Mutat Res, 1987 Oct, 180(2), 137 - 46 Base substitution mutagenesis by terminal transferase: its role in somatic mutagenesis; Snow ET et al.; We have addressed the possibility of terminal transferase involvement in somatic mutagenesis and the creation of N-region diversity, by measuring the ability of TdT to enhance single-base substitution mutagenesis during in vitro DNA synthesis . Using 3 independent assays we find that terminal transferase produces only a small increase in base-substitution mutagenesis when assayed in the presence of DNA polymerase-beta . In the presence of either polymerase-alpha or E . coli polymerase-I, however, no detectable increase in TdT-induced mutagenesis is seen . Furthermore, in an assay capable of detecting a variety of mutational events, terminal transferase primarily produces complex addition/deletion mutations, as well as a few multiple, tightly-clustered, single-base mutations . We conclude that the majority of the scattered single-base changes that occur during antibody gene differentiation are not catalyzed by terminal transferase, but instead result from another error-prone DNA synthetic process (possibly utilizing DNA polymerase-beta). Avian Dis, 1987 Oct-Dec, 31(4), 809 - 13 Antigenic relatedness and partial amino acid sequences of pili of Escherichia coli serotypes O1, O2, and O78 pathogenic to poultry; Suwanichkul A et al.; Pilus proteins from Escherichia coli serotypes O1, O2, and O78 pathogenic to poultry were compared with regard to their antigenic relatedness and partial amino acid sequences . Agglutination, immunodiffusion, and immunoblot assays with polyclonal antibodies to these pili showed that these pili not only share some common antigens but also contain antigens unique to each pilus . The partial amino-terminal amino acid sequences support our earlier findings that the pili are different but contain some structural homologies. Gut, 1987 Oct, 28(10), 1283 - 90 Organ culture of rabbit ileum as a model for the investigation of the mechanism of intestinal damage by enteropathogenic Escherichia coli; Batt RM et al.; Organ culture of rabbit ileum has been established as a model for the investigation of the mechanism of intestinal damage by enteropathogenic Escherichia coli (EPEC) . Loops of rabbit ileum were filled in vivo with saline, non-enteropathogenic P-fimbriate E coli (PFEC), or EPEC . After 45 minutes the loops were washed, then mucosal biopsies were taken and cultured for up to 48 hours . The earliest changes discernable by electron microscopy were observed at 18 hours postinfection, at which time EPEC were closely adherent to the surface of enterocytes at the base of microvilli, some of which were elongated . By 24 hours postinfection there were large areas of brush border effacement with pedestal formation around the EPEC . No such damage was seen in biopsies from the control loops (saline, PFEC), and intracellular ultrastructure was extremely well preserved in all preparations for up to 48 hours . While there were no differences at eight hours, biochemical analyses at 24 hours postinfection showed a marked increase in the release of brush border enzymes into the culture medium from EPEC-infected explants compared to explants from the control loops . These findings provide morphological and biochemical evidence for damage to the microvillus membrane by EPEC, and validate organ culture of rabbit ileum as a model for the investigation of EPEC-pathogenicity. Proc Natl Acad Sci U S A, 1987 Oct, 84(20), 6980 - 4 Reconstitution of the lysosomal proton pump; D'Souza MP et al.; Lysosomal membrane proteins solubilized with octyl beta-D-glucopyranoside were reconstituted into proteoliposomes using acetone/ether-washed phospholipids from Escherichia coli . Assays of the quenching of acridine orange fluorescence showed that addition of both ATP and valinomycin to K+-loaded proteoliposomes led to the formation of a pH gradient that was acidic inside . ATP-driven acidification took place in the absence of permeant anions and was inhibited by the "protonophore", carbonylcyanide p-trifluoromethoxyphenylhydrazone, indicating that only H+ was transported actively . Proton translocation was readily blocked by N-ethylmaleimide (10 microM gave 50% inhibition of fluorescence quenching) but was unaffected by oligomycin (50 nM), orthovanadate (50 microM), or ouabain (0.5 mM); similarly, only N-ethylmaleimide affected ATP hydrolysis by proteoliposomes (88% inhibition) . Other work showed that reconstitution of ATP-driven proton translocation required the presence of glycerol during protein solubilization and that optimal recovery depended on the use of both glycerol and phospholipid at this stage . We conclude that acidification of the lysosome is mediated by an ATPase capable of electrogenic H+ translocation without molecular coupling to other ionic species. Infect Immun, 1987 Oct, 55(10), 2465 - 70 Construction and characterization of Bordetella pertussis toxin mutants; Black WJ et al.; Pertussis toxin is one of the major virulence determinants produced by Bordetella pertussis . The DNA encoding the structural genes for pertussis toxin was cloned in Escherichia coli, and pertussis toxin subunit S4 was expressed under the control of the tac promoter . Mutations were introduced into the cloned toxin genes, and a conjugative shuttle vector system was devised for delivering the mutations from E . coli back into B . pertussis . The mutations were introduced by allelic exchange into the chromosome of B . pertussis resulting in a series of B . pertussis strains which were isogenic except at the loci encoding the structural genes for pertussis toxin . These B . pertussis strains were utilized to study the biogenesis of pertussis toxin . Polar mutations in the S1 gene led to a lack of detectable S2 or S4 subunits in whole-cell lysates, suggesting a polycistronic arrangement for these genes . Mutations in the S5 subunit gene resulted in a truncated S1 subunit, while mutations in the S4 gene resulted in a lack of detectable S2 subunit, suggesting that physical relationships among the toxin subunits are directly reflected in the stable biogenesis of the subunits. Bioorg Khim, 1987 Oct, 13(10), 1351 - 7 {Nucleotide sequence of the archaebacterial mobile genetic element ISH S1}; Zviaga TA et al.; The complete primary structure (1449 b . p.) of mobile genetic element ISH S1 from Halobacterium halobium has been elucidated using the dideoxy/M13 sequencing procedure . Computer analysis of the structure reveals similarity in overall structural organization of ISH S1 and other known transposable genetic elements of halobacteria and makes it possible to propose a hypothetical model of halobacterial promoter. Antimicrob Agents Chemother, 1987 Oct, 31(10), 1640 - 1 Norfloxacin resistance in a clinical isolate of Escherichia coli; Aoyama H et al.; Analysis of DNA gyrase supercoiling and of norfloxacin uptake in Escherichia coli GN14176, a moderately norfloxacin-resistant clinical isolate, indicated that resistance was associated with both an altered drug target and a reduction in drug uptake. Mol Gen Genet, 1987 Oct, 209(3), 518 - 25 Transcriptional repression of the dnaA gene of Escherichia coli by dnaA protein; Wang QP et al.; The promoter region of the dnaA gene and of a gene which encodes a 16 kDa protein contain sites which are recognized and bound by dnaA protein . Using assays of run-off transcription of restriction fragments, purified dnaA protein specifically repressed transcription from both dnaA promoters and from the promoter for the 16 KD gene to almost undetectable levels . This repressive effect was observed at levels of dnaA protein required for specific binding of dnaA protein to restriction fragments containing the promoters for these genes . These results indicate that transcription of these genes is regulated by binding of dnaA protein to the promoter regions of these genes. Mol Gen Genet, 1987 Oct, 209(3), 494 - 8 Effects of modulation of RNase H production on the recovery of DNA synthesis following UV-irradiation in Escherichia coli; Casaregola S et al.; The requirements for the recovery of DNA synthesis in UV-irradiated Escherichia coli were analysed in strains having varied levels of RNase H and RecA protein . We have previously shown (Khidhir et al . 1985) that the recovery of DNA synthesis in E . coli following UV treatment is an inducible SOS function requiring protein synthesis . We proposed that this reflected the need for the synthesis of specific induced replisome reactivation factor(s) for recovery . In this study we now show that recovery of DNA synthesis can in fact take place in the absence of protein synthesis in a mutant lacking RNase H and having high (constitutive) levels of RecA protein . We also show that expression of rnh is inhibited during the SOS response in recA+ but not in a recA- strain . The results are discussed in relation to the mechanism of recovery of DNA synthesis following UV irradiation in E . coli. Mol Gen Genet, 1987 Oct, 209(3), 481 - 8 Cloning and nucleotide sequencing of the genes rimI and rimJ which encode enzymes acetylating ribosomal proteins S18 and S5 of Escherichia coli K12; Yoshikawa A et al.; The rimI gene of Escherichia coli K12, which encodes an enzyme catalysing acetylation of the N-terminal alanine of ribosomal protein S18, has been cloned into a mini-F plasmid pRE432 and characterized at the molecular level . Similarly, the rimJ gene, which encodes another acetylating enzyme that is specific for ribosomal protein S5, has been cloned and characterized . From the nucleotide sequence data for the two genes the RimI enzyme was deduced to contain 161 amino acid residues with a calculated molecular weight (Mr) of 18232 and the RimJ enzyme contains 194 amino acid residues with a calculated Mr of 22687 . The proteins produced from the two genes in maxi-cells were identified by electrophoresis on acrylamide gels and their operon structure was analysed by insertional mutagenesis with transposon gamma delta (Tn1000) and by measuring the size of their transcripts . Their structural homology was analysed by DNA hybridization and by calculation with computer programs . There is only a low level of overall homology between the two genes except for a 3' terminal region in which a significant degree of homology was noticed. Mol Gen Genet, 1987 Oct, 209(3), 453 - 7 Role of replication in IS102-mediated deletion formation; Bernardi F et al.; Intramolecular transposition produces replicon dissociation in a bireplicon; this reaction is homologous to the well-characterized IS-associated deletions in the case of a monoreplicon . However the frequencies at which these two reactions occur differ by a factor of more than 10(2) in favor of deletion formation . This raises the question of how these deletions occur . We show that the presence of a productive replication on the fragment to be deleted interferes with deletion formation . Our results also suggest that the deleted fragment is not degraded during deletion formation. Mol Biol Med, 1987 Oct, 4(5), 291 - 305 High-level production of fully active human alpha 1-antitrypsin in Escherichia coli; Johansen H et al.; The human alpha-1-antitrypsin (A1AT) gene expressed in Escherichia coli as a full-length, non-fusion gene product accumulates to a relatively low level approaching less than or equal to 0.1% of total cellular protein . In contrast, deletion of the first 5, 10 or 15 codons leads to production of truncated A1AT derivatives at levels between 10 and 30% of total cellular protein . The protein with the largest truncation was insoluble and inactive following solubilization by chaotropic agents . In contrast, the two derivatives with the smaller truncations were found to be soluble, and exhibit identical specific activities in both trypsin and elastase inhibition assays to authentic human A1AT . The expression of the full-length A1AT was also optimized by making silent third position mutations within its first 15 codons . These mutations were chosen to optimize codon usage and minimize the possibility of RNA secondary structure formation in this region . Via this approach, expression of full-length, authentic, fully active A1AT was increased at least 20-fold to 2% of total cellular protein . Optimal expression was obtained using as few as three silent mutations in the first five codons, confirming the importance of this 5'-terminal region as had been defined by our deletion mutants . Both the full-length derivatives as well as the small N-terminal deletion derivative can be readily purified from bacterial extracts in fully active form suitable for the examination of their potential therapeutic application. J Appl Physiol, 1987 Oct, 63(4), 1526 - 32 Effect of polyethylene glycol-superoxide dismutase and catalase on endotoxemia in pigs; Olson NC et al.; We hypothesized that superoxide anion (O2-.) and hydrogen peroxide (H2O2) might be important mediators of endotoxin-induced acute respiratory failure (ARF) in pigs . As specific scavengers of O2- . and H2O2, we infused polyethylene glycol-superoxide dismutase (PEG-SOD; 2,000 IU/kg) and PEG-catalase (CAT; 15,000 IU/kg), respectively . Escherichia coli endotoxin (055-B5) was infused intravenously into anesthetized 10- to 14-wk-old pigs at 5 micrograms/kg the 1st h, followed by 2 micrograms.kg-1.h-1 for 3.5 h . During phase 1 (i.e., 0-2 h) and 2 (i.e., 2-4.5 h), endotoxin decreased cardiac index (CI) and lung dynamic compliance, and increased mean pulmonary arterial pressure (Ppa), pulmonary vascular resistance (PVR), total peripheral resistance (TPR), alveolar-arterial O2 gradient, and hematocrit . Endotoxemia also caused granulocytopenia and increased the postmortem bronchoalveolar lavage fluid (BALF) albumin concentration and wet-to-dry ratio of bloodless lung . During endotoxemia, PEG-SOD failed to significantly alter any measured or calculated parameter . On the other hand, PEG-CAT attenuated the early (i.e., 0-1 h) endotoxin-induced decrease in CI and increases in Ppa, PVR, and TPR, but failed