Escherichia coli

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----O2 . A model of the mechanism of four-electron chemistry for oxidation of ubiquinol-8 and formation of H2O by the cytochrome d complex is presented.

Biochemistry, 1987 Sep 8, 26(18), 5796 - 803
Enzyme II of the Escherichia coli phosphoenolpyruvate-dependent phosphotransferase system: protein-protein and protein-phospholipid interactions; Robillard GT et al.; The mannitol-specific enzyme II (EII), purified free of phospholipid, exhibits a concentration dependence in its specific activity with P-HPr and mannitol as the donor and acceptor substrates, respectively . This concentration dependence, previously observed only in the case of mannitol----mannitol phosphate exchange reaction, indicates that an oligomeric form of the enzyme is responsible for catalyzing the phosphorylation reaction (P-HPr + mannitol----mannitol-P + HPr) as well as the exchange reaction . Kinetic analysis revealed that the monomeric enzyme has a much lower specific activity than the associated species . The specific activity can be increased by raising the steady-state level of phosphorylation of EII and also by adding phospholipid, demonstrating that phosphorylation and the binding of phospholipid facilitate the association process . Kinetic measurements and fluorescence energy transfer measurements demonstrate a strong preference of EII for phospholipids with specific head group and fatty acid composition.

Biochemistry, 1987 Sep 8, 26(18), 5666 - 71
Proton nuclear magnetic resonance studies on the wild-type and single amino acid substituted tryptophan synthase alpha-subunits; Yutani K et al.; In order to elucidate the effect of single amino acid substitutions on the conformation of the tryptophan synthase alpha-subunit from Escherichia coli in solution, 1H NMR spectra of the wild-type and mutant proteins were measured at various pHs . Two of the four His C2-proton resonances of the alpha-subunit were assigned to two His residues at positions 92 and 146 by using a mutant protein with Thr substituted for the His at position 92 . The replacement did not affect the conformation of the protein significantly . The proton resonances of all the Tyr residues in the aromatic region could be picked up from other resonance peaks, employing the wild-type alpha-subunit deuterated at all of the Phe residues . On comparison of the spectra of the wild-type protein with those of the mutant protein with Met substituted for the Glu at position 49, it was concluded that the substitution affects only the residues close to the substituted residue at acidic pH but that a larger part of the protein is affected at alkaline pH . NOE experiments showed that the five Tyr residues, four of which are located in the proximity of position 49, are close to one another . The present results are discussed in the light of the conformational stability of the protein.

Biochemistry, 1987 Sep 8, 26(18), 5657 - 65
Effects of mutation on the downfield proton nuclear magnetic resonance spectrum of the 5S RNA of Escherichia coli; Gewirth DT et al.; The imino proton spectra of several mutants of the 5S RNA of Escherichia coli are compared with that of the wild type . Three of the variants discussed are point mutations, and the fourth is a deletion mutant lacking bases 11-69 of the parent sequence, all obtained by site-directed mutagenesis techniques . The spectroscopic effects of mutation are limited in all cases, and the differences between normal and mutant spectra can be used to make or confirm the assignments of resonances . Several new assignments in the 5S spectrum are reported . Spectroscopic differences due to sequence differences permit the products of single genes within the 5S gene family to be distinguished and their fates followed by NMR.

