in direct contact to the protein L25 within the ribosome falls into the sequence region previously proposed as L25 binding site from studies with isolated 5S RNA-protein complexes. Nucleic Acids Res, 1985 Jun 11, 13(11), 4085 - 96 Essential structure of E . coli promoter II . Effect of the sequences around the RNA start point on promoter function; Aoyama T et al.; Starting from a synthetic E . coli promoter with the consensus sequences at -35 and -10 regions, a sequence CAT frequently occurred in the RNA start points of natural promoters was introduced in the downstream of the consensus sequences, and the sequences around the RNA start points as well as the relative positions of CAT from the consensus sequences were altered . Analysis of the RNA start points and strength of these synthetic promoters in an in vitro transcription system provided evidence that the RNA start point was principally fixed by distance from the -10 consensus sequence (TATAAT) . Neither the promoter strength nor the RNA start point was significantly influenced by the CAT sequence . The sequences around the RNA start points rather seemed to exert influence on the response of promoter to temperature and salts. Mol Gen Mikrobiol Virusol, 1985 Jun, (6), 33 - 6 {The role of the gene umuC and plasmid muc genes in mutagenesis induced by dioxidine in E . coli K12}; Bakai TS et al.; The mutability induced by dioxidine in E . coli cells has been shown to be stringently dependent on a function of chromosomal umuC+ gene . Suppression of an umuC mutation by plasmids pKM101 or ColIb, restoring the dioxidine induced mutability, proves the possibility of umuC gene functional complementation by the plasmid muc+ genes. Biochimie, 1985 Jun, 67(6), 637 - 41 Methionyl-tRNA synthetase from E . coli: direct evidence for exchange of protomers in the dimeric enzyme by using deuteration and small-angle neutron scattering; Dessen P et al.; Direct demonstration of the reversible dissociation of native dimeric methionyl-tRNA synthetase from E . coli has been obtained using small angle neutron scattering and deuterated enzyme . Structural parameters of the fully deuterated dimer are very similar to the hydrogenated one . Analysis of the variations of the intensity and of the radius of gyration of a stoichiometric mixture of the two types of dimer (hydrogenated and deuterated), as a function of D2O content in the solvent, enabled us to characterize an hybrid dimer, having both hydrogenated and deuterated protomers . By separating the contribution of each protomer to the scattering, the radius of gyration of the protomer in situ and the distance between the centers of mass of each protomer in the dimer are determined. Zentralbl Bakteriol Mikrobiol Hyg {B}, 1985 Jun, 181(1-2), 93 - 7 {An improved lactose-peptone medium for the cultivation of chlorine-damaged E . coli bacteria}; Schubert R et al.; A description of a casein-soya-lactose broth (CSLB) for the cultivation of chlorine-impaired E . coli bacteria is given . In this liquid medium the recovery rates of chlorine impaired E . coli are superior or at least equal to recovery rates observed when casein-soya-broth (CSB) is used . Differences are regularly seen when lactose-pepton-broth (LPB) according the German Standards (DEV.K6) is used between direct inoculation into a liquid enrichment medium and inoculation into the same medium following membrane filtration are no longer found when CSSL-broth is used instead of LPB. Zentralbl Bakteriol Mikrobiol Hyg {B}, 1985 Jun, 181(1-2), 87 - 92 {Culturing of chlorine-damaged E . coli bacteria using the membrane filteration technic}; Schubert R et al.; The number of chlorine-impaired E . coli organisms obtainable with the aid of the membrane-filtration-Endo-agar-method is so reduced that this method cannot be considered equivalent to liquid enrichment . Notwithstanding the well documented difficulties that arise from differences between batches of the same filters e . g . from problems to assure their standardization, the main obstacles do not stem from the questionable suitability of membrane filters but from the toxicity of the Endo-agar . The use of lactose-pepton (L.P.)-broth (German Standard Methods 1971) equally does not lead to satisfactory results if compared to the use of casein-soya (C.S.)-broth. J Biochem (Tokyo), 1985 Jun, 97(6), 1827 - 30 X-ray diffraction studies on the complex of E . coli DNA with synthetic thermospermine; Takeda Y; X-ray studies on the complex of E . coli DNA with synthetic thermospermine in fibers were carried out . This complex showed the ordinary transition, except that it yielded a semicrystalline B-form at 66% r.h., suggesting good side-to-side interactions of the material due to some number of cross-bridges of tetramine . An idea of thymine methyl-neighbor base short contacts in A-form DNA is proposed to understand these structural changes of double helical DNA . On the assumption that the unstable A-conformation needs lateral interactions, the present X-ray results are well explained . This unstable factor is potentially of considerable biological interest in relation with DNA compaction. EMBO J, 1985 Jun, 4(6), 1605 - 8 Mutations within the 23S rRNA coding sequence of E . coli which block ribosome assembly; Skinner RH et al.; We have used site specific mutagenesis in vitro to construct a set of deletion mutations within the 5' region of a cloned 23S rRNA gene . In contrast to previously studied mutations in this gene, some of these deletions prevent the incorporation of 23S rRNA into ribosomal particles . This result is discussed in terms of a model in which interaction with the assembly initiator protein, L24, is perturbed. EMBO J, 1985 Jun, 4(6), 1575 - 82 Molecular cloning and characterization of the yeast RAD10 gene and expression of RAD10 protein in E . coli; Weiss WA et al.; A plasmid designated pNF101 was isolated by transforming rad10 mutants with a yeast genomic library and screening transformed cells for enhanced resistance to killing by u.v . radiation . Plasmid pNF101 fully complements the u.v . sensitivity of rad10 mutant strains and was shown to contain the RAD10 gene by genetic analysis of integrant strains . The nucleotide sequence of the RAD10 gene was determined . The coding region consists of 195 codons and could encode a polypeptide of calculated mol . wt . 22 616 daltons . RAD10 protein expressed in Escherichia coli maxicells has a mol . wt of approximately 30 kd measured by gel electrophoresis . The RAD10 gene was localized to chromosome XIII of Saccharomyces cerevisiae by hybridization of the cloned gene to yeast chromosomes resolved by electrophoresis, and by genetic analysis. EMBO J, 1985 Jun, 4(6), 1589 - 92 Secondary structure of a channel-forming protein: porin from E . coli outer membranes; Kleffel B et al.; Porin from Escherichia coli outer membranes has been analysed by high angle diffuse X-ray diffraction, and by attenuated total reflection infrared spectroscopy . These methods demonstrate independently that the majority of the polypeptide backbone is arranged in anti-parallel beta-pleated sheet structure . The average length of the beta-strands, which are oriented nearly normal to the membrane plane, is estimated to be 10-12 residues, independent of the method used . Although the details of strand arrangement (beta-barrels or stacked sheets) are not as yet known, porin represents the first transmembrane protein for which beta-structure has been established unequivocally. Cell, 1985 Jun, 41(2), 577 - 85 A bidirectional rho-independent transcription terminator between the E . coli tonB gene and an opposing gene; Postle K et al.; We identified an Escherichia coli gene, designated P14, that is adjacent to and in the opposite orientation to the tonB gene . The 36 base pair intercistronic region between tonB and P14 contains a novel rho-independent transcription terminator that functions bidirectionally, both in vivo and in vitro, to terminate tonB and P14 transcription . Transcription of tonB and P14 terminates at symmetrically equivalent nucleotides, such that the 3' ends of tonB and P14 transcripts are complementary . The terminator is 70% efficient in both directions in vitro . Interestingly, relative rates of in vivo RNA synthesis, immediately prior to and following the terminator, appear to indicate that it is more efficient in the tonB direction (95%) than in the P14 direction (70%) . We discuss the possibility that this gene arrangement has regulatory consequences for the expression of tonB. EMBO J, 1985 Jun, 4(6), 1583 - 7 Localization of functional domains in E . coli K-12 outer membrane porins; Tommassen J et al.; The genes ompC and phoE of Escherichia coli K-12 encode outer membrane pore proteins that are very homologous . To study the structure-function relationship of these proteins, we have constructed a series of ompC-phoE hybrid genes in which the DNA encoding part of one protein is replaced by the corresponding part of the other gene . These hybrid genes were easily obtained by using in vivo recombination . The fusion sites in the hybrid genes were localized by restriction enzyme mapping . The hybrid gene products were normally expressed and they were characterized with respect to functions and properties in which the native OmpC and PhoE proteins differ, such as pore characteristics, the receptor activity for phages and the binding of specific antibodies . Three regions within the N-terminal 130 amino acids were localized which determine pore characteristics and a segment between residues 75 and 110 contains amino acids which determine specificity for PhoE phages . A major cell surface-exposed region is located between residues 142 and 267 . This region contains residues which are required for the binding of monoclonal antibodies directed against