to modify these parameters during phase 2 . PEG-CAT also attenuated the endotoxin-induced granulocytopenia and the increased BALF albumin concentration . In the presence of inactivated PEG-CAT, these protective effects were reversed . We conclude that O2- . does not directly contribute to endotoxin-induced lung injury and that H2O2 (or a subsequent metabolite) contributes to the early endotoxin-induced hemodynamic changes, granulocytopenia, and increased permeability of the alveolar-capillary membrane. EMBO J, 1987 Oct, 6(10), 3163 - 9 Expression of proteins essential for IS1 transposition: specific binding of InsA to the ends of IS1; Zerbib D et al.; The insertion sequence IS1 displays a complex array of open reading frames (ORF) . In an attempt to identify those which encode polypeptide products, we have systematically placed each ORF under the control of the P1 promoter of phage lambda . In the expression system we used, only the product of the insA gene was present in high enough amounts to be detected by polyacrylamide gel electrophoresis . The production of InsA was further increased in a first codon hook-up to phage T7 transcriptional and translational initiation signals . Cell extracts from InsA overproducers display a DNA binding activity specific for the ends of IS1 . This activity was identified as the InsA protein itself. EMBO J, 1987 Oct, 6(10), 3145 - 53 The interaction of the recognition helix of lac repressor with lac operator; Lehming N et al.; We have constructed a system which allows systematic testing of repressor--operator interactions . The system consists of two plasmids . One of them carries a lac operon in which lac operator has been replaced by a unique restriction site into which synthetic operators can be cloned . The other plasmid carries the gene coding for the repressor, in our case a semisynthetic lacI gene of which parts can be exchanged in a cassette-like manner . A galE host allows us to select for mutants which express repressors with altered specificities . Here we report the change of specificity in the lac system by changing residues 1 and 2 of the recognition helix of lac repressor . The specificity changes are brought about cooperatively by the change of both residues . Exchanges of just one residue broaden the specificity . Our results hint that the recognition helix of lac repressor may possibly have the opposite orientation to those in Lambda cro protein or 434 CI repressor. Proc Natl Acad Sci U S A, 1987 Oct, 84(20), 7213 - 7 Insertion and deletion mutagenesis of the human cytomegalovirus genome; Spaete RR et al.; Studies on human cytomegalovirus (CMV) have been limited by a paucity of molecular genetic techniques available for manipulating the viral genome . We have developed methods for site-specific insertion and deletion mutagenesis of CMV utilizing a modified Escherichia coli lacZ gene as a genetic marker . The lacZ gene was placed under the control of the major beta gene regulatory signals and inserted into the viral genome by homologous recombination, disrupting one of two copies of this beta gene within the L-component repeats of CMV DNA . We observed high-level expression of beta-galactosidase by the recombinant in a temporally authentic manner, with levels of this enzyme approaching 1% of total protein in infected cells . Thus, CMV is an efficient vector for high-level expression of foreign gene products in human cells . Using back selection of lacZ-deficient virus in the presence of the chromogenic substrate 5-bromo-4-chloro-3-indolyl beta-D-galactoside, we generated random endpoint deletion mutants . Analysis of these mutants revealed that CMV DNA sequences flanking the insert had been removed, thereby establishing this approach as a means of determining whether sequences flanking a lacZ insertion are dispensable for viral growth . In an initial test of the methods, we have shown that 7800 base pairs of one copy of L-component repeat sequences can be deleted without affecting viral growth in human fibroblasts. J Clin Microbiol, 1987 Oct, 25(10), 1962 - 5 Detection of enterotoxigenic Escherichia coli by colony hybridization with biotinylated enterotoxin probes; Kirii Y et al.; By using biotinylated enterotoxin DNA probes, a method to detect enterotoxigenic Escherichia coli by colony hybridization was developed . The treatment of colonies on nitrocellulose membrane filters with proteinase K and Triton X-100 was essential for obtaining the specific hybridization . A total of 200 E . coli strains isolated from travelers with diarrhea were tested for colony hybridization by using a probe encoding heat-labile toxin (LT) type h . All strains (86 of 86) that produced LT, but none of the non-LT producers, hybridized with 32P-labeled and biotinylated LT type h probes . A total of 36 strains chosen randomly from the 200 isolates were tested for colony hybridization by using heat-stable enterotoxin (ST) probes . All but two strains that hybridized with the 32P-labeled ST type Ia probe also hybridized with the biotinylated ST type Ia probe . All strains that hybridized with the 32P-labeled ST type Ib probe also hybridized with the biotinylated ST type Ib probe . Thus, almost all E . coli strains tested were judged to be the same by colony hybridization with biotinylated or 32P-labeled enterotoxin probes . These results demonstrate that the biotinylated enterotoxin probes are useful in the diagnosis of enterotoxigenic E . coli strains by colony hybridization. Arch Biochem Biophys, 1987 Oct, 258(1), 95 - 100 Sulfhydryl groups of glucosamine-6-phosphate isomerase deaminase from Escherichia coli; Altamirano MM et al.; Glucosamine-6-phosphate isomerase deaminase (2-amino-2-deoxy-D-glucose-6-phosphate ketol isomerase (deaminating), EC 5.3.1.10) from Escherichia coli is an hexameric homopolymer that contains five half-cystines per chain . The reaction of the native enzyme with 5',5'-dithiobis-(2-nitrobenzoate) or methyl iodide revealed two reactive SH groups per subunit, whereas a third one reacted only in the presence of denaturants . Two more sulfhydryls appeared when denatured enzyme was treated with dithiothreitol, suggesting the presence of one disulfide bridge per chain . The enzyme having the exposed and reactive SH groups blocked with 5'-thio-2-nitrobenzoate groups was inactive, but the corresponding alkylated derivative was active and retained its homotropic cooperativity toward the substrate, D-glucosamine 6-phosphate, and the allosteric activation by N-acetyl-D-glucosamine 6-phosphate . Studies of SH reactivity in the presence of enzyme ligands showed that a change in the availability of these groups accompanies the allosteric conformational transition . The results obtained show that sulfhydryls are not essential for catalysis or allosteric behavior of glucosamine-6-phosphate deaminase. Virology, 1987 Oct, 160(2), 433 - 44 The transforming protein of avian reticuloendotheliosis virus is a soluble cytoplasmic protein which is associated with a protein kinase activity; Walro DS et al.; We have identified the product (p57v-rel) of the transforming gene, v-rel, of avian reticuloendotheliosis virus (REV-T) using antisera generated against nonoverlapping sequences representing the middle and carboxy-terminal regions of the v-rel protein expressed in Escherichia coli (N.K . Herzog and H.R . Bose, Jr., 1986, Proc . Natl . Acad . Sci . USA 83, 812-816) . The amino-terminal region of the v-rel protein was also expressed in E . coli and used to generate antisera . The immunoglobulin-enriched fractions of these antisera were used to determine the subcellular location of p57v-rel in REV-T transformed lymphoid cells . Cells were fractionated into nuclear, mitochondrial, microsomal, and cytoplasmic fractions . The majority of p57v-rel was found in the cytoplasm . Examination of REV-T transformed lymphoid cells labeled with 32Pi revealed that the majority of the phosphorylated form of the v-rel protein was also found in the cytoplasm . Indirect immunofluorescence of REV-T transformed cells gave a diffuse cytoplasmic pattern indicating that p57v-rel was not associated with any discrete cellular organelle . The distribution of p57v-rel was similar in REV-T transformed lymphoid cells labeled with {35S}methionine for short and long periods of time, suggesting that p57v-rel is a soluble cytoplasmic protein throughout its lifetime . The v-rel protein was phosphorylated when immune complexes precipitated from transformed cells with the immunoglobulin fractions obtained from antisera against the amino-terminal, middle, and carboxy-terminal regions of v-rel were incubated with {gamma-32P}ATP and Mn2+ . The phosphorylation of p57v-rel in the in vitro immune complex kinase assay was inhibited when the immunoglobulin-enriched fraction of these antisera was preincubated with the homologous v-rel fusion proteins . Preincubation with heterologous proteins did not block the phosphorylation of p57v-rel . These observations suggest that p57v-rel is associated with a protein kinase activity . Most of the kinase activity was found in the soluble cytoplasmic fraction of transformed cells . The transforming protein encoded by v-rel is a relatively stable protein with a half-life of approximately 7 to 8 hr in transformed lymphoid cells. Proc Natl Acad Sci U S A, 1987 Oct, 84(19), 6677 - 81 Molecular cloning and amino acid sequence of leukotriene A4 hydrolase; Funk CD et al.; A cDNA clone corresponding to leukotriene A4 hydrolase was isolated from a human lung lambda gt11 expression library by immunoscreening with a polyclonal antiserum . Several additional clones from human lung and placenta cDNA lambda g11 libraries were obtained by plaque hybridization with the 32P-labeled lung cDNA clone . One of these clones has an insert of 1910 base pairs that contains the complete protein-coding region . From the deduced primary structure, leukotriene A4 hydrolase is a 610 amino and protein with a calculated molecular weight of 69,140 . No apparent homologies with microsomal epoxide hydrolases were found . RNA blot analysis indicated substantial amounts of a discrete mRNA of approximately equal to 2250 nucleotides in lung tissue and leukocytes. Mutat Res, 1987 Oct, 180(2), 155 - 61 Inhibitory effect of heavy water on mutability of chemically injured Escherichia coli cells; Hakura A et al.; After E . coli cells (WP2 and WP2uvrA) were treated with chemical mutagens (methyl methanesulfonate, MMS; N-methyl-N-nitrosourea, MNU; 4-nitroquinoline 1-oxide, 4NQO) in 1/15 M phosphate buffer, the mutability of the treated cells plated on a D2O-agar plate was compared with that plated on an ordinary H2O-agar plate . The mutation frequency decreased more or less on the D2O-agar plate . The D2O-substitution effects, as termed by the relative mutation frequencies (MFD2O/MFH2O), are 0.92 for MMS, 0.29 for MNU, and 0.42 for 4NQO in WP2, and 0.68 for MMS, 0.49 for MNU, and 0.16 for 4NQO in WP2uvrA . The D2O effect seemed to be partly related to the function of the uvrA gene-associated products . The pH dependence of mutability was discussed in connection with the D2O-substitution effect. J Bacteriol, 1987 Oct, 169(10), 4765 - 9 Sequence, expression, and localization of the immunity protein for colicin M; Olschlager T et al.; Escherichia coli strains carrying the cmi locus on plasmids are immune against colicin M, which primarily inhibits murein biosynthesis, followed by lysis of cells . The nucleotide sequence of the cmi region was determined . It contains an open reading frame for a polypeptide with a molecular weight of 19,227 . However, the major protein band observed on polyacrylamide gels after transcription and translation in an in vitro system or in minicells had an apparent molecular weight between 15,000 and 16,000 . The nucleotide sequence contained internal ATG codons, two of which could serve for the synthesis of polypeptides with molecular weights of 15,349 and 15,996, respectively . A subclone with a DNA fragment that encoded these two shorter polypeptides exhibited full immunity . The colicin M immunity protein was found in the cytoplasmic membrane . The colicin M activity and immunity genes were transcribed in opposite directions . Both properties are typical of the channel-forming colicins and are in contrast to the colicins with endonuclease activities . However, colicin M does not form channels and exhibits no structural similarity to channel-forming colicins. J Bacteriol, 1987 Oct, 169(10), 4716 - 21 Structural and functional analysis of a cloned segment of Escherichia coli DNA that specifies proteins of a C4 pathway of serine biosynthesis; Ravnikar PD et al.; The plasmid pDR121 is a pBR322 derivative that contains a 3.7-kilobase-pair EcoRI fragment of DNA from the 81.2-min region of the Escherichia coli chromosome . The genomic insert encodes threonine dehydrogenase and at least one other protein . Several physical and kinetic properties of threonine dehydrogenase, overproduced in cells harboring pDR121, are identical to those of pure threonine dehydrogenase from a haploid mutant of E . coli K-12 that produces this enzyme constitutively . Tester strains with serB or glyA mutations harboring pDR121 are prototrophs . The ability to confer prototrophy on such tester strains is associated with elevated levels of threonine dehydrogenase . The functional roles of various segments of the 3.7-kilobase-pair insert of pDR121 were analyzed by constructing specific deletions and insertions . Certain subclones retained the ability to specify threonine dehydrogenase without conferring prototrophy on tester strains . This suggests that at least one other protein encoded within pDR121 plays an essential role in the conversion of threonine to serine. J Bacteriol, 1987 Oct, 169(10), 4703 - 9 DnaA protein is required for replication of the minimal replicon of the broad-host-range plasmid RK2 in Escherichia coli; Gaylo PJ et al.; The minimal origin of replication of the broad-host-range plasmid RK2 has two potential recognition sequences for the DnaA protein of Escherichia coli . DNA transfer by transformation into a dnaA-null mutant of E . coli showed that DnaA protein is needed for replication or maintenance of mini-RK2 . We isolated and purified DnaA protein as a chimeric protein, covalently attached to a piece of collagen and beta-galactosidase . The hybrid protein specifically bound to restriction fragments from the oriV region of RK2, which contained the two dnaA boxes . Deletion of the second dnaA box inactivated the origin and abolished the binding of the hybrid protein to the DNA fragment that had suffered the deletion . When the second dnaA box was replaced with an EcoRI linker of identical length, origin activity was restored . Binding experiments showed that the linker provided a weak dnaA box . An alternative explanation was that the linker restored proper spacing between sequences on either side of the deleted box, thus restoring origin activity. J Bacteriol, 1987 Oct, 169(10), 4637 - 45 dnaA, an essential host gene, and Tn5 transposition; Yin JC et al.; Mutations in dnaA, an essential gene in Escherichia coli, decrease the frequency of transposition of Tn5 . An insertion mutation in the dnaA gene does not affect Tn5 gene expression . Therefore, the DnaA protein plays a role either in the transposition reaction itself or in some type of cellular regulation of transposition . Analysis of a mutation in the DnaA box, found at the outside end of IS50, is consistent with a direct interaction of the protein through these bases . IS50 transposition, which utilizes only one end containing a DnaA box, is not affected by dnaA mutations . Overproduction of the DnaA protein does not increase transposition frequencies in wild-type cells, even when the transposase is also overproduced. J Bacteriol, 1987 Oct, 169(10), 4570 - 6 Alterations at the carboxyl terminus change assembly and secretion properties of the B subunit of Escherichia coli heat-labile enterotoxin; Sandkvist M et al.; The gene encoding the B subunit of heat-labile enterotoxin (etxB) was mutated at its 3' end by targeted addition of random nucleotide sequences . Gene products from five mutated etxB genes, all of which were shown to encode B subunits with short carboxy-terminal amino acid extensions, were analyzed with respect to a range of functional and structural properties . One class of altered B subunits, exemplified by EtxB124 and EtxB138, which both have seven extra amino acid residues, were found to be specifically defective in their ability to stably associate with A subunits and form holotoxin . Other altered B subunits were less subtlely affected by extensions at their C termini and were, in addition to their failure to associate with A subunits, unable to translocate into the periplasm of Escherichia coli, to pentamerize, or to bind to GM1 ganglioside . This suggests that the carboxy-terminal domain of EtxB mediates A subunit-B subunit interaction. Infect Immun, 1987 Oct, 55(10), 2518 - 25 Hypochlorous acid-promoted loss of metabolic energy in Escherichia coli; Barrette WC Jr et al.; Oxidation of Escherichia coli by hypochlorous acid (HOCl) or chloramine (NH2Cl) gives rise to massive hydrolysis of cytosolic nucleotide phosphoanhydride bonds, although no immediate change occurs in either the nucleotide pool size or the concentrations of extracellular end products of AMP catabolism . Titrimetric curves of the extent of hydrolysis coincide with curves for loss of cell viability, e.g., reduction in the adenylate energy charge from 0.8 to 0.1-0.2 accompanies loss of 99% of the bacterial CFU . The oxidative damage caused by HOCl is irreversible within 100 ms of exposure of the organism, although nucleotide phosphate bond hydrolysis requires several minutes to reach completion . Neither HOCl nor NH2Cl reacts directly with nucleotides to hydrolyze phosphoanhydride bonds . Loss of viability is also accompanied by inhibition of induction of beta-galactosidase . The proton motive force, determined from the distribution of 14C-radiolabeled lipophilic ions, declines with incremental addition of HOCl after loss of respiratory function; severalfold more oxidant is required for the dissipation of the proton motive force than for loss of viability . These observations establish a causal link between loss of metabolic energy and cellular death and indicate that the mechanisms of oxidant-induced nucleotide phosphate bond hydrolysis are indirect and that they probably involve damage to the energy-transducing and transport proteins located in the bacterial plasma membrane. Carcinogenesis, 1987 Oct, 8(10), 1517 - 20 DNA synthesis is blocked by cigarette tar-induced DNA single-strand breaks; Borish ET et al.; DNA single-strand breaks are caused by aqueous extracts of cigarette tar, due to the reduction of oxygen to superoxide by tar and the subsequent production of hydroxyl radicals . The action of DNA metabolism enzymes on these single-strand breaks has been studied to probe the consequences of these lesions for DNA repair . Our results demonstrate that cigarette tar-induced nicks are blocked at the 3' terminus since they are totally incapable of activating DNA for DNA synthesis by Escherichia coli DNA polymerase I . The 3' termini of these tar-induced nicks are activated, however, for DNA synthesis by E . coli exonuclease III or by the 3' phosphatase activity of T4 polynucleotide kinase . Because of the inability of tar-induced lesions to support DNA synthesis, they probably require a multi-step process for repair in vivo . As a consequence, the overall likelihood of mutation is increased due to the possibility for error at each step of the repair process. Mol Gen Genet, 1987 Oct, 209(3), 585 - 91 Analysis of structure-function relationships in Escherichia coli K12 outer membrane porins with the aid of ompC-phoE and phoE-ompC hybrid genes; van der Ley P et al.; To study structure-function relationships in the outer membrane pore proteins OmpC and PhoE of Escherichia coli K12, we have constructed a series of phoE-ompC hybrid genes in which DNA encoding part of one protein is replaced by the homologous part of the other gene . The hybrid gene products were incorporated normally into the outer membrane, allowing their functional characterization . Combined with previous studies, the present results permit the identification of regions involved in determining functions and properties in which the native PhoE and OmpC proteins differ, such as pore characteristics, receptor activity for phages and binding of monoclonal antibodies . Most of these properties were found to be determined by multiple regions clearly separated in the primary structure . The combined phage and antibody binding data have demonstrated that at least five distinct regions in PhoE and OmpC are exposed at the cell surface . The locations of these regions are in agreement with a previously proposed model for porin topology. Immunopharmacology, 1987 Oct-Nov, 14(2), 93 - 9 Physical dependence to morphine diminishes the interferon response in mice; Lorenzo P et al.; Morphine pellet implantation in mice was demonstrated to diminish resistance to encephalomyocarditis virus infections . The variations in the response to three different interferon (IFN) inducers--Newcastle disease virus, Escherichia coli lipopolysaccharide and a tilorone analogue--were evaluated . A close relationship between morphine dependence and IFN response was detected . A clear inhibition in IFN induction appeared as a concomitant phenomenon with the syndrome of morphine dependence . In the response intensity, the mice strain tested was more important than the total drug dose in the pellet . This effect of morphine on IFN responses presented a characteristic age-related pattern and, perhaps, may also be influenced by the H-2 murine phenotype. Biol Chem Hoppe Seyler, 1987 Oct, 368(10), 1413 - 25 Synthesis, cloning and expression of recombinant aprotinin; Auerswald EA et al.; Synthetic DNA fragments containing the coding sequence for the serine proteinase inhibitor aprotinin, also known as bovine pancreatic trypsin inhibitor (BPTI) a Kunitz type inhibitor were fused to form a synthetic aprotinin gene by the method of Khorana and cloned into E . coli . The synthetic gene is characterized by the presence of certain restriction sites . These restriction sites are unique within the used cloning system . Therefore, a great number of modifications can be achieved easily by exchange of appropriate restriction fragments . Using this method the variant {Glu52}aprotinin was obtained starting from the aprotinin gene . Both genes were successfully expressed in E . coli as fusion proteins with beta-galactosidase using vector pUR 278 . No translation products could be detected in four other expression system (pUR 108, pDR 540, pKK 223-3 and pUC 8) . {Glu52}aprotinin was purified and renatured after cyanogen bromide cleavage of the fusion protein . This recombinant {Glu52}aprotinin shows exactly the same trypsin-inhibitory profile as natural aprotinin. Mol Biochem Parasitol, 1987 Oct, 25(3), 247 - 55 Cloning of diagnostic 31/32 kilodalton antigens of Schistosoma mansoni; Klinkert MQ et al.; A cDNA library derived from messenger RNA of adult Schistosoma mansoni was constructed in lambda gt11 and schistosome antigens were expressed as fusions with the amino terminus of the beta-galactosidase of Escherichia coli . Using mouse and human infection sera, recombinant clones specific for a 31/32 kDa doublet having a potential in the immunodiagnosis of schistosomiasis were selected . The specificity of the clones was verified by their reactivity with monoclonal antibodies . The identity of the cloned epitopes with those of the native proteins was confirmed by Western blot analysis of total schistosome proteins, using both antibodies affinity purified from the fusion proteins and antisera raised in rabbits against the fusions . The reactivity of the cloned antigens with human infection sera suggests their usefulness for the immunodiagnosis of human schistosomiasis. J Virol Methods, 1987 Oct, 18(1), 1 - 12 Synthesis of long viral complementary DNA from 7.5 Kb poly A+ RNA templates; Frankel G et al.; The poly A+ RNA of the WW and GDVII virus isolates, belonging to the Theiler's murine encephalomyelitis virus group, were used as templates for cDNA synthesis . Since several secondary structures were present along these viral RNAs the reverse transcriptase was prematurely displaced from the RNA templates and only short cDNA molecules could be synthesized . Therefore a reliable and reproducible procedure for the synthesis of long cDNA transcripts, that can be directly used for cloning into respective plasmid or phage vectors, was developed . The precise conditions and kinetics of the several enzymatic reactions were studied . The use of methylmercury hydroxide for first strand synthesis, a correct choice of Klenow polymerase for second strand synthesis and the use of vertical gel electrophoresis in combination with zone centrifugation for removal of the excess linkers were found to be of paramount importance for the synthesis of long, up to intact, 7.5 Kb cDNA transcripts. J Med Virol, 1987 Oct, 23(2), 101 - 7 Protective immunisation against hepatitis B with an internal antigen of the virus; Murray K et al.; Preparations of hepatitis B virus (HBV) core antigen (HBcAg) synthesised in Escherichia coli have been shown previously to confer partial immunity against infection by the virus {Murray, Bruce, Hinnen, Wingfield, van Eerd, de Reus, and Schellekens: EMBO Journal 3:645-650, 1984} . In a further experiment reported here, immunisation of chimpanzees with a similar preparation of HBcAg that had been treated with sodium dodecyl sulphate in order to expose e antigen epitopes was found to protect one animal completely and another quite substantially upon challenge with the virus . The results are used to support the argument for trials in humans of a vaccine against HBV based upon or containing HBcAg and its e antigen derivative, and in discussion of a more general role for internal antigens in generating immunity against viral infection. Hybridoma, 1987 Oct, 6(5), 489 - 507 Monoclonal antibodies to human tumor necrosis factors alpha and beta: application for affinity purification, immunoassays, and as structural probes; Bringman TS et al.; Monoclonal antibodies were produced against two structurally related tumor necrosis factors (TNFs), TNF-alpha (previously called tumor necrosis factor) and TNF-beta (previously called lymphotoxin) . The potential of these antibodies for the purification of TNFs, the development of specific immunoassays, and for defining the antigenic and functional domains of these cytokines was investigated . None of the monoclonal antibodies cross-reacted with both TNF-alpha and TNF-beta, or reacted with synthetic peptides which represented several of the regions of homology between these cytokines . Neutralizing monoclonal antibodies were utilized as immunoadsorbents to purify recombinant TNF-alpha and TNF-beta from E . coli lysates . TNFs purified by this method were greater than 98 percent pure by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and exhibited specific activities that were the same as TNFs isolated from natural sources using conventional chromatographic techniques . In addition, specific ELISA assays were developed that could detect less than 1 ng/ml of TNF-alpha or TNF-beta, and in contrast to bioassays, could discriminate between these related cytokines. Anal Biochem, 1987 Oct, 166(1), 188 - 93 Purification and separation of various plasmid forms by exclusion chromatography; Moreau N et al.; A chromatographic method for the rapid isolation of preparative amounts of plasmid DNA without the use of cesium chloride centrifugation is described . The protocol uses the alkaline extraction procedure and an exclusion column of Fractogel TSK 75S . From a clear lysate it is possible to obtain plasmid DNA completely free of proteins, RNA, and chromosomal DNA . From partially purified plasmid the procedure allows the separation of the different forms . This technique was successfully applied to different plasmids ranging in size from 2.9 to 17.5 MDa . It is a preparative method yielding easily 500 micrograms of pBR322 from 1 liter of amplified culture . The plasmid is suitable for topoisomerase I, topoisomerase II, and EcoRI assays. Proc Natl Acad Sci U S A, 1987 Oct, 84(20), 7251 - 5 Interferon beta 2/B-cell stimulatory factor type 2 shares identity with monocyte-derived hepatocyte-stimulating factor and regulates the major acute phase protein response in liver cells; Gauldie J et al.; One of the oldest and most preserved of the homeostatic responses of the body to injury is the acute phase protein response associated with inflammation . The liver responds to hormone-like mediators by the increased synthesis of a series of plasma proteins called acute phase reactants . In these studies, we examined the relationship of hepatocyte-stimulating factor derived from peripheral blood monocytes to interferon beta 2 (IFN-beta 2), which has been cloned . Antibodies raised against fibroblast-derived IFN-beta having neutralizing activity against both IFN-beta 1 and -beta 2 inhibited the major hepatocyte-stimulating activity derived from monocytes . Fibroblast-derived mediator elicited the identical stimulated response in human HepG2 cells and primary rat hepatocytes as the monocyte cytokine . Finally, recombinant-derived human B-cell stimulatory factor type 2 (IFN-beta 2) from Escherichia coli induced the synthesis of all major acute phase proteins studied in human hepatoma HepG2 and primary rat hepatocyte cultures . These data demonstrate that monocyte-derived hepatocyte-stimulating factor and IFN-beta 2 share immunological and functional identity and that IFN-beta 2, also known as B-cell stimulatory factor and hybridoma plasmacytoma growth factor, has the hepatocyte as a major physiologic target and thereby is essential in controlling the hepatic acute phase response. Virology, 1987 Oct, 160(2), 323 - 9 Molecular characterization of a polymerase mutant human immunodeficiency virus; Gendelman HE et al.; A cell line (8E5) containing a single defective copy of human immunodeficiency virus proviral DNA and producing noninfectious viral particles lacking reverse transcriptase (RT) and endonuclease proteins has recently been described (Folks, et al., (1986b) J . Exp . Med . 164, 280-290) . In this report, the mutation in a full-length molecular clone of the provirus (p8E5) was mapped to a 1931-bp region of the pol gene encoding RT . The nucleotide sequence of this segment revealed a 1-base deletion 301 codons from the common amino terminus of the 64- and 51-kDa RTs . Expression of the p8E5 RT segment in Escherichia coli generated an enzymatically inactive and truncated 33-kDa protein. J Exp Med, 1987 Oct 1, 166(4), 1084 - 97 Biochemical characterization of a gamma interferon-inducible cytokine (IP-10); Luster AD et al.; An IFN-gamma-inducible protein, IP-10, has previously been described to belong to a gene family of chemotactic and mitogenic proteins, associated with inflammation and proliferation . Biochemical characterization of this predicted protein has been pursued through the development of polyclonal monospecific antisera to recombinant protein and synthetic peptides . These reagents establish that the IP-10 protein is secreted from a variety of cells (endothelial, monocyte, fibroblast, and keratinocyte) in response to IFN-gamma . Posttranslational processing occurs in the biosynthesis of this protein, resulting in a 6-7-kD species, which may reflect COOH-terminal cleavage . Pulse-chase studies indicate that this processing is a rapid event in the primary cell lines studied, completed in the 30-min labeling period . A model is presented for the processing and secondary structure of this protein . In an accompanying study, Kaplan, et al . using these antisera, demonstrate that the IP-10 protein is associated, in vivo, with a delayed-type hypersensitivity response. J Bacteriol, 1987 Oct, 169(10), 4722 - 30 Role of micF in the tolC-mediated regulation of OmpF, a major outer membrane protein of Escherichia coli K-12; Misra R et al.; Mutation in the tolC locus greatly reduces normal synthesis of OmpF, a major porin protein of Escherichia coli K-12 . Experiments that use ompF-ompC chimeric genes demonstrate that a tolC mutation exerts its effect at either the promoter or the amino-terminal end of the ompF gene . Direct analysis of ompF mRNA from tolC+ and tolC strains showed that the amount of ompF transcript in the latter was greatly reduced . We have also observed that, in addition to reducing the amount of OmpF, a tolC mutation increases the level of OmpC protein to a much greater extent than occurs in an OmpF mutant and also increases micF RNA synthesis as shown by increased beta-galactosidase synthesis in a micF-lacZ fusion strain . Based on these observations, we suggest that an increased expression of the micF gene in a tolC mutant results in the reduced expression of ompF and that a major effect of the tolC mutation may be to push the porin-regulating system to favor ompC and micF to a greater extent than under high-osmolarity conditions. J Bacteriol, 1987 Oct, 169(10), 4608 - 13 DNA polymerase III requirement for repair of DNA damage caused by methyl methanesulfonate and hydrogen peroxide; Hagensee ME et al.; The pcbA1 mutation allows DNA replication dependent on DNA polymerase I at the restrictive temperature in polC(Ts) strains . Cells which carry pcbA1, a functional DNA polymerase I, and a temperature-sensitive DNA polymerase III gene were used to study the role of DNA polymerase III in DNA repair . At the restrictive temperature for DNA polymerase III, these strains were more sensitive to the alkylating agent methyl methanesulfonate (MMS) and hydrogen peroxide than normal cells . The same strains showed no increase in sensitivity to bleomycin, UV light, or psoralen at the restrictive temperature . The sensitivity of these strains to MMS and hydrogen peroxide was not due to the pcbAl allele, and normal sensitivity was restored by the introduction of a chromosomal or cloned DNA polymerase III gene, verifying that the sensitivity was due to loss of DNA polymerase III alpha-subunit activity . A functional DNA polymerase III is required for the reformation of high-molecular-weight DNA after treatment of cells with MMS or hydrogen peroxide, as demonstrated by alkaline sucrose sedimentation results . Thus, it appears that a functional DNA polymerase III is required for the optimal repair of DNA damage by MMS or hydrogen peroxide. Infect Immun, 1987 Oct, 55(10), 2428 - 35 Characterization of an immunoprotective protein complex of Anaplasma marginale by cloning and expression of the gene coding for polypeptide Am105L; Barbet AF et al.; Immunization with an Anaplasma marginale surface protein complex containing two polypeptides (Am105U and Am105L), each having a molecular weight of 105,000, protected cattle against challenge with virulent organisms . These polypeptides were immunoprecipitated together from detergent extracts of A . marginale by a neutralizing monoclonal antibody . After surface radioiodination of intact parasites, both Am105U and Am105L contained the radiolabel . To define the structural and antigenic relationships between Am105U and Am105L and to determine individual efficacies as protective immunogens, we cloned and expressed A . marginale DNA in Escherichia coli . We identified recombinant bacteria which expressed a novel protein of 105,000 molecular weight as a major cellular component . The recombinant protein was structurally and antigenically homologous to Am105L . There were multiple, partially homologous copies of the cloned DNA sequence in the rickettsial genome. Gastroenterology, 1987 Oct, 93(4), 734 - 43 Pilus-mediated interactions of the Escherichia coli strain RDEC-1 with mucosal glycoproteins in the small intestine of rabbits; Sherman PM et al.; Escherichia coli strain RDEC-1 (serotype 015:NM) is an effacing adherent enteropathogen that binds to the intestine of rabbits in a manner morphologically identical to the binding of human enteropathogenic E . coli strains to human intestine . The rabbit enteropathogen adheres to mucosal enterocytes in vivo and to microvillus membranes in vitro . Binding of RDEC-1 to ileal brush borders and to M cells overlying Peyer's patches is mediated by pili (fimbriae) expressed on the cell surface of bacteria . To examine whether similar binding occurs to glycoproteins present in the intestinal lumen, RDEC-1 was fed to rabbits and the intestinal luminal contents were examined for in vivo colonization by RDEC-1 . In addition, preparations of rabbit luminal glycoproteins were tested for their ability to agglutinate RDEC-1 in vitro . After the infection of rabbits, RDEC-1 organisms were found in the intestinal lumen associated with glycoproteins, as defined by positive histochemical staining of luminal material by periodic acid-Schiff and Alcian blue . In vitro aggregation of RDEC-1 by luminal glycoproteins of rabbit intestine, a luminal glycoprotein fraction purified on column chromatography and rabbit ileal microvillus membranes, was determined using a microtiter plate assay and an aggregometer . Nonpiliated RDEC-1 did not interact with either of the intestinal glycoprotein preparations or microvillus membranes . RDEC-1 expressing mannose-resistant AF/R1 pili or mannose-sensitive type 1 pili coaggregated with both rabbit luminal glycoprotein preparations and rabbit ileal microvillus membranes at equivalent protein concentrations . These studies demonstrate that RDEC-1 are associated with luminal glycoproteins during in vivo infection of rabbits, and that piliated RDEC-1, but not nonpiliated, coaggregate with rabbit glycoprotein preparations in vitro . Luminal glycoproteins contained within the mucus layer may serve as a site for replication and colonization of bacteria before bacterial enteroadherence. Biochem Biophys Res Commun, 1987 Sep 30, 147(3), 1153 - 61 Biosynthesis of reovirus-specified polypeptides . Molecular cDNA cloning and nucleotide sequence of the reovirus serotype 1 Lang strain s2 mRNA which encodes the virion core polypeptide sigma 2; George CX et al.; Human reovirus serotype 1 Lang strain s2 mRNA, which encodes the virion inner capsid core polypeptide sigma 2, was cloned as a cDNA:mRNA heteroduplex in Escherichia coli using phage M13 . A complete consensus nucleotide sequence was determined . The Lang strain s2 mRNA is 1331 nucleotides in length and possesses an open reading frame with a coding capacity of 335 amino acids, sufficient to account for a sigma 2 polypeptide of 37,682 daltons . Comparison of the serotype 1 Lang s2 sequence derived from cDNA clones of s2 mRNA with the serotype 3 Dearing S2 sequence derived from cDNA clones of the S2 dsRNA genome segment reveals 86 percent homology at the nucleotide level . The predicted sigma 2 polypeptides of the Lang and Dearing strains display 98 percent homology at the amino acid level . Of 147 silent nt differences in the translated region, 136 were in the third base position of codons. Biochem Biophys Res Commun, 1987 Sep 30, 147(3), 1105 - 12 Incorporation of the carbocyclic analogue of (E)-5-(2-bromovinyl)-2'-deoxyuridine 5'-triphosphate into a synthetic DNA; Sagi J et al.; The carbocyclic analogue of (E)-5-(2-bromovinyl)-2'-deoxyuridine, C-BVDU, is a very potent and selective anti-herpes-virus compound . In order to synthesize and study the properties of a DNA that contains C-BVDU, the 5'-triphosphate, C-BVDUTP was prepared and evaluated as a potential substrate of the E . coli Klenow DNA polymerase enzyme . Although C-BVDUTP proved to be a very poor substrate also of this enzyme, it could be incorporated up to 3.6% into the synthetic DNA, poly(dA-dT, C-BVDU) . This level of substitution decreased significantly the template activity for DNA and RNA polymerases, as compared to that of poly(dA-dT). Biochem Biophys Res Commun, 1987 Sep 30, 147(3), 1077 - 81 Bovine mitochondrial ribosomes: effect of cations and heterologous dissociation factors on subunit interactions; Spremulli L et al.; The effects of cations and ribosome dissociation factors on the equilibrium between the bovine mitochondrial ribosome and its subunits has been investigated . As observed with other ribosomes, Mg2+ ions promote subunit association while monovalent cations promote subunit dissociation . E . coli IF-3 will prevent the reassociation of mitochondrial 28 S and 39 S subunits . However, at least 5-fold higher concentrations of IF-3 are required with mitochondrial subunits than are required with bacterial subunits . The cytoplasmic factor eIF-6, has no detectable activity in preventing mitochondrial ribosomal subunit association. FEBS Lett, 1987 Sep 28, 222(1), 199 - 203 Cloning and localization of the repressor gene (c) of the Mu-like transposable phage D108; Levin DB et al.; We have localized the D108 thermosensitive (cts) repressor gene to a region of DNA approx . 