Biochemistry, 1987 Sep 8, 26(18), 5616 - 25
Homology-dependent changes in adenosine 5'-triphosphate hydrolysis during recA protein promoted DNA strand exchange: evidence for long paranemic complexes; Schutte BC et al.; As a first step in DNA strand exchange, recA protein forms a filamentous complex on single-stranded DNA (ssDNA), which contains stoichiometric (one recA monomer per four nucleotides) amounts of recA protein . recA protein monomers within this complex hydrolyze ATP with a turnover number of 25 min-1 . Upon introduction of linear homologous duplex DNA to initiate strand exchange, this rate of ATP hydrolysis drops by 33% . The decrease in rate is complete in less than 2 min, and the rate of ATP hydrolysis then remains constant during and subsequent to the strand exchange reaction . This drop is completely dependent upon homology in the duplex DNA . In addition, the magnitude of the drop is linearly dependent upon the length of the homologous region in the linear duplex DNA . Linear DNA substrates in which pairing is topologically restricted to a paranemic joint also follow this relationship . Taken together, these properties imply that all of the available homology in the incoming duplex DNA is detected very early in the DNA strand exchange reaction, with the linear duplex DNA paired paranemically with the homologous ssDNA in the complex throughout its length . The results indicate that paranemic joints can extend over thousands of base pairs . We note elsewhere {Pugh, B . F., & Cox, M . M . (1987b) J . Biol . Chem . 262, 1337-1343} that this duplex acquires resistance to digestion by DNase with a much slower time course (30 min), which parallels the progress of strand exchange . Together these results imply that the duplex DNA is paired with the ssDNA but remains outside the nucleoprotein filament . Finally, the results also support the notion that ATP hydrolysis occurs throughout the recA nucleoprotein filament.

J Mol Biol, 1987 Sep 5, 197(1), 131 - 40
Kinetics of the spontaneous transient unfolding of a native protein studied with monoclonal antibodies . Monomer/dimer transition in the tryptophan-synthase beta 2 subunit; Chaffotte AF et al.; Included in a series of monoclonal antibodies obtained after immunization with the native holo beta 2 subunit of tryptophan synthase of Escherichia coli (EC 4.2.1.20), are some that interact preferentially with a denatured state of the antigen (Friguet et al., 1984) . A study of the equilibrium and kinetic characteristics of the interaction of one of these antibodies with native apo beta 2 (i.e . free of pyridoxal 5'-phosphate) and with one of its proteolytic domains is reported here . The antibody is shown to interact strongly with the isolated domain in accordance with a simple equilibrium . In the presence of native beta 2, the antibody binds exclusively to the dissociated beta-monomer . The interaction of this antibody with native apo beta 2 is used to determine the equilibrium and kinetic constants of the monomer-dimer equilibrium . The values obtained are 4.5 X 10(-8) M for the equilibrium constant and 7.9 X 10(-3) s-1 for the rate constant of the dissociation of apo beta 2 into beta-monomers.

J Biol Chem, 1987 Sep 5, 262(25), 12325 - 31
NAD(P)H:flavin oxidoreductase of Escherichia coli . A ferric iron reductase participating in the generation of the free radical of ribonucleotide reductase; Fontecave M et al.; The active form of one subunit of Escherichia coli ribonucleotide reductase (protein B2) contains an organic free radical localized to tyrosine 122 of its polypeptide chain . When this radical is scavenged, e.g . by treatment with hydroxyurea, the enzyme is inactivated (protein B2/HU) . E . coli contains an enzyme system consisting of at least three proteins that in the presence of NADPH, FMN, dithiothreitol, and oxygen introduce the tyrosyl radical into B2/HU (Eliasson, R., Jornvall, H., and Reichard, P . (1986) Proc . Natl . Acad . Sci . U . S . A . 83, 2373-2377) . One of the three proteins was identified as superoxide dismutase . We now identify a second protein, previously provisionally named Fraction c, as an NAD(P)H:flavin oxidoreductase (flavin reductase) . After 4,000-fold purification the protein moved as a single band on sodium dodecyl sulfate gel electrophoresis with a molecular weight of 28,000-29,000 . The enzyme contained no flavin but reduced riboflavin, FMN, and FAD by NADH, or riboflavin and FMN by NADPH . It is a powerful ferric iron reductase . We propose that its complementing activity during radical generation involves participation in the reduction of the ferric iron center of protein B2/HU . Radical formation is then linked to the reoxidation of iron by oxygen . The flavin reductase may also participate in other aspects of iron metabolism of E . coli.

J Biol Chem, 1987 Sep 5, 262(25), 12323 - 4
A preliminary crystallographic study of recombinant human interleukin 1 beta; Gilliland GL et al.; Recombinant human interleukin 1 beta which is expressed in Escherichia coli has been crystallized by the method of vapor diffusion using ammonium sulfate as the precipitant . The space group is P4(1) or P4(3) with a = b = 55.0 A and c = 77.1 A and one molecule in the asymmetric unit . The crystals diffract to beyond 2.4 A and are suitable for a three-dimensional x-ray structure determination.