the cell surface-exposed part of PhoE and residues which determine specificity for OmpC phages. Nucleic Acids Res, 1985 May 24, 13(10), 3699 - 710 Evidence that pheV, a gene for tRNAPhe of E . coli is transcribed from tandem promoters; Caillet J et al.; A DNA fragment of 487 bp containing a gene for tRNAPhe has been sequenced . Although the tRNAPhe sequence is identical to that of pheU (which maps at 94.5 min) the surrounding sequences are quite different . This sequence is thus that of a second gene for tRNAPhe (which we shall call pheV) . In vitro transcription experiments and S1 mapping in vivo show the existence of two promoters separated by about 60 nucleotides . The second transcript starts only 3 nucleotides 5' from the tRNAPhe structural sequence . A DNA sequence characteristic of a rho-independent terminator is located 30 nucleotides 3' of the end of the structural gene and is shown to function efficiently in vitro. Nucleic Acids Res, 1985 May 24, 13(10), 3685 - 97 Replacement and insertion of nucleotides at the anticodon loop of E . coli tRNAMetf by ligation of chemically synthesized ribooligonucleotides; Doi T et al.; Insertion of the four major nucleotides at the 5'-side of the anticodon triplet of E . coli tRNAMetf was performed by joining of the half molecules obtained by limited digestion with RNase A and the chemically synthesized tetranucleotide pN-C-A-U using RNA ligase . Insertion of U-U at the 5'-side or A and A-A at the 3'-side of the anticodon were also performed using U-U-C-A-U, C-A-U-A and C-A-U-A-A . The constant U next to the 5'-side of the anticodon was replaced with A and C by ligation of A-C-A-U and C-C-A-U to the 5'-half molecule which had been treated with periodate plus lysine, followed by joining to the 3'-half . These modified tRNAs were tested for their ability to accept methionine with the methionyl-tRNA synthetase of E . coli . The affinity of these analogs for the synthetase decreased more extensively when the insertion was at the 3'-side of the anticodon triplet . Insertion of mononucleotides at the 5'-side or replacement of the constant U next to the 5'-side of the anticodon did not affect aminoacylation drastically . This may mean that the 3'-side of the anticodon loop of tRNA is one of the major recognition sites for the methionyl-tRNA synthetase. Biofizika, 1985 May-Jun, 30(3), 446 - 9 {Permeability of the E . coli cell membrane for thiourea, dimethylsulfoxide and glycerol}; Efimov VB et al.; Permeability of cell envelope of Escherichia coli was investigated by the method of osmotic shock for thiourea, dimethylsulfoxide and glycerol . Characteristic times were obtained for passive permeation of these reagents . Variation of r in consequence dimethylsulfoxide less than thiourea less than glycerol was determined . The dependence of tau on the concentration of permeant reagents was observed and it was found that tau was decreased with increasing concentration of the permeant solute which was connected with the modification of the cytoplasmic membrane. EMBO J, 1985 May, 4(5), 1327 - 32 Effect of dam methylation on the activity of the E . coli replication origin, oriC; Messer W et al.; Methylation of GATC sites by the dam methylase is required for efficient initiation of DNA replication at the replication origin, oriC, of Escherichia coli . This is demonstrated by the inability of minichromosomes to be maintained in dam mutant strains . The requirement for methylated GATC sites is less stringent in vitro than in vivo . The time required for complete methylation of the origin region apparently determines the minimal spacing of replication forks on the chromosome. EMBO J, 1985 May, 4(5), 1287 - 92 The HBV HBX gene expressed in E . coli is recognised by sera from hepatitis patients; Kay A et al.; We have cloned the X gene (HBx) and the HBc antigen (HBc Ag) gene of human hepatitis B virus (HBV) in Escherichia coli as fusion products with beta-galactosidase . Both HBV genes are expressed in E . coli strain CSR 603 . Expression is detected by u.v . irradiation of the bacteria, metabolic labelling and electrophoresis of the labelled extracts on SDS-polyacrylamide gels . The HBc Ag protein produced in bacteria can be recognised by anti-HBc sera and peptides derived from the protein are also recognised by anti-HBe sera . The HBx protein is recognised by some, but not all, sera which are anti-HBe positive . HBx Ag is also recognised by a woodchuck antibody similar to anti-HBe (anti-WHe) . These results constitute the first proof that the open reading frame X is a true viral gene and is expressed during HBV (and WHV) infection and that an HBx/anti-HBx system, which may have important biological implications, can exist in parallel with the classic HBe/anti-HBe system. Biokhimiia, 1985 May, 50(5), 814 - 9 {Proteins firmly bound to DNA in the E . coli nucleoid}; Gaziev AI et al.; The nucleoid isolated from E . coli cells was subjected to further deletion by treatment with 2 M NaCl . After disintegration of this nucleoid by ultrasonication, two fractions were obtained, i . e., a rapidly (RS) and slowly sedimenting (SS) ones . The protein, RNA and DNA patterns in the RS fraction are similar to that of the eukaryotic cell nuclear matrix . Electrophoretic analysis of total non-dissociating by 2 M NaCl proteins revealed that the RS and SS fractions predominantly contain proteins with Mr 31,27 and 23 kD . The protein with Mr = 31 kD is firmly bound to DNA, does not dissociate in the guanidine hydrochloride (4 M)-urea (5 M) mixture as well as in solution of 1% sodium-dodecyl sulphate and may be responsible for the chromosome binding to the E . coli membrane. Mol Gen Mikrobiol Virusol, 1985 May, (5), 13 - 9 {Characteristics of RecA-independent recombination of plasmids in E . coli cells producing restriction endonuclease EcoRI}; Strikhanov SN et al.; The restriction endonuclease EcoRI dependent recombination of compatible plasmids has been studied in RecA cells of Escherichia coli . Plasmid RP4 and the isogenic ColE1 type plasmids pSA14 or pSA25, differing in restriction-modification RM EcoRI genes, have been used to study this type of recombination . EcoRI dependent recombination of plasmids is demonstrated in RecA cells and, thus, is independent of general system of homologous recombination . The classes of recombinant plasmids isolated from RecA cells differ from the classes isolated from wild type cells . Levels of tetracycline resistance conferred by plasmid RP4 are shown to be dependent on the alleles of RecA+ gene, being extremely low in RecA cells . This property is demonstrated to be useful for obtaining the multicopy recombinant plasmids resulting from EcoRI dependent recombination in RecA cells of Escherichia coli. Somat Cell Mol Genet, 1985 May, 11(3), 223 - 38 Shuttling of integrated vectors from mammalian cells to E . coli is mediated by head-to-tail multimeric inserts; Lutfalla G et al.; With the aim of producing nonviral shuttle vectors for mammalian cells, we have constructed mouse mitochondrial DNA derivatives comprising the xanthine-guanine phosphoribosyltransferase gene as a selectable marker . Complete or subcomplete mitochondrial genomes were inserted into the plasmid pBB3 and transferred into hepatoma cells in order to generate, in vivo, new recombinant molecules . A second- and a third-generation vector, p12.2b and p delta respectively, were thus isolated for their ability to shuttle from mammalian cells to recA+ E . coli . Transfection of rodent fibroblasts and hepatoma cells showed that, contrary to our expectations, p12.2b and p delta are not self-replicating episomes; their shuttling from mammalian cells to recA+ E . coli is mediated by tandem integrated copies . The relevant property of p12.2b and p delta is a ubiquitous propensity to form head-to-tail multimeric structures when they integrate into mammalian host chromosomes . This ability is missing in pBB3 and appears only following the insertion of various mitochondrial or nuclear DNA fragments into the plasmid . These data are discussed in terms of homologous recombination and shuttling of integrated vectors. Mutat Res, 1985 May, 145(3), 113 - 8 Genetic control of heat resistance and thermotolerance by recA and uvrA in E . coli K12; Grecz N et al.; Several recA and uvrA derivatives of E . coli K12 AB1157 develop a transient increase in heat resistance, i.e . induced thermotolerance after a brief exposure to 43.5 degrees C (less than 1 h) . Thermotolerance was identified from the appearance of an inflection in the survival curve or from the loss of heat resistance in the presence of chloramphenicol (CAM) or rifampicin . Heat resistance and induced thermotolerance were enhanced by recA and uvrA gene functions and their contribution was roughly as follows: AB1157 (recA+ uvrA+) greater than AB2463 (recA- uvrA+) greater than AB1886 (recA+ uvrA-) greater than AB2480 (recA- uvrA-) . In heat resistance, uvrA and recA contributed approximately equally and their effects were additive . Induced thermotolerance developed sooner and was maintained at a higher level in the presence of uvrA as compared with recA . Since uvrA-dependent excision repair is scheduled prior to recA-dependent (postreplication) repair, induction of thermotolerance may be linked to DNA repair . Although recA and uvrA play a distinct role, they are not essential, and thermotolerance can develop in the absence of either one or both of these gene functions . Furthermore, since thermotolerance can be induced in recA mutants (AB2463 and AB2480), its biochemical pathway must be different from that of the recA-dependent SOS system. Biokhimiia, 1985 May, 50(5), 749 - 54 {Effect of 5-azacytidine on E . coli cells with different DNA-methylases}; Venozhinskis MT et al.; A correlation was found between the bacteriocide effect of 5-aza-C and the amount of cytosine DNA-methylases in E . coli cells . 