600 base pairs (bp) in length by sub-cloning an RsaI restriction endonuclease fragment (bp 200 to bp 802 from the left-end of the D108 genome) . We determined that the gene product from this fragment appears to be the same size (19 kDa) as that expressed from clones containing larger fragments of D108 DNA . Results from in vitro gel electrophoresis band-retardation and in vivo immunity assays show that the sub-cloned repressor appears to be fully functional. J Biol Chem, 1987 Sep 25, 262(27), 12889 - 92 Transcriptional control of rat heme oxygenase by heat shock; Shibahara S et al.; A heat shock element is located in the 5'-flanking region of the rat heme oxygenase gene (HO gene) . The incubation of rat glioma cells at 42 degrees C or with hemin at 37 degrees C increased the levels of heme oxygenase mRNA within 1 h and produced a maximum at 3 h (at least a 20-fold increase) . In both treatments, the heme oxygenase activity started to increase after a lag period of about 1 h and reached a maximum value at 5 h . There was an apparent additive effect of both treatments on the heme oxygenase induction . Studies with actinomycin D and cycloheximide suggested that both heat shock and hemin acted at the transcriptional level to induce heme oxygenase . Therefore, we analyzed the transient expression of chimeric fusion genes harboring the promoter of the rat HO gene ligated to the Escherichia coli gene gpt in rat glioma cells and in K1735 mouse amelanotic melanoma cells . The 5'-flanking region of the rat HO gene bearing the heat shock element conferred the heat inducibility of gpt RNA production in both cell lines; however, hemin treatment did not induce gpt RNA . These results indicate that rat heme oxygenase is a heat shock protein and that hemin induces heme oxygenase through a different mechanism from heat shock. J Chromatogr, 1987 Sep 25, 420(2), 253 - 62 Alternative procedure for the purification of the heat-stable enterotoxin of enterotoxigenic Escherichia coli pathogenic for calves; Altmann K et al.; A method for purification of the heat-stable enterotoxin (ST) of enterotoxigenic Escherichia coli (ETEC) strains (C1444 and B41) pathogenic for calves and some physiochemical properties of the ST are described . The method involved ultrafiltration on PM-10 and UM-2 Diaflo membranes, acetone fractionation, ion-exchange chromatography on AG 1-X2, chromatofocusing and a combination of hydrophobic interaction chromatography on octyl-Sepharose CL-4B and gel-permeation on Bio-Gel P-2 . Polyacrylamide gel electrophoresis in sodium dodecyl sulphate of fluorescamine-labeled purified, reduced and alkylated ST preparations revealed a single band with approximate molecular masses of 2500 and 2200 for the C1444 and B41 STs, respectively . For the C1444 ST, the final purification achieved was approximately 27,000-fold on the basis of absorbance at 280 nm per mouse effective dose . However, it was 2000-fold when calculated on the basis of mg protein per effective dose (5 ng) . Amino acid composition of the C1444 ST was found to be different from that of the B41 ST suggesting that the ST produced by bovine isolates may be heterogeneous in their structure. Nucleic Acids Res, 1987 Sep 25, 15(18), 7443 - 50 Nucleotide sequence of the transcriptional repressor gene korB which plays a key role in regulation of the copy number of broad host range plasmid RK2; Theophilus BD et al.; The product of the korB gene of broad host range plasmid RK2 is one of at least two proteins which repress transcription of the essential replication gene trfA . We report here the nucleotide sequence of korB and the properties of its predicted polypeptide product KorB which has a molecular weight of 39,011 Da . KorB is likely to be a soluble protein with an overall net negative charge . However, consistent with a role in transcriptional regulation, there is a region with extensive homology to the alpha helix-turn-alpha helix motif of many DNA binding proteins . This region shows no significant homology to equivalent regions of the TrfB protein which is the primary transcriptional repressor of RK2 and which binds to an operator whose half sites show considerable homology to the half sites of the korB operator. J Biol Chem, 1987 Sep 25, 262(27), 13258 - 62 Probing the structure of gal operator-repressor complexes . Conformation change in DNA; Majumdar A et al.; The gal operon is regulated by binding of Gal repressor to two operator loci, OE and OI, which are separated by 114 base pairs (bp) . We have probed the actual operator DNA segments with and without Gal repressor occupation by characterizing the regions protected by repressor from DNase I digestion and dimethyl sulfate methylation . The segments which are protected from DNase I digestion in both OE and OI are about 22 bp long and seem to include 2-3 extra bp on either side of a 16-bp similar sequence containing an approximate dyad symmetry, with a consensus half-symmetry sequence GTG(G/T)AA-C . Repressor occupation hinders the reactivity of the consensus guanines in the four half-symmetry sequences, as shown by retardation of methylation at the N-7 positions by dimethyl sulfate owing to repressor binding . The protected guanines are symmetrically located . Since a dimeric Gal repressor affects symmetrically located bases, it is consistent with the notion that each half-operator is occupied by a repressor subunit . Because the N-7 positions of methylation of guanines lie in the major grooves and the protected guanines are located at positions 1, 3, 8 and the rotational 1', 3', and 8' in the 16-bp dyad symmetry, we suggest that Gal repressor establishes direct contacts with bases at 1, 3, 1', and 3' through two major grooves lying on one face of an operator helix and prevents reactivity of the guanines at 8 and 8' of a third major groove on the opposite face by changing the DNA helical structure at this position . Contacts at other positions are also discussed. J Biol Chem, 1987 Sep 25, 262(27), 13241 - 5 The internal signal sequence of Escherichia coli leader peptidase is necessary, but not sufficient, for its rapid membrane assembly; Dalbey RE et al.; Leader peptidase of Escherichia coli, a protein of 323 residues, has three hydrophobic domains . The first, residues 1-22, is the most apolar and is followed by a polar region (23-61) which faces the cytoplasm . The second hydrophobic domain (residues 62-76) spans the membrane . The third hydrophobic domain, which has a minimal apolar character, and the polar, carboxyl-terminal two-thirds of the protein are exposed to the periplasm . Deletion of either the amino terminus (residues 4-50) or the third hydrophobic region (residues 83-98) has almost no effect on the rate of leader peptidase membrane assembly, while the second hydrophobic domain is essential for insertion (Dalbey, R., and Wickner, W . (1987) Science 235, 783-787) . To further define the roles of these domains, we have replaced the normal, cleaved leader sequence of pro-OmpA and M13 procoat with regions containing either the first or second apolar domain of leader peptidase . The second apolar domain supports the translocation of OmpA or coat protein across the plasma membrane, establishing its identity as an internal, uncleaved signal sequence . In addition to this sequence, we now find that leader peptidase needs either the amino-terminal domain or the third hydrophobic domain to permit its rapid membrane assembly . These results show that, although a signal sequence is necessary for rapid membrane assembly of leader peptidase, it is not sufficient. J Biol Chem, 1987 Sep 25, 262(27), 13188 - 97 Mechanism of damage recognition by Escherichia coli DNA photolyase; Husain I et al.; Escherichia coli DNA photolyase binds to DNA containing pyrimidine dimers with high affinity and then breaks the cyclobutane ring joining the two pyrimidines of the dimer in a light- (300-500 nm) dependent reaction . In order to determine the structural features important for this level of specificity, we have constructed a 43 base pair (bp) long DNA substrate that contains a thymine dimer at a unique location and studied its interaction with photolyase . We find that the enzyme protects a 12-16-bp region around the dimer from DNase I digestion and only a 6-bp region from methidium propyl-EDTA-Fe (II) digestion . Chemical footprinting experiments reveal that photolyase contacts the phosphodiester bond immediately 5' and the 3 phosphodiester bonds immediately 3' to the dimer but not the phosphodiester bond between the two thymines that make up the dimer . Methylation protection and interference experiments indicate that the enzyme makes major groove contacts with the first base 5' and the second base 3' to the dimer . These data are consistent with photolyase binding in the major groove over a 4-6-bp region . However, major groove contacts cannot be of major significance in substrate recognition as the enzyme binds equally well to a thymine dimer in a 44-base long single strand DNA and protects a 10-nucleotide long region around the dimer from DNase I digestion . It is therefore concluded that the unique configuration of the phosphodiester backbone in the strand containing the pyrimidine dimer, as well as the cyclobutane ring of the dimer itself are the important structural determinants of the substrate for recognition by photolyase. J Biol Chem, 1987 Sep 25, 262(27), 13180 - 7 DNase I footprint of ABC excinuclease; Van Houten B et al.; The incision and excision steps of nucleotide excision repair in Escherichia coli are mediated by ABC excinuclease, a multisubunit enzyme composed of three proteins, UvrA, UvrB, and UvrC . To determine the DNA contact sites and the binding affinity of ABC excinuclease for damaged DNA, it is necessary to engineer a DNA fragment uniquely modified at one nucleotide . We have recently reported the construction of a 40 base pair (bp) DNA fragment containing a psoralen adduct at a central TpA sequence (Van Houten, B., Gamper, H., Hearst, J . E., and Sancar, A . (1986a) J . Biol . Chem . 261, 14135-14141) . Using similar methodology a 137-bp fragment containing a psoralen-thymine adduct was synthesized, and this substrate was used in DNase I-footprinting experiments with the subunits of ABC excinuclease . It was found that the UvrA subunit binds specifically to the psoralen modified 137-bp fragment with an apparent equilibrium constant of K8 = 0.7 - 1.5 X 10(8) M-1, while protecting a 33-bp region surrounding the DNA adduct . The equilibrium constant for the nonspecific binding of UvrA was Kns = 0.7 - 2.9 X 10(5) M-1 (bp) . In the presence of the UvrB subunit, the binding affinity of UvrA for the damaged substrate increased to K8 = 1.2 - 6.7 X 10(8) M-1 while the footprint shrunk to 19 bp . In addition the binding of the UvrA and UvrB subunits to the damaged substrate caused the 11th phosphodiester bond 5' to the psoralen-modified thymine to become hypersensitive to DNase I cleavage . These observations provide evidence of an alteration in the DNA conformation which occurs during the formation of the ternary UvrA.UvrB.DNA complex . The addition of the UvrC subunit to the UvrA.UvrB.DNA complex resulted in incisions on both sides of the adduct but did not cause any detectable change in the footprint . Experiments with shorter psoralen-modified DNA fragments (20-40 bp) indicated that ABC excinuclease is capable of incising a DNA fragment extending either 3 or 1 bp beyond the normal 5' or 3' incision sites, respectively . These results suggest that the DNA beyond the incision sites, while contributing to ABC excinuclease-DNA complex formation, is not essential for cleavage to occur. J Biol Chem, 1987 Sep 25, 262(27), 13147 - 54 Fluorescence resonance energy transfer studies on the proximity relationship between the intrinsic metal ion and substrate binding sites of Escherichia coli RNA polymerase; Wu FY et al.; DNA-dependent RNA polymerase from Escherichia coli contains 2 mol of zinc/mol of holoenzyme (alpha 2 beta beta' sigma) with one zinc each in the beta and beta' subunits . A new method to substitute selectively the zinc in the beta subunit was developed by the inactivation of RNA polymerase with 0.25 M NaNO3, 1 M NaCl, 1 mM diaminocyclohexane tetraacetic acid, and 0.1 mM dithiothreitol followed by reconstitution with Co(II), Cd(II), or Cu(II) . The hybrid Co-Zn, Cd-Zn, or Cu-Zn RNA polymerase thus obtained retains, respectively, 91, 88, and 50% enzyme activity of the reconstituted Zn-Zn RNA polymerase . Co-Zn RNA polymerase exhibits absorption maxima at 395 and 465 nm, and Cu-Zn RNA polymerase at 637 nm (epsilon = 815 M-1 cm-1) . 1-Aminonaphthalene-5-sulfonic acid (AmNS) derivatives of ATP, UTP, and dinucleoside monophosphates (diNMPs), UpA or ApU, were synthesized with AmNS attached to NTP via a gamma-phosphoamidate bond or to diNMPs via a 5'-secondary amine linkage . Since the fluorescence emission maxima of (5'-AmNS)UpA, (gamma-AmNS)ATP, and (gamma-AmNS)UTP at 445, 464, and 464 nm, respectively, when excited at 340 nm, overlap the 465-nm absorption band of Co-Zn RNA polymerase, the spatial relationship between fluorescence substrate analogs and the intrinsic Co(II) in Co-Zn RNA polymerase was studied by fluorescence resonance energy transfer technique . The fluorescence of the initiator, (5'-AmNS)UpA, and elongator, (gamma-AmNS)UTP, of the RNA chain, was quenched 20.3 and 7.1%, by the addition of saturation concentration of Zn-Zn RNA polymerase, and 21.3 and 14.7%, respectively, by the addition of template, poly(dA-dT) . The fluorescence of (5'-AmNS)UpA and (gamma-AmNS)UTP was quenched 81.8 and 80.6%, respectively, by the addition of the saturation concentration of Co-Zn RNA polymerase in the absence of template, and 82.7 and 82.9% in the presence of template . On the basis of respective Ro values of 21.3 and 21.9 A for the (5'-AmNS)UpA-Co and (gamma-AmNS)UTP-Co pairs, the distances from Co(II) to the initiation site and to the elongation site were calculated to be 17.4 and 17.5 A, respectively, in the absence and 17.2 and 17.4 A in the presence of template. J Biol Chem, 1987 Sep 25, 262(27), 13081 - 5 A mutation that alters the nucleotide specificity of elongation factor Tu, a GTP regulatory protein; Hwang YW et al.; A single amino acid substitution (Asp to Asn) at position 138 of Escherichia coli elongation factor Tu (EF-Tu) was introduced in the tufA gene clone by oligonucleotide site-directed mutagenesis . The mutated tufA gene was then expressed in maxicells . The properties of {35S}methionine-labeled mutant and wild type EF-Tu were compared by in vitro assays . The Asn-138 mutation greatly reduced the protein's affinity for GDP; however, this mutation dramatically increased the protein's affinity for xanthosine 5'-diphosphate . The mutant protein forms a stable complex with Phe-tRNA and xanthosine 5'-triphosphate, which binds to ribosomes, whereas it does not form a complex with Phe-tRNA and GTP (10 microM) . These results suggest that in EF-Tu.nucleoside diphosphate complexes, amino acid residue 138 must interact with the substituent on C-2 of the purine ring . Thus, in wild type EF-Tu, Asp-138 would hydrogen bond to the 2-amino group of GDP, and in the mutant EF-Tu, Asn-138 would form an equivalent hydrogen bond with the 2-carbonyl group of xanthosine 5'-diphosphate . Aspartic acid 138 is conserved in the homologous sequences of all GTP regulatory proteins . This mutation would allow one to specifically alter the nucleotide specificity of other GTP regulatory proteins. J Biol Chem, 1987 Sep 25, 262(27), 13044 - 9 Construction of a lethal mutation in the synthesis of the major acidic phospholipids of Escherichia coli; Heacock PN et al.; In order to determine if the major acidic phospholipids of Escherichia coli are essential to the organism, we constructed a null allele (pgsA30) of the pgsA gene thus rendering the organism incapable of synthesizing phosphatidylglycerol or cardiolipin . In strains carrying the pgsA30 allele cell viability, synthesis of gene product and the ability to synthesize the two major acidic phospholipids were dependent on the presence of a functional copy of the pgsA gene carried on a plasmid which was temperature-sensitive for replication . Growth ceased at the temperature restrictive for plasmid replication when the acidic phospholipid content dropped to about 10% of wild type levels which is slightly higher than the level reported in cells carrying the pgsA3 allele in a genetic background derived from strain SD12; the latter cells, which are capable of synthesizing low levels of acidic phospholipids, were previously shown to have no abnormal growth phenotype (Miyazaki, C., Kuroda, M., Ohta, A., and Shibuya, I . (1985) Proc . Natl . Acad . Sci . U . S . A . 