J Biol Chem, 1987 Sep 5, 262(25), 11920 - 6
The role of translocation in ribosomal accuracy . Translocation rates for cognate and noncognate aminoacyl- and peptidyl-tRNAs on Escherichia coli ribosomes; Gast FU et al.; The ribosomal translocation, as measured in vitro by peptide formation on poly(U)-programmed Escherichia coli ribosomes in the presence of ternary complex, deacylated tRNA or N-acetyl-Phe-tRNA, and elongation factor G, is the rate-limiting step of protein synthesis . Elongation factor G stimulates the spontaneous translocation by a factor of about 500 . N-Acetyl-Phe-Phe-tRNA(Phe E . coli) is translocated with a rate constant of 1-2 s-1 at 25 degrees C . Translocation of N-acetyl-Phe-Phe-tRNA(Phe yeast) and N-acetyl-Phe-Leu-tRNA(Leu E . coli) under identical conditions proceeds with a rate by about a factor of 2 and 10, respectively, more slowly . The translocation rate, therefore, is influenced by the nature of the tRNAs in the A-site . We can show, furthermore, that also the tRNA in the P-site, and presumably in the E-site as well, influences the rate of translocation . Reduced rates of translocation of noncognate peptidyl-tRNAs are accompanied by preferential dissociation of these tRNAs at the beginning of the translation of a mRNA.

J Biol Chem, 1987 Sep 5, 262(25), 12337 - 43
Primary structure of the Escherichia coli folC gene and its folylpolyglutamate synthetase-dihydrofolate synthetase product and regulation of expression by an upstream gene; Bognar AL et al.; The nucleotide sequence of the gene for folylpoly-gamma-glutamate synthetase-dihydrofolate synthetase (folC) has been determined . The deduced amino acid sequence codes for a protein of Mr 45,380 and contains regions with homology to the A and B regions of ATP-binding sites . The folC gene is adjacent to a gene located 70 base pairs upstream of the initiation codon for folC . The nucleotide sequence of this upstream gene was also determined . Deletion of the upstream gene sequences from recombinant plasmids containing the folC gene results in about a 100-fold decrease in plasmid-dependent folylpolyglutamate synthetase activity, suggesting that the major promoter for folC is 5' to the upstream gene . The upstream gene codes for a protein of Mr 33,346, which is expressed in maxicells and amplified in cells containing the upstream gene in recombinant pUC8 plasmids . Expression of the upstream gene in maxicells was greater than that of folC, as determined by the intensity of 35S-labeled proteins after sodium dodecyl sulfate-gel electrophoresis . A region of dyad symmetry exists between the coding sequences of the two genes which may code for a transcription termination signal and be responsible for the attenuation of the expression of the folC gene relative to the upstream gene . The folC gene is located about 1 kilobase upstream of the purF gene region at 50 min on the Escherichia coli map . The function of the upstream gene product is unknown . It contains sequences with homology to metal-binding domains in nucleic acid-binding proteins . A new purification procedure for obtaining large quantities of folylpolyglutamate synthetase-dihydrofolate synthetase is described.

J Biol Chem, 1987 Sep 5, 262(25), 12332 - 6
The function of superoxide dismutase during the enzymatic formation of the free radical of ribonucleotide reductase; Fontecave M et al.; An enzyme system from Escherichia coli activates an inactive form of ribonucleotide reductase by transforming a tyrosine residue of the enzyme into a cationic free radical . The process requires NAD(P)H, a flavin, dithiothreitol, and oxygen and at least three proteins . After purification to near homogeneity two of the proteins were identified as superoxide dismutase and NAD(P)H:flavin oxidoreductase (Fontecave, M., Eliasson, R., and Reichard, P . (1987) J . Biol . Chem . 262, 12325-12331) . The nature of the third protein, provisionally named Fraction b, is unknown . The flavin reductase is believed to reduce the ferric iron center of the ribonucleotide reductase as a prerequisite for radical generation . Here we demonstrate that the flavin reductase under aerobic conditions generates superoxide anions which inactivate ribonucleotide reductase . Superoxide dismutase protects the enzyme or a sensitive intermediate formed during the generation of the tyrosyl radical from the harmful effects of superoxide . Hydrogen peroxide, formed by superoxide dismutase, is also harmful . In this case, catalase present in Fraction b might protect the system . Fraction b has, however, an additional unknown function in the overall process of radical generation.