5-Aza-C-DNA induced partial or complete inhibition of bacterial DNA-methylases with different site specificity; cytosine DNA-methylases were inhibited by the DNA more effectively than adenine DNA-methylase Eco dam . The inhibitory influence of 5-aza-C-DNA on cytosine DNA-methylases was due to the formation of stable inactive complexes between the enzyme and the non-methylating cytosine analog in the recognition sites . Cytosine DNA-methylase Eco RII formed a relatively firm bond with 5-aza-C-DNA, which could be disrupted by 1 M KCl; this disruption restores the DNA-methylase activity and the inhibiting capacity of 5-aza-C-DNA . Thus, the binding of cytosine DNA-methylase to 5-aza-C in DNA is noncovalent; the inhibition of the enzyme by 5-aza-C-DNA is reversible. Nucleic Acids Res, 1985 Apr 25, 13(8), 2683 - 98 Methyl phosphotriesters in alkylated DNA are repaired by the Ada regulatory protein of E . coli; McCarthy TV et al.; The E . coli ada+ gene product that controls the adaptive response to alkylating agents has been purified to apparent homogeneity using an overproducing expression vector system . This 39 kDa protein repairs 0(6)-methylguanine and 0(4)-methylthymine residues in alkylated DNA by transfer of the methyl group from the base to a cysteine residue in the protein itself . The Ada protein also corrects one of the stereoisomers of methyl phosphotriesters in DNA by the same mechanism, while the other isomer is left unrepaired . Different cysteine residues in the Ada protein are used as acceptors in the repair of methyl groups derived from phosphotriesters and base residues. FEBS Lett, 1985 Apr 22, 183(2), 313 - 6 On the recovery of Cys-containing peptides during peptide mapping by HPLC . Tryptic peptides of Trp-tRNA synthetase of E.coli; Koeppe RE 2nd et al.; Conditions are presented for separating the major tryptic peptides of E.coli tryptophanyl-transfer RNA synthetase by reversed-phase liquid chromatography using a water-methanol gradient in the presence of 0.1% trifluoroacetic acid . Three of the peptides contain cysteine and are recovered in good yields if alkylated, but otherwise cannot be detected . A convenient post-digestion alkylation procedure is appropriate for use with small samples of protein which can be digested under reducing conditions . These results will be of interest for studies of the labeling of sulfhydryl groups in other proteins. Appl Biochem Biotechnol, 1985 Apr, 11(2), 133 - 40 Immobilized E . coli alkaline phosphatase . Its properties, stability, and utility in studying the dephosphorylation of proteins; Basheeruddin K et al.; We have immobilized E . coli alkaline phosphatase (EC 3.1.3.1) by linking it covalently to sepharose 4B . This preparation has several advantages over the soluble enzyme . The immobilized enzyme is easily separable from other constituents in incubation mixtures . The immobilized enzyme can be reused repeatedly and is more stable than the soluble enzyme to heat treatment in the presence of 10 mM Mg2+ . The insoluble and soluble phosphatases removed 75 and 77%, respectively, of the inorganic phosphorus from casein . The immobilized enzyme inactivated two enzymes believed to be active in the phosphorylated state, acyl-CoA:cholesterol acyltransferase (ACAT) by 39% and NADPH-cytochrome P-450 reductase by 89% . The utility of immobilized alkaline phosphatase for studying the phosphorylation and dephosphorylation of soluble or membrane-bound enzymes and proteins is discussed. Zentralbl Bakteriol Mikrobiol Hyg {A}, 1985 Apr, 259(2), 252 - 67 {Immunity of the skin--infection and percutaneous immunization of rabbits with E . coli ATCC 13676}; Hammerschmidt W et al.; After intracutaneous infection of rabbits with a suspension of E . coli which was followed by a transient local inflammation, the local and systemic immune responses were determined using the lymphocyte stimulation test (LTT) and the hemolysis plaque assay (HPA) . Lymphocytes of the lymphatic system draining the infected skin area and blood lymphocytes were used . With lymphocytes derived from the local lymph nodes, a substantial increase of specific stimulation in the LTT was detected beginning at day 3 after infection and lasting up to the termination of the experiment (3 weeks) . Blood lymphocytes were stimulated at a lower level: The activity showed a peak at day 4 and an elevated level only during a 10-day period . After the intracutaneous infection with E . coli, increasing numbers of antibody-releasing lymph node cells were detected in the HPA . The antibody-secreting cells of the IgM and IgG classes clearly showed an increasing specificity for E . coli lipopolysaccharide coupled to sheep red blood cells . As with the LTT, the highest activities (values of specificity and number of plaque-forming lymphocytes) were observed at the end of the experimental period . An emulsified preparation of a heat-inactivated E . coli culture (E . coli-BKS) which had been applied locally onto the artificially altered skin evoked a similar immunological response after a 2 or 3-weeks treatment . In such animals an increased activity of lymph node cells could be registered by LTT and HPA as compared to reactions from placebo-treated control animals . However, the topical immunization with nonviable E . coli stimulated not only lymphocytes which produced antibodies directed specifically against E . coli lipopolysaccharide as demonstrated by the HPA . An increased number of lymphocytes reacted even with native sheep red blood cells . This observations is discussed in respect of a polyclonal B-cell activation by lipopolysaccharide of the E . coli-BKS. Mol Gen Mikrobiol Virusol, 1985 Apr, (4), 15 - 21 {Plasmid recombination stimulated by restriction endonuclease EcoRI in vivo: formation of recombinant plasmids in recA+-cells of E . coli}; Aleshkin GI et al.; The possible participation of restriction endonuclease EcoRI in recombination of compatible nonhomologous plasmids in E . coli cells has been studied . To study the process, plasmids RP4 and R245 have been transferred by conjugation into the recipient cells of E . coli harbouring one of isogenic plasmids, pSA14 and pSA25, different for the genes coding restriction endonuclease EcoRI . The genetic analysis of transconjugant phenotypes, coded by the plasmids, has permitted to register the recombinant plasmids after compatibility of parent plasmids in E . coli cells . Recombination of plasmid RP4 with the plasmid pSA14, carrying EcoRI genes, has been registered in E . coli cells, producing the restriction endonuclease, while plasmid recombination has not been found in the cells harbouring plasmid pSA25, isogenic for all genes, except for EcoRI genes, with plasmid pSA14 . Restriction endonuclease EcoRI is concluded to stimulate site specific recombination of nonhomologous compatible plasmids in vivo . EcoRI-mediated recombination of plasmid R245 with plasmid pSA14 is discussed. EMBO J, 1985 Apr, 4(4), 1041 - 7 Failure of E . coli K-12 to transport PhoE-LacZ hybrid proteins out of the cytoplasm; Tommassen J et al.; A phoE-lacZ hybrid gene encoding the N-terminal 300 amino acid residues of pre-PhoE protein, fused to an almost complete beta-galactosidase molecule was constructed in vitro . Cell fractionation experiments suggested that the hybrid gene product is transported to the outer membrane . However, by using immuno-cytochemical labelling on ultra-thin cryosections it was shown that the hybrid protein accumulated in the cytoplasm . Thus, it appears that: (i) data on the localization of hybrid proteins merely based on cell fractionation experiments are not reliable, and (ii) either the C-terminal 15% of PhoE protein contain information which is essential for transport, or PhoE-LacZ hybrid proteins can never be transported out of the cytoplasm . The implications of these results for current models on the translocation of outer membrane proteins are discussed. Eur J Immunol, 1985 Apr, 15(4), 345 - 50 Constraints in T-B cooperation related to epitope topology on E . coli beta-galactosidase . I . The fine specificity of T cells dictates the fine specificity of antibodies directed to conformation-dependent determinants; Manca F et al.; Experiments to test the relationship between the epitopes on a protein antigen recognized by T and B cells in their collaboration to produce antibody cannot rely solely on hapten-carrier models . In the present work we used E . coli beta-galactosidase, a molecule whose tertiary and quaternary epitopes have been well characterized, as the model antigen . T helper cells were raised by stimulating mice with the intact or the denatured molecule or with any of several beta-galactosidase cyanogen bromide peptides . In a series of in vitro helper T cell assays we confronted the various T populations with B cells preimmunized with the native antigen, and we tested their capacity to help production of (a) binding antibodies and (b) antibodies directed to single conformational epitopes, characterized by their capacity to protect the enzyme from heat denaturation or to activate defective beta-galactosidase . According to our results, (a) equivalent T cell help can be provided by T helper cells primed with native or denatured antigen, even for the production of "conformational" antibodies; (b) one of the peptides (CB-18) is most efficient in raising help for binding antibodies; and (c) two peptides (CB-20 and CB-21) rank highest in priming T helper cells for the eventual production of protecting and activating antibodies, respectively . Thus, not every beta-galactosidase-specific T helper cell is useful in providing help to B cells specific for any particular epitope on the molecule, but rather preferential pairings exist, possibly