82, 7530-7534) . The pgsA30 allele, unlike the pgsA3 allele, could not support growth in strain SD12 . Neither allele could support growth in two other independently derived strains of E . coli . Therefore, there is a direct dependence of cell viability on a functional pgsA gene product . Strain SD12 appears to contain a suppressor which allows cells with a reduced capability to synthesize acidic phospholipid (pgsA3 allele) to grow, but cannot support growth in cells with a complete lack of synthetic capability (pgsA30 allele). J Biol Chem, 1987 Sep 25, 262(27), 12926 - 9 Tertiary structure of histidine-containing protein of the phosphoenolpyruvate:sugar phosphotransferase system of Escherichia coli; el-Kabbani OA et al.; The tertiary structure of the histidine-containing phosphocarrier protein (HPr) of the Escherichia coli phosphoenolpyruvate:sugar phosphotransferase system has been determined by x-ray diffraction at 2.8-A resolution . Initially, a partial structure was fitted to the multiple isomorphous replacement map and then least-squares refined by the Konnert/Hendrickson restrained parameter method (Konnert, J . H., and Hendrickson, W . A . (1980) Acta Crystallogr . A36, 344-350) and finally, a subsequent map was computed by use of the phase combination method of Read (Read, R . J . (1986) Acta Crystallogr . A42, 140-149) . More of the protein structure was located in the latter map . The procedure of model building, least-squares refinement, and electron density map recalculation was repeated until the tertiary structure of HPr was obtained . The overall structure of HPr consists of four beta-strands, three helical regions, and four beta-turns . At the active center, the His15 imidazole interacts with one oxygen atom of the alpha-carboxyl C terminus of the polypeptide chain; the conserved Arg17 side chain interacts with the other oxygen atom of the alpha-carboxyl C terminus as well as with the side chain of Glu85 . This is the first x-ray analysis of a protein of the phosphoenolpyruvate:sugar phosphotransferase system . Furthermore, this work represents a protein structure which has been solved by starting with a model that represented only one-third of the scattering matter. Science, 1987 Sep 25, 237(4822), 1614 - 8 Evidence for dispensable sequences inserted into a nucleotide fold; Starzyk RM et al.; Previous experimental results along with the structural modeling presented indicate that a nucleotide fold starts in the amino-terminal part of Escherichia coli isoleucyl-transfer RNA synthetase, a single chain polypeptide of 939 amino acids . Internal deletions were created in the region of the nucleotide fold . A set of deletions that collectively span 145 contiguous amino acids yielded active enzymes . Further extensions of the deletions yielded inactive or unstable proteins . The three-dimensional structure of an evidently homologous protein suggests that the active deletions lack portions of a segment that connects two parts of the nucleotide fold . Therefore, the results imply that removal of major sections of the polypeptide that connects these two parts of the fold does not result in major perturbation of the nucleotide binding site. J Immunol Methods, 1987 Sep 24, 102(2), 183 - 6 A rapid immunological spot test for the identification of proteins in covalently linked protein-nucleic acid complexes; Gulle H et al.; A method is described for the rapid immunological identification of proteins in studies of multicomponent systems . In this case the system is the E . coli ribosome, and the ribosomal proteins to be identified are covalently attached to fragments of labelled ribosomal RNA as a result of chemical cross-linking procedures . Antisera raised against the individual ribosomal proteins are spotted onto a nitrocellulose sheet, and an aliquot of the covalent complex under test is added to each antibody spot . After suitable washing procedures, a positive reaction with one or other of the antisera is visualized by autoradiography of the labelled RNA moiety attached to the antibody via the ribosomal protein . Amounts of protein as low as 10 pg can readily be detected. Biochim Biophys Acta, 1987 Sep 24, 915(2), 188 - 98 Immunochemical properties of D-amino-acid oxidase; Gavazzi E et al.; Antiserum against homogeneous hog kidney D-amino-acid oxidase (D-amino-acid: oxygen oxidoreductase (deaminating), EC 1.4.3.3) was elicited in rabbits, and monospecific antibodies were prepared by affinity chromatography . The antibodies inhibited up to 90% of hog D-amino-acid oxidase activity, and 100% of the enzyme could be immunoprecipitated . The antibodies inhibited both holoenzyme and reconstituted apoprotein to a similar degree, indicating that they did not interfere with the FAD-binding site of the protein . The antibodies inhibited D-amino-acid oxidase activity from other mammalian species to a similar degree, while the enzyme activities from birds, amphibians, fishes and yeast were inhibited and immunoprecipitated to lower extents . In immunoblotting experiments, after SDS-polyacrylamide gel electrophoresis, the antibodies recognized a single band of about 40 kDa in all the species analyzed, and the entity of the signal was inversely related to the phylogenetic distance from mammals . The antibodies did not inhibit D-alanine dehydrogenase activity from Escherichia coli, but gave positive bands in immunoblotting. Biochemistry, 1987 Sep 22, 26(19), 5997 - 6004 Limited proteolysis alters the photoaffinity labeling of adenosine 3',5'-monophosphate dependent protein kinase II with 8-azidoadenosine 3',5'-monophosphate; Bubis J et al.; Photoaffinity labeling of the regulatory subunits of cAMP-dependent protein kinase with 8-azidoadenosine 3',5'-monophosphate (8-N3cAMP) has proved to be a very specific method for identifying amino acid residues that are in close proximity to the cAMP-binding sites . Each regulatory subunit contains two tandem cAMP-binding sites . The type II regulatory subunit (RII) from porcine heart was modified at a single site, Tyr-381 {Kerlavage, A., & Taylor, S.S . (1980) J . Biol . Chem . 255, 8483-8488} . When a proteolytic fragment of this RII subunit was photolabeled with 8-N3cAMP, two sites were covalently modified . One site corresponded to Tyr-381 and, thus, was analogous to the native RII . The other site of modification was identified as Tyr-196, which is not labeled in the native protein . Photoaffinity labeling was carried out in the presence of various analogues of cAMP that show a preference for one of the two tandem cAMP-binding sites . These studies established that the covalent modification of Tyr-381 was derived from 8-N3cAMP that was bound to the second cAMP-binding site (domain B) and that covalent modification to Tyr-196 was due to 8-N3cAMP that was bound to the first cAMP-binding site (domain A) . These sites of covalent modification have been correlated with a model of each cAMP-binding site on the basis of the crystal structure of the catabolite gene activator protein (CAP), which is the major cAMP-binding protein in Escherichia coli. Biochemistry, 1987 Sep 22, 26(19), 6188 - 94 Promoter recognition by Escherichia coli RNA polymerase: effects of base substitutions in the -10 and -35 regions; Szoke PA et al.; We have constructed the PRM promoter of phage lambda and eight variants, which represents intermediates in the conversion of this promoter to one that has complete homology to the consensus sequences in the -10 and -35 regions . The in vivo activity of these promoters was determined from the beta-galactosidase or galactokinase activities in cells harboring plasmids, in which the cloned promoters were driving the expression of these genes . Additionally, the kinetics of the interaction of Escherichia coli RNA polymerase with the same series of promoters was measured as a function of RNA polymerase concentration . This allowed the overall rate of functional or open complex formation to be dissected into the equilibrium constant for binding of the polymerase to form a closed promoter complex and the rate of subsequent isomerization to yield the open complex . The following conclusions can be drawn from the data presented: (1) The consensus sequence is optimal for promoter function both in vivo and in vitro . (2) Alterations of the -10 and -35 regions have similar effects on the kinetics of RNA polymerase binding in vitro; with one exception, the same holds for promoter activity in vivo . (3) The in vitro rate of RNA polymerase binding to a promoter is solely determined by the number of positions at which its -10 and -35 regions match the consensus promoter sequence . The functional importance of a match does not appear to be determined by the sequence conservation at the particular position . (4) The extent to which a particular base change affects the kinetic parameters depends on the sequence of the promoter into which it is introduced. Biochemistry, 1987 Sep 22, 26(19), 5989 - 96 Thermodynamics of active-site ligand binding to Escherichia coli glutamine synthetase; Ginsburg A et al.; Active-site ligand interactions with dodecameric glutamine synthetase from Escherichia coli have been studied by calorimetry and fluorometry using the nonhydrolyzable ATP analogue 5'-adenylyl imidodiphosphate (AMP-PNP), L-glutamate, L-Met-(S)-sulfoximine, and the transition-state analogue L-Met-(S)-sulfoximine phosphate . Measurements were made with the unadenylylated enzyme at pH 7.1 in the presence of 100 mM KCl and 1.0 mM MnCl2, under which conditions the two catalytically essential metal ion sites per subunit are occupied and the stoichiometry of active-site ligand binding is equal to 1.0 equiv/subunit . Thermodynamic linkage functions indicate that there is strong synergism between the binding of AMP-PNP and L-Met-(S)-sulfoximine (delta delta G' = -6.4 kJ/mol) . In contrast, there is a small antagonistic effect between the binding of AMP-PNP and L-glutamate (delta delta G' = +1.4 kJ/mol) . Proton effects were negligible (less than or equal to 0.2 equiv of H+ release or uptake/mol) for the different binding reactions . The binding of AMP-PNP (or ATP) to the enzyme is entropically controlled at 303 K with delta H = +5.4 kJ/mol and delta S = +150 J/(K.mol) . At 303 K, the binding of L-glutamate (delta H = -22.2 kJ/mol) or L-Met-(S)-sulfoximine {delta H = -45.6 kJ/mol with delta Cp approximately equal to -670 +/- 420 J/(K.mol)} to the AMP-PNP.Mn.enzyme complex is enthalpically controlled with opposing delta S values of -29 or -46 J/(K.mol), respectively . The overall enthalpy change is negative and the overall entropy change is positive for the simultaneous binding of AMP-PNP and L-glutamate or of AMP-PNP and L-Met-(S)-sulfoximine to the enzyme . For the binding of the transition-state analogue L-Met-(S)-sulfoximine phosphate (which inactivates the enzyme by blocking active sites), both enthalpic and entropic contributions also are favorable at 303 K {delta G' approximately equal to -109 and delta H = -54.8 kJ/mol of subunit and delta S approximately equal to +180 J/(K.mol)}. J Mol Biol, 1987 Sep 20, 197(2), 373 - 4 Crystallographic characterization of recombinant human interleukin 2; Fujishima A et al.; Recombinant human interleukin 2 produced by Escherichia coli was purified to homogeneity and crystallized after being separated from methionyl interleukin 2 . Crystals suitable for structural studies have been obtained by the seed enlargement technique, using the method of vapor diffusion with ammonium sulfate as the precipitant at pH 4.6 . The space group is P2(1)2(1)2 with cell dimensions a = 49.2 A, b = 87.6 A and c = 32.4 A . The asymmetric unit contains one molecule of the protein . From preliminary results, the crystals are moderately stable to X-rays and produce measurable reflections to a resolution of about 2.2 A . The diffraction data for the native crystals have been collected on a diffractometer at 2.4 A resolution. J Mol Biol, 1987 Sep 20, 197(2), 195 - 204 Mechanism for chromosome and minichromosome segregation in Escherichia coli; Helmstetter CE et al.; A mechanism for the segregation of chromosomes and minichromosomes into daughter cells during division of Escherichia coli is presented . It is based on the idea that the cell envelope contains a large number of sites capable of binding to the chromosomal replication origin, oriC, and that a polymerizing DNA strand becomes attached to one of the sites at initiation of