J Mol Biol, 1987 Sep 5, 197(1), 89 - 100
ATP synthase from bovine mitochondria . The characterization and sequence analysis of two membrane-associated sub-units and of the corresponding cDNAs; Walker JE et al.; ATP synthase from bovine mitochondria is a complex of 13 different polypeptides, whereas the Escherichia coli enzyme is simpler and contains eight subunits only . Two of the bovine subunits, b and d, which had not been characterized, have been isolated from the purified enzyme . Subunits with sizes corresponding to bovine subunits b and d are evident in preparations of the enzyme from mitochondria of other species . Partial protein sequences have been determined by direct methods . On the basis of some of this information, two oligonucleotide mixtures, 17 and 18 bases in length, have been synthesized and used as hybridization probes in the isolation of clones of the cognate cDNAs . The sequences of the two proteins have been deduced from their DNA sequences . Subunit b is 214 amino acid residues in length and has a free N terminus . Subunit d is 160 amino acid residues long . Its N-terminal alanine is blocked by an N-acetyl group, as demonstrated by fast atom bombardment mass spectrometry of N-terminal peptides . The sequence near the N terminus of the b subunit is made predominantly of hydrophobic residues, whereas the remainder of the protein is mainly hydrophilic . This N-terminal hydrophobic region may be folded into an alpha-helical structure spanning the lipid bilayer . In its distribution of hydrophobic residues, this protein resembles the b subunits of ATP synthase complexes in bacteria and chloroplasts . The b subunit in E . coli forms an important structural link between the extramembrane sector of the enzyme F1, and the intrinsic membrane domain, FO . It is proposed that the bovine mitochondrial subunit b serves a similar function . If this is so, the mitochondrial enzyme, as the chloroplast ATP synthase, contains equivalent subunits to all eight of those that constitute the E . coli enzyme . Subunit d has no extensive hydrophobic sequences, and is not apparently related to any subunit described in the simpler ATP synthases in bacteria and chloroplasts.

J Mol Biol, 1987 Sep 5, 197(1), 37 - 46
High-affinity L-arabinose transport operon . Nucleotide sequence and analysis of gene products; Scripture JB et al.; The nucleotide sequence of the "high-affinity" L-arabinose transport operon has been determined 3' from the regulatory region and found to contain three open reading frames designated araF, araG and araH . The first gene 3' to the regulatory region, araF, encodes the 23-residue signal peptide and the 306-residue mature form of the L-arabinose binding protein (33,200 Mr) . The binding protein, which has been described elsewhere, is hydrophilic, soluble and found in the periplasm of Escherichia coli . This gene is followed by an intragenic space of 72 nucleotides, which contains a region of dyad symmetry 23 nucleotides long capable of forming an 11-member stem-loop . The second gene, designated araG, contains an open reading frame capable of encoding an equally hydrophilic protein containing 504 residues (55,000 Mr) . Following a 14-nucleotide spacer, which does not appear to have any secondary structure, the third open reading frame, herein designated araH, is capable of encoding a hydrophobic protein containing 329 residues (34,000 Mr) that can only be envisioned as having an integral membrane location . 3' to araH there is a T-rich region containing a 24-nucleotide area of dyad symmetry centered 55 nucleotides from the termination codon . Analysis of the derived primary sequences of the araG and araH products indicates the nature and potential features of these components . The araG protein was found to possess internal homology between its amino and carboxyl-terminal halves, suggesting a common origin . The araG gene product has been shown to be homologous to the rbsA gene product, the hisP product, the ptsB product and the malK product, all of which presumably play similar roles in their respective transport systems . Putative ATP binding sites are observed within the regions of homology . The araH gene product has been shown to be homologous to the rbsC gene product, which is the first observed homology between two purported membrane proteins.