governed by a proximity rule. Biochem Biophys Res Commun, 1985 Mar 29, 127(3), 1026 - 31 IHF stimulation of lambda cII gene expression is inhibited by the E . coli NusA protein; Peacock S et al.; The effects of E . coli proteins Integrative Host Factor (IHF) and NusA on the regulation of lambda cII gene expression are presented . As reported previously (Peacock et al . {1984} Proc . Natl . Acad . Sci . USA 81, 6009-6013), IHF stimulates the DNA-directed in vitro synthesis of cII protein or its first dipeptide, fMet-Val . Whereas NusA, by itself, has no effect on cII expression, the presence of NusA inhibits the IHF-mediated stimulation of cII synthesis. Nucleic Acids Res, 1985 Mar 25, 13(6), 1939 - 52 Cloning of the E . coli O6-methylguanine and methylphosphotriester methyltransferase gene using a functional DNA repair assay; Margison GP et al.; Alkylating agents react with various nitrogen and oxygen atoms in DNA and many of the products are substrates for repair processes . Oxygen atom derivatives such as O6-methylguanine (O6-meG) O4-methylthymine and methylphosphotriesters (MP) have been shown to undergo repair by methyl group removal . The proteins involved in the latter reaction can be considered to be methyltransferases (MT) because their action results in the transfer of the methyl group to a cysteine residue within a polypeptide . A rapid and sensitive assay for MT activity has been developed and used to screen extracts of bacteria harbouring an E . coli genomic DNA library carried in a plasmid vector . We report here the cloning of an E . coli gene coding for O6-meG and MP MT repair functions . These two activities reside on a 37Kd protein that can undergo a host-dependent cleavage to produce an 18Kd protein which contains only O6-meG MT and a 13Kd protein which contains only MP MT. FEBS Lett, 1985 Mar 25, 182(2), 429 - 34 Effects of GTP,GDP{beta S} and glucose on adenylate cyclase activity of E . coli B; Stein JM et al.; Adenylate cyclase activity was measured in suspensions of E.coli B, rendered permeable with toluene . The enzyme was activated in a dose-dependent manner by GTP and by its non-hydrolysable analogue, GTP{gamma S} . In contrast, incubation with GDP{beta S}, a non-phosphorylatable analogue of GDP, caused a dose-related inhibition of adenylate cyclase; this was partially overcome by addition of GTP . GTP did not relieve, and GDP{beta S} augmented, the non-competitive and dose-related inhibition of E.coli adenylate cyclase by glucose. Nucleic Acids Res, 1985 Mar 25, 13(6), 1829 - 40 Analysis of regulatory sequences upstream of the E . coli uvrB gene; involvement of the DnaA protein; van den Berg EA et al.; A region located upstream of the uvrB promoters P1 and P2 was found to cause high plasmid loss when cloned in multicopy vectors . Two sequence elements responsible for this phenomenon were identified by mapping of spontaneous mutations that restore plasmid maintenance: a sequence known to have in vitro promoter activity and a partially overlapping sequence that shows extensive homology to recognition sites for the DnaA protein . Accordingly alterations in the level of DnaA protein in vivo were found to affect the extent of plasmid loss . A possible role for interaction of the DnaA protein with the region of interest is discussed in relation to regulation of uvrB expression. Biochem Biophys Res Commun, 1985 Mar 15, 127(2), 464 - 72 The in vivo formation of nonbilayer lipid phase in E . coli membranes during the development of Ca2-dependent competence; Sabelnikov AG et al.; 31P-NMR studies on E . coli cells reveal the in vivo formation of nonbilayer lipid structures, presumably of H11 phase, during Ca2-dependent competence induction . The data suggest the involvement of these structures in the exogenous DNA transfer into the cells during genetic transformation and transfection . The suggestion is supported by in vitro experiments in which the liposomes composed of different phospholipid species bind 14C-DNA in DNase and wash resistant form in conditions promoting the hexagonal phase formation. Biochimie, 1985 Mar-Apr, 67(3-4), 357 - 60 The adaptive response in E . coli; Defais M; The adaptive response appears in E . coli after exposure to low levels of alkylating agents . This system is under the positive control of the ada gene . At least two enzymes are induced during the response: 3-methyladenine DNA glycosylase II and O6-methylguanine DNA methyltransferase . The latter is also the product of the ada gene. Bioorg Khim, 1985 Mar, 11(3), 358 - 69 {E . coli DNA polymerase . A study of the mechanism of primer binding using oligothymidylate analogs with ethylated internucleotide phosphate groups}; Levina AS et al.; The following individual diastereomers of oligothymidylate ethyl esters (the alkyl phosphodiester group is asymmetric with R or S configuration) have been prepared: d{(Tr)8Tp'(Et)T} (I), d{(Tp)8Tp''(Et)T} (II), d{(Tp)8Tp'(Et)TpT} (III), d{(Tp)8Tp'' X (Et)TpT} (IV) . A totally esterified analogue d{{(Tp(Et)7}T} (V) was obtained as a diastereomeric mixture . All oligothymidylate derivatives revealed substrate activity as primers of DNA polymerase with poly(dA) as a template . The values of the maximal reaction rates were equal to 14; 2,6; 68; 24 and 0,1% for oligothymidylates (I)-(V) with respect to Vm value (100%) for (Tp)9T . Km values of oligothymidylates (I)-(V), 2,7; 2,5; 0,51; 7,2 microM, were obtained in relation to Km for d{(Tp)9T} (0,4 microM) . Diastereomers (I) and (II) were not destroyed by Klenow fragment of DNA polymerase I which has only 3'----5' exonuclease activity . However, these derivatives were hydrolyzed by complete DNA polymerase I due to its 5'----3' exonuclease activity, the reaction rate being 3-10 times lower than in case of d{(Tp)9T} . The data suggest an essential contribution to the primer binding from the positive enzyme group interaction with the 3'-end negatively charged phosphate group of oligonucleotide, together with the primer complementary interaction with the template . At least two phosphodiester groups of the oligonucleotide primer are essential for the reaction of polymerization following the correct binding. Mutat Res, 1985 Mar, 142(3), 93 - 7 The specificity of base-pair substitution induced by the mutL and mutS mutators in E . coli; Choy HE et al.; The Escherichia coli mutator alleles, mutL and mutS, produced transversion as well as transition base-pair substitutions with the trpA reversion system . Transversions, however, were generally mutator-induced at a lower level than transitions and the specific type of transversion and its nucleotide position appeared to strongly affect its level of enhancement . These results are interpreted to mean that mutL- and mutS-dependent mismatch correction is generally more effective at correcting transition mispairings than transversion mispairings . Correction of transversion mispairings is probably dependent upon site of occurrence and type of mismatch. J Steroid Biochem, 1985 Mar, 22(3), 377 - 85 Influence of E . coli endotoxin on ACTH induced adrenal cell steroidogenesis; Garcia R et al.; The effect of endotoxin (lipopolysaccharide from E . coli) on isolated adrenocortical cells was examined . Lipopolysaccharide decreased the ACTH-induced steroidogenesis . This effect was shown by all corticotropin concentrations studied, and the longer the incubation time, the higher the effect produced . The rate of decrease of ACTH-induced steroidogenesis was dependent on the concentration of lipopolysaccharide in the medium . Binding of {125I}ACTH to adrenocortical cells was modified by lipopolysaccharide; this modification was related to a decrease of the ACTH-induced steroidogenesis . This effect supports the hypothesis of a direct interaction between lipopolysaccharide and the cell membrane with a concomitant distortion of the cell surface affecting the ACTH receptor sites of their environment . {14C}Lipopolysaccharide binds to isolated adrenocortical cells . Binding specificity was investigated by competitive experiments in the presence of various types of endotoxins, polypeptide hormones and proteins . Unlabelled lipopolysaccharide from the same bacterial strain and isolated under identical conditions than the labelled lipopolysaccharide exerted the strongest inhibitory activity . Unlabelled lipopolysaccharide of various strains different from that originating the labelled lipopolysaccharide exerted the less displacement . It would imply a certain kind of specificity but the decrease in the binding of lipopolysaccharide produced by ACTH and glucagon suggests the existence of non-specific interactions between lipopolysaccharide and cell membrane. Biochimie, 1985 Mar-Apr, 67(3-4), 365 - 9 Mechanism of SOS-induced targeted and untargeted mutagenesis in E . coli; Maenhaut-Michel G; This paper retraces the evolution of hypotheses concerning mechanisms of SOS induced mutagenesis . Moreover, it reports some recent data which support a new model for the mechanism of targeted and untargeted mutagenesis in E . coli . In summary, the SOS mutator effect, which is responsible for untargeted mutagenesis and perhaps for the misincorporation step in targeted mutagenesis, is believed to involve a fidelity function associated with DNA polymerase III and does not require the umuC gene product . umuC and umuD gene products are probably required specifically for elongation of DNA synthesis past blocking lesions, i.e . to allow mutagenic replication of damaged DNA. Am J Trop Med Hyg, 1985 Mar, 34(2), 257 - 65 Identification of pathogenic Leishmania promastigotes by DNA: DNA hybridization with kinetoplast DNA cloned into E . coli plasmids; Lawrie JM et al.; We report the characterization of Leishmania (L . infantum, L . donovani, and L . major) kinetoplast DNA (kDNA) by the use of