a round of replication . The attachment sites are distributed throughout the actively growing cell envelope, i.e . lateral envelope and septum, but not in the existing cell poles . This asymmetric distribution of oriC attachment sites accounts for the experimentally observed non-random chromosome and minichromosome segregation, and for the variation in the degree of non-random segregation with cell strain and growth rate . The multi-site attachment concept also accounts for the unstable maintenance of minichromosomes. Biochem Pharmacol, 1987 Sep 15, 36(18), 2945 - 50 Phosphorolytic cleavage of 2-fluoroadenine from 9-beta-D-arabinofuranosyl-2-fluoroadenine by Escherichia coli . A pathway for 2-fluoro-ATP production; Huang P et al.; 2-Fluoroadenine (F-Ade) is a metabolite of 9-beta-D-arabinofuranosyl-2-fluoroadenine (F-ara-A) that may be involved in the development of toxic side effects from this anticancer drug . The liberation of F-Ade from F-ara-A has been examined in different biological systems . Extracts of Escherichia coli but not mammalian cells or tissues catalyzed the conversion of F-ara-A to F-Ade with apparent Km and Vmax values of 1350 microM and 7.7 nmol/min/mg protein respectively . This reaction depended on the presence of phosphate and was inhibited by purine nucleosides in a competitive manner, indicating that the enzyme responsible for the conversion is purine nucleoside phosphorylase . After incubation of intact bacteria with 100 microM {3H}F-ara-A, {3H}F-Ade was the same percentage of cellular radioactivity as in the medium, but it was only one-tenth the concentration of F-ara-A in the cells . In contrast, the cellular concentration of 2-fluoro-ATP was 20-fold greater than that of F-ara-A-5'-triphosphate . These results suggest that F-ara-A entered the bacteria intact and was phosphorolytically cleaved to liberate F-Ade, which would have been either anabolized to the toxic triphosphate or excreted . The latter pathway would provide a route by which F-Ade might be absorbed into the host circulation. Biochem Biophys Res Commun, 1987 Sep 15, 147(2), 565 - 71 Pyridoxal 5'phosphate binding site of Escherichia coli beta cystathionase and cystathionine gamma synthase comparison of their sequences; Martel A et al.; The phosphopyridoxyl peptides of beta cystathionase and cystathionine gamma synthase of Escherichia Coli were identified after reduction, carboxymethylation and proteolysis of the holoenzymes . Their comparison with those obtained from rat liver gamma cystathionase (Fearon, C.W., Rodkey, J.A . and Abeles R.H . 1982 . Biochemistry 21 3790-3794.) showed a high degree of homology between the three PLP binding sites with the presence of the tripeptide sequence: Thr-Lys(Pxy)-Tyr in their structure . This homology suggests that these enzymes of methionine metabolism have probably the same origin. J Biol Chem, 1987 Sep 15, 262(26), 12812 - 20 Electron microscopic visualization of the RecA protein-mediated pairing and branch migration phases of DNA strand exchange; Register JC 3rd et al.; The RecA protein of Escherichia coli will drive the pairing and exchange of strands between homologous DNA molecules in a reaction stimulated by single-stranded binding protein . Here, reactions utilizing three homologous DNA pairs which can undergo both paranemic and plectonemic joining were examined by electron microscopy: supertwisted double-stranded (ds) DNA and linear single-stranded (ss) DNA, linear dsDNA and circular ssDNA, and linear dsDNA and colinear ssDNA . Several major observations were: (i) with RecA protein bound to the DNA, plectonemic joints were ultrastructurally indistinguishable from paranemic joints; (ii) complexes which appeared to be joined both paranemically and plectonemically were present in these reactions in roughly equal numbers; and (iii) in complexes undergoing strand exchange, both DNA partners were often enveloped within a RecA protein filament consisting of hundreds of RecA protein monomers and several kilobases of DNA . These observations suggest that, following RecA protein-ssDNA filament formation, strand exchange proceeds by a pathway that can be divided structurally into three phases: pairing, envelopment/exchange, and release of the products. J Biol Chem, 1987 Sep 15, 262(26), 12647 - 53 Purification and properties of the MalT protein, the transcription activator of the Escherichia coli maltose regulon; Richet E et al.; Expression of the Escherichia coli maltose regulon is controlled by MalT, a transcriptional activator (Mr = 102,288) encoded by the malT gene . Activation of transcription depends on the presence of the inducer, maltotriose . Using an in vitro transcription/translation assay to monitor the protein, we have purified MalT in native form from MalT-overproducing bacteria . The purified protein is able to promote transcription from different MalT-controlled promoters in well-defined in vitro systems . Maltotriose and the MalT protein suffice to stimulate initiation of transcription at malPp by the E . coli RNA polymerase holoenzyme . In contrast, both MalT protein and cAMP receptor protein are required with their respective effectors, maltotriose and cyclic AMP, for activation of malEp . These data are in agreement with in vivo observations . In addition, we present evidence that MalT is an ATP-binding protein, a result suggesting that ATP may play a role in transcription initiation. J Biol Chem, 1987 Sep 15, 262(26), 12434 - 7 Expression of cDNAs encoding the precursor and the mature form of chicken mitochondrial aspartate aminotransferase in Escherichia coli; Jaussi R et al.; Both the precursor and the mature form of chicken mitochondrial aspartate aminotransferase were synthesized in Escherichia coli . The precursor was found to sediment quantitatively together with insoluble cell material . In contrast, mature mitochondrial aspartate aminotransferase could be readily extracted from the cells and was indistinguishable from the enzyme isolated from chicken heart in all respects tested: specific activity 230 units mg-1; Mr 2 X 45,000; pI greater than 9; NH2-terminal sequence SSWWSHVEMG, the initiator methionine having been removed by the bacteria . Thus, the polypeptide chain representing mature mitochondrial aspartate aminotransferase is an autonomous folding unit which attains its functional spatial structure independently of the presence of the prepiece, trans-membrane passage, and proteolytic processing. J Biol Chem, 1987 Sep 15, 262(26), 12403 - 5 Tyrosine 70 increases the coenzyme affinity of aspartate aminotransferase . A site-directed mutagenesis study; Toney MD et al.; The crucial step in enzymatic transamination is the tautomerization of aldimine/ketimine intermediates, formed between the pyridoxyl coenzyme and the amino/keto acid substrate, which is catalyzed primarily by the active site residue Lys-258 (Malcolm, B . A., and Kirsch, J . F . (1985) Biochem . Biophys . Res . Commun . 132, 915-921; W . L . Finlayson and J . F . Kirsch, in preparation) . Tyr-70 is localized in close proximity to Lys-258 and, in addition, forms a hydrogen bond with the coenzyme phosphate . Tyr-70 has been postulated to have an important role in the tautomerization (Kirsch, J . F., Eichele, G., Ford, G . C., Vincent, M . G., Jansonius, J . N., Gehring, H., and Christen, P . (1984) J . Mol . Biol . 174, 497-525) . This hypothesis has now been tested by the construction and analysis of a mutant Escherichia coli aspartate aminotransferase in which Tyr-70 has been changed to Phe (Y70F) . Y70F retains at least 15% of the maximal activity of the wild type enzyme (WT) (kcat = 170 +/- 15 s-1 for WT versus greater than or equal to 26 +/- 3 s-1 for Y70F and shows increased Michaelis constants for both substrates (KmAsp = 2.5 +/- 0.4 mM; Km alpha Kg = 0.59 +/- 0.08 mM for WT versus KmAsp = 3.9 +/- 0.3 mM; Km alpha Kg = 2.70 +/- 0.02 mM for Y70F (where alpha Kg is alpha-ketoglutarate) ) . The spectrophotometrically determined pK a values of the internal aldimines formed between pyridoxal 5'-phosphate (PLP) and Lys-258 are identical for WT and Y70F . In assays where excess L-aspartate and excess PLP are incubated with either WT or Y70F, the mutant enzyme converts the free PLP to free pyridoxamine 5'-phosphate 80-fold faster than WT (k = (3.75 +/- 0.23) X 10(-2)s-1 for Y70F versus (4.90 +/- 0.02) X 10(-4)s-1 for WT) . Y70F also converts free pyridoxamine 5'-phosphate to free PLP faster than WT . Thus, Y70F dissociates coenzyme more readily than does WT . It therefore appears that the role of Tyr-70 is mainly in preventing the dissociation of the coenzyme from the enzyme . Tyr-70 does not function in an essential chemical step. J Biol Chem, 1987 Sep 15, 262(26), 12722 - 7 Monoclonal antibodies specific for the tau subunit of the DNA polymerase III holoenzyme of Escherichia coli . Use to demonstrate that tau is the product of the dnaZX gene and that both it and gamma, the dnaZ gene product, are integral components of the same enzyme assembly; Hawker JR Jr et al.; We have established two murine hybridoma cell lines that secrete monoclonal antibodies directed against the tau subunit of the DNA polymerase III holoenzyme of Escherichia coli . Both antibodies have been purified and identified to be of the IgG1 class . Competition assays indicate that they bind to two distinct portions of the tau subunit . These antibodies have been used to demonstrate that tau is an integral part of all DNA polymerase III holoenzyme assemblies and that tau is the product of the dnaZX gene . Both of the antibodies react only with tau, not with gamma, the other protein product of the dnaZX gene . Immunoprecipitation studies demonstrated that tau is contained within the same enzyme assemblies as gamma (dnaZ protein) . This observation is discussed in the light of the DNA polymerase III holoenzyme functioning as an asymmetric dimer, capable of coordinating leading with lagging strand replication. Int J Cancer, 1987 Sep 15, 40(3), 403 - 7 Protection of cynomolgus monkeys against infection by human T-cell leukemia virus type-I by immunization with viral env gene products produced in Escherichia coli; Nakamura H et al.; Protection against human T-cell leukemia virus type-I (HTLV-I) infection in cynomolgus monkeys, achieved by immunizing the animals with env gene products of HTLV-I produced in Escherichia coli, was evaluated . Four monkeys that had been immunized with the env product produced antibody against HTLV-I gp68 and gp46, and their sera were found to cause strong inhibition of syncytium formation of a cat fibroblast cell line induced by HTLV-I . Immunized and non-immunized monkeys were challenged with live MT-2 cells, a high HTLV-I-producer cell line . After challenge, all the control non-immunized monkeys were infected with HTLV-I, as judged by the frequent detection of HTLV-I-antigens in cultures of their peripheral blood mononuclear cells (PBMC), whereas no antigens were recovered from PBMC of immunized monkeys . These results indicate that humoral immunity against HTLV-I-envelope protein elicited by immunization with the polypeptides synthesized in bacteria protected the monkeys against primary infection with HTLV-I. Eur J Biochem, 1987 Sep 15, 167(3), 533 - 40 Reaction of tryptophanyl-tRNA synthetase from beef pancreas with periodate-oxidized ATP; Fournier M et al.; Tryptophanyl-tRNA synthetase from beef pancreas reacts with periodate-oxidized ATP according to biphasic kinetics . A rapid phase involves two groups of the protein, presumably lysine side-chains . The slow phase corresponds to the reaction of a larger number of groups . The time-course of the partial losses of the ATP-PPi isotopic exchange and of the aminoacylation activities of the enzyme follow the labelling of the two fast-reacting groups . However, the ability of the enzyme to form a bis(tryptophanyladenylate)-enzyme complex is not lost after reaction of these two groups with the reagent . The affinity for ATP is also unaffected by this initial labelling of the protein, as seen from the Km values of this substrate in the ATP-PPi isotopic exchange reaction . These data suggest that, in this fast initial reaction, oxidized ATP reacts neither with specific ATP-binding groups of the enzyme nor with any major catalytic residue of the tryptophan-activation site . In contrast with this first step, the further slow labelling of lysine residues leads to a disappearance of the aminoacylation ability of the enzyme, while it does not further affect the ATP-PPi exchange activity . The behaviour of beef tryptophanyl-tRNA synthetase during derivatization with oxidized ATP is therefore at variance with that which has been described for the homologous E . coli enzyme. Biochem Biophys Res Commun, 1987 Sep 15, 147(2), 778 - 86 Role of superoxide in radiation-killing of Escherichia coli and in thymine release from thymidine; Lin WS et al.; The role of superoxide and hydroxyl radicals in gamma-radiation-killing of Escherichia coli K12 was studied in aerated suspensions supplemented with formate, phosphate, superoxide dismutase, catalase and saturated with nitrous oxide . Nitrous oxide, which converts e-aq to .OH, caused decreased radiosensitivity . On the other hand, formate, which results in conversion of .OH to .O2-, resulted in an increased radiosensitivity . The results implicated .O2- as a major cause of radiation-mediated cell-killing . The addition of the enzymes, superoxide dismutase or catalase to the E . coli suspensions prior to and during irradiation had no effect on cell survival, indicating that the biologically significant site of generation and action of .O2- is an intracellular one . Further studies were undertaken to examine the role of superoxide in DNA damage . The release of thymine from the DNA base, thymidine was studied as a result of gamma-irradiation and of chemically generated superoxide (using KO2 in dimethyl sulfoxide) . Thymine was identified by HPLC and mass spectrometry . C-13 NMR analysis of the reaction mixture of thymidine with KO2 in dimethyl sulfoxide provided evidence for