J Mol Biol, 1987 Sep 5, 197(1), 27 - 35
High-affinity L-arabinose transport operon . Gene product expression and mRNAs; Horazdovsky BF et al.; Various portions of the "high-affinity" L-arabinose transport operon were cloned into the plasmid expression vector pKK223-3 and the operon-encoded protein products were identified . The results indicate that three proteins are encoded by this operon . The first is a 33,000 Mr protein that is the product of the promoter-proximal L-arabinose binding protein coding sequence, araF . A 52,000 Mr protein is encoded by sequence 3' to araF and has been assigned to the araG locus . The sequence 3' to araG encodes a 31,000 Mr protein that has been assigned to the araH locus . Both the araG and araH gene products are localized in the membrane fraction of the cell, implying a role in the membrane-associated complex of the high-affinity L-arabinose transport system . Nuclease S1 protection studies indicate that two operon message populations are present in the cell, a full-length operon transcript and a seven- to tenfold more abundant binding protein-specific message . The relative abundance of these two message populations correlates with the differential expression of the binding protein and the membrane-associated proteins of the transport system.

J Biol Chem, 1987 Sep 5, 262(25), 11916 - 9
Novobiocin inhibits Xenopus transcription factor IIIA-DNA interactions; Fiser-Littell RM et al.; Novobiocin and coumermycin, inhibitors of the B subunit of Escherichia coli DNA gyrase, inhibit the binding of Xenopus transcription factor IIIA (TFIIIA) to the 5 S RNA gene . Nalidixic acid and oxolinic acid, inhibitors of the A subunit of DNA gyrase, have no effect on the TFIIIA-5 S RNA gene interaction . Novobiocin and coumermycin inhibit TFIIIA-dependent DNA renaturation . Novobiocin dissociates TFIIIA.5 S RNA gene complexes and TFIIIA.5 S RNA complexes (7 S particles) . Novobiocin induces TFIIIA aggregation, a phenomenon likely to be responsible for the inhibition of TFIIIA-DNA interactions . Novobiocin inhibition of TFIIIA can be reversed by dilution.

Science, 1987 Sep 4, 237(4819), 1197 - 201
Chemical conversion of a DNA-binding protein into a site-specific nuclease; Chen CH et al.; The tryptophan gene (trp) repressor of Escherichia coli has been converted into a site-specific nuclease by covalently attaching it to the 1,10-phenanthroline-copper complex . In its cuprous form, the coordination complex with hydrogen peroxide as a coreactant cleaves DNA by oxidatively attacking the deoxyribose moiety . The chemistry for the attachment of 1,10-phenanthroline to the trp repressor involves modification of lysyl residues with iminothiolane followed by alkylation of the resulting sulfhydryl groups with 5-iodoacetamido-1,10-phenanthroline . The modified trp repressor cleaves the operators of aroH and trpEDCBA upon the addition of cupric ion and thiol in a reaction dependent on the corepressor L-tryptophan . Scission was restricted to the binding site for the repressor, defined by deoxyribonuclease I footprinting . Since DNA-binding proteins have recognition sequences approximately 20 base pairs long, the nucleolytic activities derived from them could be used to isolate long DNA fragments for sequencing or chromosomal mapping.

Nature, 1987 Sep 3-9, 329(6134), 86 - 7
Site-directed mutation affecting polyomavirus capsid self-assembly in vitro; Garcea RL et al.; Nonequivalent bonding of identical protein subunits occurs in the polyomavirus capsid were identical pentameric capsomeres occupy both hexavalent and pentavalent positions in the icosahedral surface lattice . The polyomavirus major capsid protein VP1, purified after expression of the recombinant gene in Escherichia coli, has been isolated as capsomeres that self-assemble into capsid-like structures in vitro . The ability to switch bonding specificity in different symmetry environments therefore must be intrinsic to the VP1 molecule . In vitro self-assembly provides an assay for VP1 mutations affecting capsomere and capsid formation . We report here that a directed mutation in the VP1 expression vector, leading to a protein truncated at the carboxy terminus, results in a mutant VP1 that forms capsomeres, but not capsids, in the in vitro assembly assay . The carboxy terminus of VP1 therefore appears to be involved in the specific bonding responsible for the non-equivalent association of capsomeres.