restriction endonuclease digestion patterns and Southern hybridizations . Overall, the sizes and fragment patterns of MspI restriction endonuclease-produced DNA fragments vary from species to species . However, kDNA isolates from different species and strains cross-reacted to a great extent in Southern hybridization experiments . Only kDNA isolated from L . infantum and L . major had little homology during hybridization reactions . To prepare DNA probes that would differentiate between species of Leishmania, minicircle kDNA was digested with restriction enzymes and ligated to an E . coli plasmid . Several plasmids were isolated that specifically detect in hybridization experiments as few as 5 X 10(3) L . donovani or L . infantum promastigotes lysed on nitrocellulose filters. Biochem Biophys Res Commun, 1985 Feb 28, 127(1), 49 - 55 Characterization of the fluorescent bimane derivative of E . coli initiator transfer RNA (tRNAfMet); Pande C et al.; The invariant modified base 4-thiouridine of the E . Coli initiator tRNA was chemically modified using a sulfhydryl specific fluorogenic probe, monobromobimane . The modified tRNAfMet is virtually indistinguishable biochemically from the native form in the aminoacylation and formylation reactions, and in its binding behavior to the ribosomal P site . Fluorescence quenching by I- increases 40% when the modified tRNA is charged with formylmethionine, even at this relatively well-shielded position in the tRNA elbow . Most important, the fluorescence polarization increases by a factor of 2, to almost the irrotational value, when fMet-tRNAfMet binds to the ribosomal P site, providing a useful tool for studying fMet-tRNAfMet-ribosome interaction equilibria and kinetics. FEBS Lett, 1985 Feb 25, 181(2), 381 - 4 Isolation of the aspartokinase domain of bifunctional aspartokinase I-homoserine dehydrogenase I from E.coli K12; Veron M et al.; A proteolytic fragment (Mr approximately 25 000) carrying only the aspartokinase activity has been purified by chromatofocusing after limited proteolysis of aspartokinase I-homoserine dehydrogenase I from E.coli K12 . The NH2-terminal sequence shows that it corresponds to the amino terminal peptide of the native enzyme . The results confirm a previous hypothesis about the organization of native aspartokinase I-homoserine dehydrogenase I. Schweiz Med Wochenschr, 1985 Feb 16, 115(7), 235 - 8 {Effect of interferon-alpha 2 (E . coli) in hairy cell leukemia}; Hofmann V et al.; Recombinant interferon-alpha 2 (E . coli) produced a clinically significant improvement in hemoglobin, granulocytes and platelets in 7 of 8 patients with hairy cell leukemia . Response to treatment was already noticeable in the fourth treatment week . In one case without improvement after 120 days, treatment was stopped . So far only one complete remission has been documented . Because of the remarkable improvement in the peripheral blood values, the induction of a complete remission may not be the ultimate goal of interferon treatment . The side effects of this subcutaneous low-dose treatment consisted mainly of mild flu-like symptoms of short duration . The results obtained with recombinant interferon-alpha 2 confirm the initial observation by Quesada et al . with partially purified leukocyte-interferon . In our experience, these results are superior to those obtained in similar conditions with chlorambucil. Monatsschr Kinderheilkd, 1985 Feb, 133(2), 82 - 5 {Chemotaxis defect and recurrent E . coli meningitis}; Herpertz B et al.; The increased susceptibility to bacterial infections due to a decreased chemotaxis of neutrophil granulocytes was investigated in a premature small for gestational age baby, who such suffered from recurrent E . coli-meningitis . Causes as anatomic lesions, brain abscess, septic granuloumatosus disease and the Chediak-Higashi-anomaly were ruled out . At the age of 9 and 13 weeks chemotaxis function compared to age-matched and adult controls was diminished . Addition of serum from healthy adults increased directed migration without reaching normal levels . The study of the complement status revealed a diminution of CH50, the alternate pathway components and C6-C9 . Thus, decreased chemotaxis is believed to originate from a combined intrinsic-extrinsic defect . No relapses of infection were observed during prophylactic treatment with ascorbate and cotrimoxazole . Intrinsic as well as extrinsic chemotaxis became normal at the age of 7 months . Simultaneously, complement status was found to approach normal levels . The relevance of haematologic-immunologic evaluation in cases of relapsing severe bacterial infections in preterm newborns is discussed. Cancer Biochem Biophys, 1985 Feb, 7(4), 325 - 31 Ultraviolet radiation-induced derepression of the lactose operon of E . coli; Spodheim-Maurizot M et al.; The effect of ultraviolet irradiation of a regulatory protein, the lac repressor, on its interactions with operator DNA is investigated by spectroscopic and electrophoresis methods . A second set of experiments is performed to assay the capacity of the system containing the irradiated repressor to be induced by IPTG . The protein-nucleic acid interactions are modified upon ultraviolet irradiation of the repressor . The inducer becomes ineffective and repressor stays "locked" to DNA in conditions in which the native repressor is released from the system . These facts are discussed in terms of genes repression and of promotion step in ultraviolet induced carcinogenesis. EMBO J, 1985 Feb, 4(2), 527 - 32 In vivo evidence that the nusA and infB genes of E . coli are part of the same multi-gene operon which encodes at least four proteins; Nakamura Y et al.; Previous work has shown that the Escherichia coli nusA gene codes for a protein which regulates transcription termination . The 16.0-kb EcoRI DNA fragment that includes the nusA gene, codes for at least eight bacterial proteins of mol . wts . 48 000 (argG), 21 000 (p21), 64 000 (nusA), 120 000 (IF2 alpha)-(infB), 91 000 (IF2 beta)(infB), 15 000 (p15), 10 000 (rpsO) and 85 000 (pnp) . We have constructed several deletion and fusion derivatives from this cloned DNA and examined in vivo the structure and expression of these genes . First, the promoter functional in vivo for the nusA gene was mapped at approximately 800 bp upstream of the nusA structural gene . Second, the synthesis of five proteins, p21, NusA, IF2 alpha, IF2 beta (and p15) proteins, was affected by the deletion of the nusA promoter . Third, these same five proteins were hyperproduced after fusion of the DNA fragment to the lambda pL promoter . In addition, subcloning experiments revealed that the p15 gene is expressed by the read-through transcription from the infB gene . These results lead us to conclude that the genes coding for the p21, NusA, InfB (IF2 alpha and IF2 beta), and p15 proteins form a single-transcriptional unit ('nusA-infB operon') in vivo and that rpsO and pnp genes do not belong to the same operon . The in vivo attenuation site of this operon is described. Biokhimiia, 1985 Feb, 50(2), 307 - 11 {Effect of the redox state of glutathione on acetate kinase activity in E . coli}; Kulis IuIu et al.; Oxidized glutathione inhibits acetate kinase (EC 2.7.2.1) of E . coli . The rate of inactivation depends on ATP concentration . The rate constant for the glutathione-induced inhibition is 0.17 min-1, Ki is 4.2 mM (pH 7.2, 25 degrees C) . The inhibition of acetate kinase by glutathione is reversible, the equilibrium constant being equal to 4.4 or 0.09 at saturating concentrations of ATP (pH 8.0, 25 degrees C) . The physiological level of reduced and oxidized glutathione can modulate the acetate kinase activity in vivo. Zentralbl Bakteriol Mikrobiol Hyg {A}, 1985 Feb, 259(1), 59 - 70 {Adherence of E . coli strains with various adhesins to macrophages from bone marrow and the cell line P 388D1}; Harff V et al.; The adherence of E . coli with different hemagglutination patterns to mouse-bone-marrow-derived macrophages and macrophages of the cell line P 388D1 was investigated . The bacterial strains used showed different adherence to macrophages or red blood cells . MS-adhesins identified by hemagglutination tests were also involved in the attachment of bacteria to macrophages . In addition, strains containing both, MS and MR adhesins, simultaneously showed a participation of their MR-adhesins in adherence to macrophages . This could be shown by inhibition experiments with alpha-D-mannose . Bacteria of strain D 133 failed to induce hemagglutination of any source of erythrocytes tested, though MR-adherence to macrophages could be found . In contrast, other strains known to carry MR-hemagglutinins on their cell surface, did not attach to macrophages, even if much higher numbers of bacteria were used . A linear correlation between the amount of bacteria used and the number of adherent bacteria/macrophage was detectable . The number of bacteria found on the macrophages differed according to the population of macrophages studied, indicating differences in the expression of corresponding receptors in the macrophage plasma membrane . In order to investigate the role of fimbriae in adherence, bacteria were used which had been grown under fimbriae suppressing conditions . Some of the bacterial strains showed a 10 to 30 fold reduction in adherence to macrophages upon this treatment, indicating the importance of fimbriae-associated adhesins in the interaction of bacteria and phagocytes . On the other hand, three bacterial strains could be identified, whose adherences was not influenced by such culture conditions . This means that beside fimbrial adhesins even membrane bound adhesins could be involved in the phagocytic process. Nucleic Acids Res, 1985 Jan 25, 13(2), 501 - 19 Expression of polyoma early gene