attack of .O2 at the ribosyl Cl' atom. J Biol Chem, 1987 Sep 15, 262(26), 12843 - 50 DNA damage at thymine N-3 abolishes base-pairing capacity during DNA synthesis; Huff AC et al.; 3-Methylthymine was synthesized into DNA copolymers and deoxynucleoside triphosphate to study its effect on DNA synthesis by the Klenow fragment of Escherichia coli polymerase I and avian myeloblastosis virus reverse transcriptase . Both polymerases were greatly inhibited by template 3-methylthymine . In response to 3-methylthymine, misincorporation of dTTP increased slightly, but occurred only at low levels consistent with spontaneous misincorporation in vitro . Surprisingly, template 3-methylthymine resulted in a striking decrease in background misincorporation, relative to normal incorporation by the Klenow fragment, of dGTP and, to a lesser extent, of dATP and dCTP . The incorporation of 3-methyl-dTTP into DNA was studied using DNA sequencing technology . The Klenow fragment failed to incorporate 3-methyl-dTTP even at 1 mM . Reverse transcriptase incorporated 3-methyl-dTTP opposite adenine, cytosine, and thymine, but at only about 1/40,000th the efficiency of complementary deoxynucleoside triphosphate incorporation . Furthermore, synthesis generally stalled at sites of 3-methyl-thymine incorporation . From these results, we conclude that damage at the central hydrogen-bonding position of thymine abolishes its base-pairing capabilities during DNA synthesis. J Biol Chem, 1987 Sep 15, 262(26), 12541 - 9 Enzymatic conversion of glutamate to delta-aminolevulinic acid in soluble extracts of Euglena gracilis; Mayer SM et al.; Glutamate was converted to the chlorophyll and heme precursor delta-aminolevulinic acid in soluble extracts of Euglena gracilis . delta-Aminolevulinic acid-forming activity depended on the presence of native enzyme, glutamate, ATP, Mg2+, NADPH or NADH, and RNA . The requirement for reduced pyridine nucleotide was observed only if, prior to incubation, the enzyme extract was filtered through activated carbon to remove firmly bound reductant . Dithiothreitol was also required for activity after carbon treatment . delta-Aminolevulinic acid formation was stimulated by RNA from various plant tissues and algal cells, including greening barley leaves and members of the algal groups Chlorophyta (Chlorella vulgaris, Chlamydomonas reinhardtii), Rhodophyta (Cyanidium caldarium), Cyanophyta (Anacystis nidulans, Synechocystis sp . PCC 6803), and Prochlorophyta (Prochlorothrix hollandica), but not by RNA derived from Escherichia coli, yeast, wheat germ, bovine liver, and Methanobacterium thermoautotrophicum . E . coli glutamate-specific tRNA was inhibitory . Several of the RNAs that did not stimulate delta-aminolevulinic acid formation nevertheless became acylated when incubated with glutamate in the presence of Euglena enzyme extract . RNA extracted from nongreen dark-grown wild-type Euglena cells was about half as stimulatory as that from chlorophyllous light-grown cells, and RNA from aplastidic mutant cells stimulated only slightly . delta-Aminolevulinic acid-forming enzyme activity was present in extracts of light-grown wild-type cells, but undetectable in extracts of aplastidic mutant and dark-grown wild-type cells . Gabaculine inhibited delta-aminolevulinic acid formation at submicromolar concentration . Heme inhibited 50% at 25 microM, but protoporphyrin IX, Mg-protoporphyrin IX, and protochlorophyllide inhibited only slightly at this concentration. J Biol Chem, 1987 Sep 15, 262(26), 12393 - 6 RNase H activity associated with bacterially expressed reverse transcriptase of human T-cell lymphotropic virus III/lymphadenopathy-associated virus; Hansen J et al.; The reverse transcriptase polymerase of the human T-cell lymphotropic virus/lymphadenopathy-associated virus has been cloned into an expression vector and expressed in Escherichia coli . Two polypeptides of 66 and 51 kDa molecular mass are detectable in polymerase-expressing bacterial lysates with human patient sera . They are processed from a short-lived 120-kDa polyprotein precursor equivalent to a region consisting of polymerase, protease, and endonuclease . The 51 kDa protein appears to originate from the 66-kDa molecule; additional processing products are 32- and 15-kDa proteins . The bacterially expressed polymerase is enzymatically active and exhibits the template specificities, ion requirements, and response to inhibitors of the authentic enzyme . It was purified by DEAE-cellulose-, phosphocellulose-, and poly(rC)-agarose column chromatography followed by glycerol density gradient centrifugation . It copurifies with an RNase H activity, suggesting the existence of a virus-coded DNA polymerase-RNase H complex . The purified bacterial enzyme allows a safe large-scale screening for inhibitors of both activities. FEBS Lett, 1987 Sep 14, 221(2), 194 - 8 Variations with position of replication errors due to exonuclease warm-up; Lecomte PJ et al.; A.A mismatch errors occurring during poly(dA) replication with the Klenow fragment of E . coli DNA polymerase I have been quantified . The A/T ratio measured for chains extended by 1-25 nucleotides decreases by a factor of at least 15 from beginning to end . The deduced true error rate may decrease by a factor of 2.5 at each successive nucleotide addition . When ddATP is used instead of dATP, the ddA/T ratio indicates little variation of the misincorporation probability with position . Thus, the accuracy improvement in the first case is due to a warm-up of the proofreading function. Nucleic Acids Res, 1987 Sep 11, 15(17), 6883 - 97 A homogeneous nucleic acid hybridization assay based on strand displacement; Vary CP; A homogeneous nucleic acid hybridization assay which is conducted in solution and requires no separation steps is described . The assay is based on the concept of strand displacement . In the strand displacement assay, an RNA "signal strand" is hybridized within a larger DNA strand termed the "probe strand", which is, in turn, complementary to the target nucleic acid of interest . Hybridization of the target nucleic acid with the probe strand ultimately results in displacement of the RNA signal strand . Strand displacement, therefore, causes conversion of the RNA from double to single-stranded form . The single-strand specificity of polynucleotide phosphorylase (EC 2.7.7.8) allows discrimination between double-helical and single-stranded forms of the RNA signal strand . As displacement proceeds, free RNA signal strands are preferentially phosphorolyzed to component nucleoside diphosphates, including adenosine diphosphate . The latter nucleotide is converted to ATP by pyruvate kinase(EC 2.7.1.40) . Luciferase catalyzed bioluminescence is employed to measure the ATP generated as a result of strand displacement. Nucleic Acids Res, 1987 Sep 11, 15(17), 6827 - 41 Random cloning of bent DNA segments from Escherichia coli chromosome and primary characterization of their structures; Mizuno T; A simple method for the selective detection of DNA segments containing a sequence-directed static bend was developed . Two-dimensional polyacrylamide gel electrophoresis performed at two different temperatures (60 degrees C and 10 degrees C) can effectively separate a bent DNA from a mixture of normal DNA . Using this method, a bank of plasmids carrying bent DNA inserts from the E . coli total chromosome was constructed . The primary characterization of a set of bent DNA segments randomly cloned from E . coli was presented. Biochim Biophys Acta, 1987 Sep 11, 925(3), 341 - 6 Studies on the mechanism of Escherichia coli heat-stable enterotoxin-induced diarrhoea in mice; Goyal J et al.; The unidirectional fluxes of Na+, Cl- and Ca2+ and activities of calmodulin in the intestinal microvillar core were studied in Escherichia coli heat-stable enterotoxin-treated mice . There was net secretion of Na+ and Cl- in toxin-treated animals, while in control animals there was net absorption of these ions . In both control and experimental animals, there was net absorption of Ca2+; however, the absorption was significantly higher (P less than 0.01) in experimental animals when compared to controls . In the presence of Ca2+-ionophore, there was a net secretion of Na+ and Cl- in controls, while the Ca2+-ionophore could not cause any change in the fluxes of these ions in experimental animals . The activity of calmodulin was significantly higher (P less than 0.01) in experimental animals . Verapamil, a calcium channel blocker, and trifluoperazine, a calmodulin inhibitor, reversed the effects of Ca2+-ionophore and heat-stable enterotoxin . These studies demonstrate that the toxin acts through Ca2+-calmodulin, and secretion of Na+ and Cl- in experimental animals is due to an increase in calcium absorption and an increase in calmodulin activity in the intestinal microvillar core. Cell, 1987 Sep 11, 50(6), 885 - 99 An antitermination protein engages the elongating transcription apparatus at a promoter-proximal recognition site; Barik S et al.; As a transcriptional activator, the N protein of phage lambda acts to suppress transcription termination by recognizing a promoter-proximal site, nut, which is separated from the terminators by thousands of base pairs . We demonstrate here that N interacts with the elongating RNA polymerase in transit through the boxB domain of nut . This interaction leads to the stable association of N as an integral component of the transcription apparatus . During subsequent elongation, N translocates along with polymerase through several defined terminators positioned beyond nut . Therefore, by being an operon-specific subunit of the transcription apparatus, N presumably prevents the interaction of polymerase with termination signals. Cell, 1987 Sep 11, 50(6), 901 - 8 Cellular factors couple recombination with growth phase: characterization of a new component in the lambda site-specific recombination pathway; Thompson JF et al.; Here we characterize FIS (factor for inversion stimulation), a new cellular component of the lambda site-specific recombination pathway . This host protein binds to a specific region in the lambda attP overlapping the Xis binding sites and can bind cooperatively with Xis to these sites . FIS stimulates lambda excision up to 20-fold in vitro in the presence of suboptimal Xis concentrations, but has no effect in the presence of saturating Xis; FIS has no effect on integrative recombination . FIS can replace one Xis molecule in a series of cooperative and competitive interactions but cannot carry out excision in the absence of Xis . FIS's role in the regulation of recombination has been inferred from in vivo modification of DNA . In exponentially growing cells the lambda FIS site is fully occupied, whereas in stationary-phase cells this binding site is vacant. Nucleic Acids Res, 1987 Sep 11, 15(17), 6813 - 26 Promotion of double-strand break repair by human nuclear extracts preferentially involves recombination with intact homologous DNA; Lopez B et al.; Parameters of DNA double strand break (dsb) repair catalysed by human nuclear extract were analysed using, as substrate, the replicative form (RF) of M13 mp8 in which a single double strand break (dsb) was introduced by restriction . After incubation with the extract, the dsb repair was estimated by the ability of the incubated RF to produce plaques following transfection into JM 109 (Rec A-) bacteria . The possibility of recombination with a purified fragment from M13 mp8 RF enhances up to 20 times the plaquing ability of the RF . The repair by recombination occurs under several conditions: i) the break in the RF must be located in the region of homology with the fragment . ii) the fragment has to be intact in the region corresponding to the break in the RF . iii) a minimal length of homology between the region surrounding the dsb in the RF, and the fragment is required . The in vitro reaction is ATP dependent and dNTP's partially dependent . Dephosphorylation of the free ends in the RF decreases the repair by ligation but is without effect on the recombination. Biochim Biophys Acta, 1987 Sep 10, 893(2), 373 - 7 Binding protein-dependent transports in 2-oxo acids dehydrogenase mutants of Escherichia coli; Richarme G; The binding protein-dependent transport of ribose, galactose and maltose are reduced in several 2-oxo acids dehydrogenase mutants of Escherichia coli . The results suggest an implication of the pyruvate dehydrogenase complex and to a lesser extent of the 2-oxoglutarate dehydrogenase complex in the energization of these transport systems. Biochim Biophys Acta, 1987 Sep 10, 893(2), 289 - 95 Electron flow and heme-heme interaction between cytochromes b-558, b-595 and d in a terminal oxidase of Escherichia coli; Hata-Tanaka A et al.; The ESR signals of the cytochromes in the Escherichia coli terminal oxidase cytochrome d complex were studied at cryogenic temperature . The intensities and g values of the rhombic high-spin signals changed when the electronic state of cytochrome d was changed from the oxidized state to the reduced or oxygen-binding or CO-binding state . These rhombic signals were therefore assigned to cytochrome b-595, which is located near cytochrome d in the oxidase complex . This assignment was supported by the finding that the Em value of the rhombic signals differed from that of cytochrome d (Hata, A . et al . (1985) Biochim . Biophys . Acta 810, 62-72) . Photolysis and ligand-exchange experiments with the reduced CO complex of the oxidase were performed in the presence of oxygen at -140 degrees C . The ESR spectra of three intermediate forms trapped by controlled low temperatures were detected . These forms were designated as the oxygen-binding intermediate I (ESR-silent), oxygen-binding intermediate II (giving ESR signals at g = 6.3, 5.5 and 2.15), and oxygen-binding intermediate III (giving signals at g = 6.3, 5.5 and 6.0) . From these results, electron flow in the cytochrome d complex is proposed to proceed in the order, cytochrome b-558----cytochrome b-595----cytochrome d--