Mol Biol (Mosk), 1987 Sep-Oct, 21(5), 1352 - 9
{Study of conformation characteristics of "X-form" of alternating polynucleotides by the method of slow 1H----3H transition}; Lesnik EA et al.; The rate constants of 1H----3H exchange between water and C8H-groups of purine residues of alternating polynucleotides: poly{d(A-C)}.poly{d(G-T)} and poly{d(A-T)}.poly{d(A-T)}, as well as Escherichia coli DNA, dAMP and dGMP, in solutions with high concentration (4.3 or 6 M) CsF, in water ethanol (60%) solution and (in comparison) in 0.15 M NaCl were determined at 25 degrees C . The 1H----3H exchange rate exchange rate constants for adenylic (kA) and guanylic (kG) residues of polynucleotides were compared with the corresponding constant for DNA and mononucleotides . It was shown that at conditions when poly{d(G-T)} and poly{d(A-T)}.poly{d(A-T)} exhibit the "X-form" CD spectrum, alteration of exchange rates in polynucleotides (approximately 2-fold increase in kA in CSF and approximately 1.5-fold decrease in kA and kG in 60% ethanol with 0.15 M NaCl) is due to the effect of solvents on the chemical reactivity of purine residues, but does not reflect a conformational transition . The analysis of these results allows us to conclude, that alternating polynucleotides under the above mentioned conditions retain roughly the conformations inherent in them in 0.15 M NaCl: poly{d(A-C)}.poly{d(G-T)} conformation in 4.3 m CsF or 60% ethanol differs only insignificantly from the "canonic" B-DNA, whereas the poly{d(A-T)}.poly{d(A-T)} conformation in 6 M CSF corresponds to B-alternating DNA.

Vaccine, 1987 Sep, 5(3), 215 - 8
Synergistic teratogenic effect produced in mice by whole cell pertussis vaccine; Au-Jensen M et al.; Pertussis whole cell bacterial vaccine was injected in mice during early pregnancy to disclose any teratogenic effect on the brain of the fetuses . Cytochalasin D by itself induced exencephaly in a dose dependent way in fetal mice . When pregnant mice received a single injection of pertussis vaccine on day 8 of gestation and a subteratogenic dose of cytochalasin D on days 8, 9 and 10 of gestation a synergistic teratogenic action of pertussis vaccine and cytochalasin D in mice was observed . When autopsy was performed after a further 9 to 10 days a significant number of brain malformations was found . In order to analyse which component in the vaccine might be responsible for the co-teratogenic effect, purified pertussis components, pertussis toxin and filamentous haemagglutinin were used in combination with cytochalasin D, but no malformations occurred . The same results were obtained by using diphtheria-tetanus-polio (DiTePol) vaccine and acellular pertussis component vaccine, whereas the use of whole cell typhoid vaccine resulted in a high rate of fetuses with exencephalies . Experiments with purified Bordetella pertussis and Escherichia coli lipopolysaccharides indicated that lipopolysaccharides in whole cell pertussis vaccine as well as in typhoid vaccine were the factors causing teratogenicity in fetal mice.

Br J Pharmacol, 1987 Sep, 92(1), 3 - 4
Increased intestinal formation of Paf in endotoxin-induced damage in the rat; Whittle BJ et al.; Platelet-activating factor (Paf) has been proposed as a mediator of the gastrointestinal damage in endotoxic shock . The formation of Paf in rat jejunal tissue, determined following extraction and bioassay on rabbit washed platelets has therefore been correlated with the induction of damage following endotoxin administration . Intravenous injection of E . coli endotoxin led to a time-dependent increase in the jejunal formation of Paf, which after 20 min was twenty fold greater than the control level . There was a significant correlation between elevated Paf release and intestinal hyperaemia and haemorrhage, thus supporting a role for Paf as a mediator of such damage.