products in E . coli; Schaffhausen B et al.; The three products of the early region of polyoma virus have been cloned for expression in E . coli using the Tac promoter . Although the identical promoter and ribosome binding site are used in each final construction, the observed level of protein expression is different for each protein . While plasmids expressing wild type T antigens as well as a plasmid expressing the truncated Py-1387T middle T antigen lacking the membrane-anchoring sequence give rise to synthesis of proteins readily detectible by 35S-methionine labeling and immunoprecipitation, only small T and the middle T of Py-1387T are made in amounts sufficient for ready detection in total cell protein . Unlike middle T expressed in animal cells, middle T produced in E . coli is not detectibly phosphorylated . Further, the E . coli protein lacks tyrosine kinase activity. Biochem Biophys Res Commun, 1985 Jan 16, 126(1), 412 - 8 Labeling of a specific arginine residue at the active site of glutamine synthetase (E.coli); Colanduoni JA et al.; Chemical modification of a specific arginine residue of Escherichia coli glutamine synthetase has been accomplished by the use of the arginine-specific reagents p-hydroxyphenylglyoxal, phenylglyoxal, and methylglyoxal . Modification of one arginine residue results in complete inactivation of the enzyme and the modified enzyme seems to be extremely stable since no reactivation is observed upon addition of free arginine or dialysis . Saturating levels of ATP but not L-glutamate, L-methionine sulfoximine, or inorganic phosphate provide substantial protection against inactivation of the enzyme suggesting the modified amino acid is at or near the ATP substrate binding site . However, an ATP affinity analog is not prevented from binding upon modification of the arginine residue indicating that the reduction in catalytic activity is not solely due to alteration in substrate binding but may also reflect a catalytic role for the arginine residue. Nature, 1985 Jan 10-18, 313(5998), 149 - 51 Synthesis in E . coli of alpha 1-antitrypsin variants of therapeutic potential for emphysema and thrombosis; Courtney M et al.; The primary function of alpha 1-antitrypsin (alpha 1-AT), an antiprotease produced by the liver, is the inhibition of neutrophil elastase, a protease capable of hydrolysing most connective tissue components . The importance of alpha 1-AT is demonstrated by the high incidence of early-onset emphysema in individuals with hereditary alpha 1-AT deficiency (Type PiZZ), in whom serum levels of alpha 1-AT are 10-20% of normal . Oxidants in tobacco smoke can inactivate alpha 1-AT in vitro, and studies have shown that alpha 1-AT from the lungs of individuals who smoke cigarettes may also be partially inactivated, perhaps explaining the high incidence of emphysema associated with cigarette smoking . Oxidative inactivation is probably due to modification of the Met residue (Met358) at the P1 subsite position of the elastase binding site of the protein . To study the possibility of modulating the biological properties of alpha 1-AT, we have introduced selected sequence modifications at the reactive site by in vitro mutation of a cloned alpha 1-AT complementary DNA . We describe here the characterization of two alpha 1-AT analogues produced in Escherichia coli . The first, alpha 1-AT(Met385----Val), is not only fully active as an elastase inhibitor but is also resistant to oxidative inactivation . The other, alpha 1-AT(Met358----Arg), no longer inhibits elastase but is an efficient thrombin inhibitor . The active site of the latter is identical to that of the alpha 1-AT (Pittsburgh) variant, which was associated with a fatal bleeding disorder. Acta Biochim Pol, 1985, 32(4), 329 - 49 Substrate selection by RNA polymerase from E . coli . The role of ribose and 5'-triphosphate fragments, and nucleotides interaction; Szafranski P 2nd et al.; Steady-state kinetic studies of the rifampicin-effected abortive initiation of transcription by E . coli RNA polymerase (EC 2.7.7.6) on the A1 T7 phage promoter were carried out with the use of ATP, UTP and a number of their appropriately modified analogues . The kinetic parameters KiA, KmB, Ki and KsB characterizing the affinity of the substrates and inhibitors of the reaction to the initiation and elongation sites of the enzyme:promoter and the enzyme:promoter:nucleoside triphosphate complexes were determined therefrom . Their comparative analysis indicated that 1) the triphosphate chain of the initiating purine nucleoside triphosphate interacts with some protein acceptor groups through the alpha- and beta-phosphate residues; the phosphates are engaged in binding of nucleoside triphosphates at the elongation site in the absence of the primer nucleotide; 2) the ribose 2'-OH of the elongating nucleotide, but neither of the ribose hydroxyl groups of the initiating nucleotide, participate in substrate recognition by protein receptors; 3) either substrate, ATP or UTP, bound to the initiation complex increases by about the same factor (greater than or equal to 10) the affinity of the other to its binding site; 4) the 3'-OH of the primer nucleotide and the gamma-phosphate of the elongating nucleotide are involved in the synergistic interaction of the substrates; alpha- and beta-phosphates of the elongating nucleotide, bound to some protein receptors, also contribute to this process . It is postulated that the interaction of substrates is mediated through an Mg2+ ion, known to be required for binding of the substrates in the elongation site, and a minimal molecular model of a PuoTP:Mg (II): nucleoside triphosphate chelate complex in the catalytic centre of the transcription initiation open complex is proposed. Nucleic Acids Symp Ser, 1985, (16), 279 - 82 Inhibitory effects of the 3'-noncoding region derived from eukaryotic cDNA on the expression of the upstream structure gene in E . coli; Nomura M et al.; The influence of 3'-noncoding region (ncr) of the cDNA derived from an eukaryotic mRNA on the preceding structure gene expression in E . coli was investigated using human immune interferon (GIF) and human tumor necrosis factor (TNF) cDNAs . Two sets of gene, with (ncr+) and without (ncr-) 3'-noncoding region, have been expressed in E . coli . In both cases, the level of ncr+ gene expression was five times lower than that of ncr- gene . When these ncrs were placed just downstream of various structure genes, the same effects were also observed . The RNA brot analysis showed that the ncr+ genes were transcribed into shorter and much less mRNA than ncr- genes . These results suggest that these ncrs accelerate the degradation of the mRNA by either causing the premature termination of transcription or the generation of sequence on the transcripts hypersensitive to E . coli endoribonucleases. Acta Chir Scand, 1985, 151(3), 205 - 11 Tissue thromboplastin generation in circulating mononuclear phagocytes and development of coagulation disorders during E . coli endotoxinaemia in pigs; Andersen OK et al.; Tissue thromboplastin generation in monocytes was studied during various stages of Escherichia coli endotoxinaemia in pigs . The pigs were monitored in halothane anaesthesia and mechanically ventilated . Blood was sampled from the superior caval vein before and during endotoxin infusion and up to 6 hours after its start . Monnuclear leukocytes were harvested with Lymphoprep separation and monocyte counts were made, using TRITC-labelled sheep erythrocytes, acridine orange and a fluorescence microscope . Thromboplastin was quantified in a two-stage assay by incubating the test sample together with purified factor X, factor VII and Ca++ . The generated factor Xa was thereafter assayed . There was statistically significant increase of tissue thromboplastin activity in monocytes after endotoxin infusion . Maximum level was reached at the end of the infusion and was maintained throughout the observation period . Decrease occurred in platelets, leukocytes, antithrombin III, fibrinogen and clotting factors V, VII and VIII, and clotting time was prolonged . These findings indicated significant disseminated intravascular coagulation . The endotoxin-stimulated monocytes with their elevated tissue thrombo-plastin activity thus may play an important part in development of the DIC which so often follows septicemia. Mutat Res, 1985 Jan-Feb, 142(1-2), 1 - 4 The resistance of E . coli cultivated in low concentrations of dichlorvos to N-methyl-N'-nitro-N-nitrosoguanidine induced mutagenesis; Alldrick AJ et al.; Cultivation of E . coli B/r strain WP2 in low concentrations of either 4-nitroquinoline N-oxide (4NQO) or N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) had no effect on the mutagenic or cytotoxic consequences of subsequent challenge with dichlorvos (DCV) . However, although the sensitivity of E . coli cells taken from cultures grown in low concentrations of DCV to the effects of 4NQO was unchanged, the cells were more resistant to the mutagenic (but not cytotoxic) consequences of MNNG challenge . This phenomenon was not observed in WP2 derivatives deficient in either error-free (uvrA-) or error-prone (lexA-) DNA-repair, suggesting that a factor common to both these repair pathways may be involved. Mol Gen Genet, 1985, 201(3), 467 - 76 Characterization and properties of very large inversions of the E . coli chromosome along the origin-to-terminus axis; Louarn JM et al.; Suppression of a dnaA46 mutation by integration of plasmid R100.1 derivatives in the termination region of chromosome replication in E . coli results in medium dependence, the suppressed bacteria being sensitive to rich medium at 42 degrees C . Derivatives of such bacteria have been selected for growth at 42 degrees C in rich medium and we have analyzed