Med Radiol (Mosk), 1987 Sep, 32(9), 26 - 31
{Comparative effectiveness of different types of radiation in the induction of gene mutations: basic and applied aspects}; Aleksandrov ID et al.; Large-scale radiation-genetic studies on bacterial cells (E . coli) and Drosophila (D . melanogaster) using methods like an analysis of mutations of some structural genes and assessment of the frequency of mutations with relation to survival have shown for the first time that the efficacy of neutrons (E = 0.85 MeV) in the induction of gene mutations in Pro- and Eukaryotae is much lower than that of weak ionizing radiation with the same survival . Some features of radiation mutagenesis in a Drosophila mutant c(3)G defective in genetic recombination were described . A low level of the frequency of radiation-induced mutations typical of this mutant was also characteristic for some E . coli rec--mutants . A fact earlier reported for bacteria consisting in a higher (than one could expect extrapolation of lethal-sublethal irradiation doses to small ones) frequency of gene mutations in the range of relatively small absorbed doses of gamma-irradiation (the survival rate being not lower than 80%) was also first established for Drosophila . The importance of these entirely new facts for the theory of mutations in radiation genetics are briefly discussed.

Cancer Lett, 1987 Sep, 36(3), 297 - 305
Azelaic acid as a competitive inhibitor of thioredoxin reductase in human melanoma cells; Schallreuter KU et al.; Azelaic acid has been shown to inhibit thioredoxin reductase (TR) at the surface of guinea pig and human skin, on cultures of human keratinocytes, melanocytes, melanoma cells, murine melanoma cells (Cloudman S91), and on purified enzymes from Escherichia coli, rat liver, and human melanoma . Human melanoma cells are more resistant to inhibition by azelaic acid than murine melanoma or human melanocytes . Kinetic studies with pure TRs indicate that azelaic acid is a reversible competitive inhibitor . Fluorescence spectroscopy has been used to show that azelaic acid does not interfere with electron transfer from NADPH to FAD on TR . However, azelaic acid does inhibit electron transfer from the dithiolate active site of this enzyme . Inhibition by azelaic acid is pH-dependent, requiring the dissociation of both carboxylate groups, and also the dissociation of the active site dithiol groups . Binding studies with {14C}azelaic acid at different pHs, indicate that inhibition is first due to the formation of a thioester on the active thiolate groups followed by transacylation of a basic amino acid residue in the active site . A comparative study of TR inhibition by C6, C9, C10 and C12 saturated dicarboxylic acids was also determined on guinea pig skin in vivo . These homologous dicarboxylic acids gave greater inhibition with increasing size (i.e . mol wt.).

Am J Physiol, 1987 Sep, 253(3 Pt 2), R425 - 33
Effect of naloxone on regional cerebral blood flow during endotoxin shock in conscious rats; Law WR et al.; Maintenance of cerebral blood flow (CBF) is vital during cardiovascular shock . Since opioids have been implicated in the pathophysiology of endotoxin shock and have been shown to alter cerebral perfusion patterns, we determined whether opioids were responsible for any of the changes in regional CBF observed during endotoxin shock and whether the use of naloxone might impair or aid in the maintenance of CBF . When blood flow (BF) is studied with microspheres in rats, the left ventricle of the heart is often cannulated via the right carotid artery . Questions have arisen concerning the potential adverse effects of this method on CBF in the hemisphere ipsilateral to the ligated artery . We measured right and left regional CBF by use of this route of cannulation . Twenty-four hours after cannulations were performed, flow measurements were made using radiolabeled microspheres in conscious unrestrained male Sprague-Dawley rats (300-400 g) before and 10, 30 and 60 min after challenging with 10 mg/kg Escherichia coli endotoxin (etx) or saline . Naloxone (2 mg/kg) or saline was given as a treatment 25 min post-etx . We found no significant differences between right and left cortical, midbrain, or cerebellar BF at any time in any treatment group . After etx, the whole brain received a large share of the depressed cardiac output . Thus global CBF was not significantly reduced below its pre-etx value, an effect unaltered by naloxone . Regionally, BF was reduced to cerebellum and midbrain by 30 min post-etx . Naloxone prevented this depression . No region was affected to a greater or lesser degree than others.(ABSTRACT TRUNCATED AT 250 WORDS)