representatives of the most frequently observed type: bacteria displaying, once cured of the suppressor plasmid, both rich-medium sensitivity and temperature sensitivity . We found, in all cases, that the chromosome had undergone a major inversion event between two inverted IS5's . One is located at 29.2 min on the chromosome map and the other at either one of two positions between 69 and 80 min . The consequences of such inversions for cell growth are discussed . Some of them result from the fact that the replication terminator T2 is located, in inverted chromosomes, close to oriC in the orientation which allows its functioning as a terminus (de Massy et al . in press) . Our observations allow an estimation of the frequency of inversions arising from recombination between pairs of inverted chromosomal IS, which could be as high as 10(-2) per cell per generation . We also found that inversion reversal occurs frequently after Hfr conjugational transfer of one of the IS5's, in its wild-type location . This led us to propose a new mechanism of recombination, in which the incoming DNA strands serve as guides to favor recombination between the resident sequences. Circ Shock, 1985, 16(3), 307 - 16 Verapamil improves cardiac function and increases survival in canine E . coli endotoxin shock; Bosson S et al.; The effects of verapamil, a calcium antagonist, on survival and on hemodynamic and metabolic parameters were studied in canines administered E . coli endotoxin . Shams, endotoxin controls, and endotoxin-shocked dogs treated with a 4-hour infusion of verapamil were studied . The animals were anesthetized, catheters and endotracheal tube were inserted, and an IV infusion was started after administration of endotoxin . All dogs were kept on a respirator for 4 hours while measurements were taken; they were then extubated and returned to their cages . Survival was considered permanent by 7 days . Eight of 13 treated dogs survived, in contrast with only one of 14 controls . Treated dogs had significantly higher cardiac index (4.64 vs 3.62 L/min/m2), pulmonary artery pressure (16 vs 13 mmHg), and left ventricular stroke work (44.3 vs 29.7 gm/m2 beat), and significantly lower heart rate and systemic vascular resistance at 4 hours . Serum glucose, acid phosphatase, pH, and Hct were also significantly improved by verapamil treatment. Folia Biol (Praha), 1985, 31(3), 213 - 34 Consensus symmetry pattern in E . coli promoter sequences; Pivec L et al.; A computer method was used to select two subgroups in 172 Escherichia coli promoter nucleotide sequences characterized by "standard" 17 bp and "non-standard" 17 +/- 2 bp spacing between the Pribnow box and -35 region . The conservation of the two-fold rotational (2f) and true palindrome (tp) symmetry relations was determined between nucleotide doublets in both promoter subgroups which represent consensus symmetry patterns . Statistically significant symmetries were primarily distinguished from those established by the nucleotide sequence conservation . The consensus symmetry pattern of standard promoters involves 45 of the 60 promoter nucleotide positions at which less strongly conserved and non-conserved sequences were mostly occupied (per se) . They also show a high level of symmetry centre conservation . The conservation of the statistically significant 2f symmetry centres at positions -4.5 and -31.5 suggests partial conservation of the Pribnow box and -35 pentamer in the codogenic strand in an opposite orientation, respectively . The non-standard promoters differ from the standard ones by the consensus symmetry pattern and by the 2f and tp symmetry centre distribution with an overall lower degree of symmetry conservation in the -35 region . It has been suggested that the conserved symmetry relations provide an additional condition necessary for the specific interaction of RNA polymerase with the promoter sequence to initiate transcription. Acta Chem Scand B, 1985, 39(4), 273 - 90 Chemical synthesis of a pentaribonucleoside tetraphosphate constituting the 3'-acceptor stem sequence of E . coli tRNAIle using 2'-O-(3-methoxy-1,5-dicarbomethoxypentan-3-yl)-ribonucleoside building blocks . Application of a new achiral and acid-labile 2'-hydroxyl protecting group in tRNA synthesis; Sandstrom A et al.; A synthesis of a pentaribonucleotide fragment constituting the residues 59-63 of 3'-terminus of E . coli tRNAIle, 5'-ApGpUpCpC-3', has been carried out using a new, easily accessible and achiral 2'-ketal protecting group . The new 2'-ketal group has an additional advantage in that it is easily functionalized to the diamide with aqueous ammonia in the penultimate step of deblocking of fully protected oligoribonucleotides . Such a functionalization of the 2'-ketal group at the penultimate step of deblocking of the fully protected tRNA molecule enhances its relative rate of removal under an acidic condition with a minimum of damage of the target tRNA molecule. Microbios, 1985, 42(168), 67 - 75 Purification and partial characterization of megamodulin, a heat-stable protein factor from E . coli and its stimulatory effect on RNA polymerase holoenzyme; Sen S et al.; Megamodulin, a heat-stable protein from Escherichia coli was isolated and purified near homogeneity as determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis . It had a molecular weight of 71,000 and pl between 3.5 and 4.0 . This factor stimulated E . coli RNA polymerase 71-fold in the presence of a synthetic template such as poly (rA).p(dT) . When TATAAA sequence was used as template, the RNA polymerase activity was increased 68 times by this factor . The possible mechanism by which this protein factor may regulate the RNA polymerase activity has been described. Microbios, 1985, 42(168), 119 - 24 Morphological changes in spherical E . coli induced by a DC electrical field; Ellis HW; Normally spherical cells of Escherichia coli C when grown on a solid minimal salts medium through which an electrical current was passed, developed as short rods, or occasionally, as longer filaments . Currents of up to 30 mA DC were used. Mol Gen Genet, 1985, 199(1), 7 - 13 Mutations in the sigma subunit of E . coli RNA polymerase which affect positive control of transcription; Hu JC et al.; The sigma subunits of bacterial RNA polymerases are required for the selective initiation of transcription . We have isolated and characterized mutations in rpoD, the gene which encodes the major form of sigma in E . coli, which affect the selectivity of transcription . These mutations increase the expression of araBAD up to 12-fold in the absence of CAP-cAMP . Expression of lac is unaffected, while expression of malT-activated operons is decreased . We determined the DNA sequence of 17 independently isolated mutations, and found that they consist of three different changes in a single CGC arginine codon at position 596 in the sigma polypeptide. Mol Gen Genet, 1985, 199(1), 133 - 40 Mechanism of transient inhibition of DNA synthesis in ultraviolet-irradiated E . coli: inhibition is independent of recA whilst recovery requires RecA protein itself and an additional, inducible SOS function; Khidhir MA et al.; The mechanism of the inhibition and of the recovery of DNA synthesis in E . coli following UV-irradiation was analysed in several mutants defective in repair or in the regulation of the RecA-LexA dependent SOS response . Several lines of evidence indicated that inhibition is not an inducible function and is probably due to the direct effect of lesions in the template blocking replisome movement . Recovery of DNA synthesis after UV was largely unaffected by mutations in the uvrA, recB or umuC genes . Resumption of DNA synthesis does however require protein synthesis and the regulatory action of recA . Experiments with a recA constitutive mutant and recA 200 (temperature sensitive RecA) demonstrated that RecA protein itself is directly required but is not sufficient for recovery of DNA synthesis . We therefore propose that recovery of DNA synthesis depends upon the concerted activity of RecA and the synthesis of an inducible Irr (induced replisome reactivation) factor under RecA control . We suggest that the mechanism of recovery involves the action of Irr and RecA to promote movement of replisomes past non-instructive lesions, uncoupled from polymerisation and/or that Irr and RecA are required to promote re-initiation of a stalled replication complex downstream of a UV-lesion subsequent to such an uncoupling step. Mol Gen Genet, 1985, 199(1), 106 - 10 Purification of alpha-hemolysin from an overproducing E . coli strain; Gonzalez-Carrero MI et al.; The genetic determinant of the alpha-hemolysin encoded by plasmid pHly152 has been cloned in both orientations in plasmid pBR322 giving rise to plasmids pSU157 and pSU158 . E . coli strains carrying either of these recombinant Hly plasmids produced about 20 times more hemolysin activity than the parental plasmid pHly152, when grown in minimal medium supplemented with hemoglobin . Thus high hemolytic activity is not lethal to the cells, contrary to previous assumptions . alpha-Hemolysin was purified from culture supernatants of strain SU100 (pSU157) by ammonium sulfate precipitation and gel filtration in Sephacryl S-200 in the presence of 6 M urea . When purified alpha-hemolysin preparations were subjected to electrophoretic analysis in denaturing conditions, a single 107 kdal polypeptide was observed . This probably corresponds to the alpha-hemolysin protein, since an isogenic E . coli strain carrying plasmid pSU161, an Hly- mutant derivative of pSU157, did not synthesize the 107 kdal polypeptide. Mutat Res, 1985 Jan-Mar, 145(1-2), 35 - 41 Repair of platinum-DNA lesions in E . coli by a pathway which does not recognize DNA damage caused by MNNG or UV light; Germanier M et al.; The adaptive response is an inducible DNA-repair system which diminishes the mutagenic and toxic effects of alkylating agents . A mutant of E . coli constitutive for adaptative repair, BS21, has been isolated . A spontaneous revertant of this strain, BS23, lacks the adaptive response . When compared to its wild-type parent, mutant BS21 showed an increased resistance to the killing and mutagenic effects of a compound which is not a classical alkylating agent, the antitumor drug cis-diamminedichloroplatinum(II) (cis-DDP) . However, this resistance to cis-DDP was also found in strain BS23 which lacks the adaptive response . cis-DDP bound to the DNA of all 3 strains with the same efficiency . In addition, we have investigated the effect of UV radiation and we failed to observe a significant difference in the survival and mutagenesis of these strains . This evidence suggests that the resistance of BS21 and BS23 strains to cis-DDP is not a consequence of the adaptive response or increased excision repair. Nippon Geka Gakkai Zasshi, 1985 Jan, 86(1), 23 - 31 {The mechanism of action of E.coli endotoxin on oxidative phosphorylation of the rat liver mitochondria}; Ishiyama S et al.; The effects of E.coli endotoxin on oxidative phosphorylation were investigated in vitro using isolated rat liver mitochondria . The respiratory control index (RCI) was significantly decreased by preincubation of mitochondria for 20 min . at 30 degrees C in the presence of the endotoxin, which repressed dose-dependently the state 3 respiration . The extent of the repression of this state 3 respiration was comparable with that of the exchange reaction of adenine nucleotide . The endotoxin, however, did not inhibit several mitochondrial enzymes including the electron transport system at the enzyme level . The exchange reaction and the number of the ADP-binding sites of the adenine nucleotide carrier were assayed using {14C} ADP and {3H} carboxyatractyloside, respectively . The exchange reaction was repressed by 72% and the number of binding site of carboxyatractyloside was decreased by 20% by the endotoxin (100 micrograms/mg mitochondrial protein) . These effects of endotoxin on the adenine nucleotide carrier were additionally enhanced by the Ca2+ ion . The occurrence of these effects was prevented by EGTA or dibucaine, a potent inhibitor of phospholipase A2 . These results suggested that the endotoxin may activate phospholipase A2 which may harm the lipid layer of the mitochondrial membrane. Int J Radiat Biol Relat Stud Phys Chem Med, 1985 Jan, 47(1), 49 - 56 The formation of photoreactivable damage by direct excitation of DNA in X-irradiated E . coli cells; Duba VV et al.; The role of the direct excitation process in the formation of photoreactivable damage (pyrimidine dimers) in E . coli WP2 hcr-exr- cells has been studied . The pyrimidine dimers were detected by photoreactivation following anoxic irradiation by X-rays (220 kVp) . The dose modifying factor (DMF) is 1.28 +/- 0.09 . A biophysical model is used for a theoretical examination of the importance of the direct excitation process in the formation of photoreactivable damage and the experimental data are consistent with this model. Mutat Res, 1985 Jan-Feb, 148(1-2), 1 - 12 Evaluation of the DNA-repair host-mediated assay . I . Induction of repairable DNA damage in E . coli cells recovered from liver, spleen, lungs, kidneys, and the blood stream of mice treated with methylating carcinogens; Kerklaan PR et al.; The DNA-repair host-mediated assay was further calibrated by determining the genotoxic activities of 4 methylating carcinogens, namely, dimethylnitrosamine (DMNA), 1,2-dimethylhydrazine (SDMH), methyl nitrosourea (MNU) and methyl methanesulphonate (MMS) in various organs of treated mice . The ranking of the animal-mediated genotoxic activities of the compounds was compared with that obtained in DNA repair assays performed in vitro . The differential survival of strain E . coli K-12/343/113 and of its DNA-repair-deficient derivatives recA, polA and uvrB/recA, served as a measure of genotoxic potency . In the in vitro assays and at equimolar exposure concentrations, MMS and MNU are the most active chemicals, followed by DMNA, which shows a slight genotoxic effect only in the presence of mouse liver homogenate; SDMH has no activity under these conditions . In the host-mediated assays, the order of genotoxic potency of the compounds was quite different: those carcinogens which require mammalian metabolic activation, namely, DMNA and SDMH, show strong effects in liver and blood, a lesser effect in the lungs and kidneys and the least effect in the spleen . The activity of MNU, a directly acting compound, is similar in all organs investigated, but it is clearly lower than that of DMNA and SDMH . MMS, also a directly acting carcinogen, causes some (barely significant) effect at the highest dose tested . A similar order of potency was observed when the compounds were tested in intrasanguineous host-mediated assays with gene mutation as an endpoint . DMNA and SDMH induce comparable frequencies of L-valine-resistant mutants in E . coli K-12/343/113 recovered from liver and spleen of treated mice, the effect in the liver being the strongest . MNU is mutagenic only at a higher dose, while MMS shows no effect . The results are discussed with respect to the literature data on organ-specific DNA adduct formation induced by the compounds . It is concluded that qualitatively there is a good correlation between the degree of genotoxic activity found in the DNA repair host-mediated assay and DNA adduct formation in the animal's own cells . This is exemplified by the finding that the relative order of genotoxic activity of the 4 methylating agents in bacteria recovered from various organs (DMNA approximately equal to SDMH greater than MNU greater than MMS) is reflected by the same order of magnitude in DNA alkylation in corresponding mammalian organs . Quantitatively, the indirectly acting agents DMNA and SDMH seem to induce fewer genotoxic effects in bacteria present in the liver than would be expected on the basis of DNA-adduct formation data. Mutat Res, 1985 Jan-Mar, 145(1-2), 43 - 8 Cross-linking studies with the uvrA and uvrB proteins of E . coli; Strike P et al.; The interactions of the uvrA and uvrB proteins with DNA have been investigated using a DNA-protein cross-linking technique . It is demonstrated that hydrolysis of ATP by the uvrA protein facilitates cross-linking of this protein to single-stranded DNA, whether the DNA is UV irradiated or not . In contrast, cross-linking to unirradiated double-stranded DNA is not facilitated by ATP hydrolysis and is in fact increased by the substitution of the non-hydrolysable analogue aTP gamma S for ATP . In the presence of ATP, a dose-dependent increase is observed in the amount of uvrA protein which can be cross-linked to UV-irradiated double-stranded DNA . Binding of uvrB protein to puvrA-DNA complexes has a stabilising effect and increases the number of complexes which can be cross-linked whether the substrate is single- or double-stranded DNA . We can find no evidence that ATP hydrolysis by uvrA protein results in unwinding of UV-damaged DNA. Mol Gen Genet, 1985, 201(2), 347 - 50 Amplification of the ArgF region in strain HfrP4X of E . coli K-12; Jessop AP et al.; In E . coli K-12 the argF gene is flanked by ISI sequences in direct repeat . Mutants that overproduce the argF-coded enzyme ornithine transcarbamylase can be selected; we have shown that in one class of these mutants there is an approximately forty five-fold amplification of the region bounded by the ISI repeats . This class of mutants has been detected only in strains in which the F-factor is integrated in cis to the region. Adv Exp Med Biol, 1985, 185, 63 - 82 Studies of TGEV spike protein gp195 expressed in E . coli and by a TGE-vaccinia virus recombinant; Hu S et al.; The gene coding for the surface spike protein gp195 of TGEV has been cloned and expressed in E . coli in the form of fusion proteins . These proteins were isolated and used to immunize laboratory animals . All animals developed antibodies cross-reacting with the TGEV virion but failed to neutralize the virus . The entire gp195 gene was also inserted into vaccinia to generate a TGEV-vaccinia recombinant virus (vTGE) that expressed TGEV gp195 . Animals vaccinated with vTGE produced neutralizing antibodies against both TGEV and vaccinia . These results suggest the potential use of the recombinant vTGE as a vaccine against TGEV infection. Mol Gen Genet, 1985, 201(1), 35 - 7 Conjugation-dependent enhancement of induced and spontaneous mutation in the lacI gene of E . coli; Christensen RB et al.; The frequency of lac mutations induced in an F'lacIS plasmid, transferred by conjugation from UV-irradiated, excision-deficient donors to excision-deficient, delta pro lac recipients, is 2-3 fold higher than that typical of nonmating cells which contain the plasmid . These additional induced mutations can probably be ascribed to errors made during the first, or repliconation, synthesis that takes place in the recipient during the course of plasmid transfer . We also find that spontaneous mutation rates are enhanced in conjugating cells, indicating that fewer errors are corrected, or more made, during transfer replication. Boll Ist Sieroter Milan, 1985, 64(1), 6 - 11 E . coli adherence to CER cells infected by vesicular stomatitis virus; Chiarini F et al.; Research was carried out on the adherence of a mannose-resistant uropathogenic E . coli strain to CER cells infected with vesicular stomatitis virus (vsv) . A decrease in the bacterial adhesion was noticed during the early phases of viral infection, probably due to a close relationship between cell receptors for VSV and E . coli, both