Virology, 1987 Sep, 160(1), 162 - 8
Analysis of functional domains on reovirus cell attachment protein sigma 1 using cloned S1 gene deletion mutants; Nagata L et al.; Previously a reovirus (serotype 3) S1 gene cDNA was inserted into the lac cloning site of pUC13 and expressed in Escherichia coli to yield a sigma 1 fusion protein (F-sigma 1) capable of binding to mouse L fibroblasts and of agglutinating human red blood cells (S.A . Masri, L . Nagata, D . C . W . Mah, and P . W . K . Lee, 1986, Virology 149, 83-90) . To probe the functional domains on the sigma 1 protein, restriction enzymes which divide the S1 gene into four segments (5'-I-II-III-IV-3') of similar size were used to generate five in-frame deletion mutants (D1-D5) . Corresponding mutant forms of sigma 1 were expressed in E . coli and were assayed for (i) host cell (mouse L fibroblasts) binding activity; (ii) glycophorin (reovirus erythrocyte receptor) binding activity (R . W . Paul and P . W . K . Lee, 1987 . Virology 159, 94-101 and (iii) recognizability by a library of neutralizing monoclonal anti-sigma 1 antibodies . It was found that mutant sigma 1 forms with segment III or segment IV deleted did not exhibit any detectable L-cell binding activity, whereas mutants with these two segments intact (but lacking segment II or segments I and II) were capable of attaching to L-cell receptors, albeit with reduced efficiencies . On the other hand, only F-sigma 1, but none of the mutants, could bind immobilized glycophorin . These data clearly suggest that the host cell binding domain of sigma 1 is distinct from its hemagglutination domain . Also, the five neutralizing anti-sigma 1 monoclonal antibodies tested were all found to recognize epitopes on either the middle segments or the carboxy-terminal half of sigma 1.

Eur J Biochem, 1987 Sep 1, 167(2), 227 - 31
cDNA cloning and in vitro synthesis of the Dolichos biflorus seed lectin; Schnell DJ et al.; The Dolichos biflorus seed lectin contains two structurally related subunits . A cDNA library was constructed using RNA isolated from D . biflorus seeds actively synthesizing the seed lectin . The library was expressed in Escherichia coli using a lambda Charon 16 vector, and lectin-specific antiserum was used to isolate a seed lectin cDNA . Hybridization of the D . biflorus seed lectin cDNA to RNA isolated from seeds actively producing both lectin subunits identifies a single-size RNA of 1100 bases . An oligodeoxyribonucleotide probe, constructed from an amino acid sequence common to both lectin subunits, detects the same size RNA . Translation of seed mRNA in vitro and immunoprecipitation of translation products using a lectin-specific antiserum yields a single polypeptide of slightly higher molecular mass than the largest seed lectin subunit . This seed lectin precursor is indistinguishable from a polypeptide synthesized from mRNA hybrid selected by the seed lectin cDNA . These data support the existence of a single polypeptide precursor for both subunit types of the D . biflorus seed lectin and suggest that differences between the subunit types arise by posttranslational processing.

Crit Care Med, 1987 Sep, 15(9), 863 - 8
Effects of intravascular volume expansion on lung fluid balance in a canine model of septic shock; D'Orio V et al.; We tested the early effects of endotoxin on both the permeability of capillary membranes and microvascular pressure . One group of dogs (n = 8) were fluid loaded (30 ml/kg dextran-40) after having been subjected to a 2-h Escherichia coli endotoxin infusion (0.25 micrograms/kg X min) . A second control group of animals (n = 6) was submitted to a similar (25 ml/kg) volume loading over an equivalent 30-min period . We estimated extravascular lung water (EVLW), calculated the effective pulmonary capillary pressure, and determined the alveolar-capillary filtration coefficient (Kf) after volume loading . Only the septic animals consistently showed elevated EVLW values consistent with pulmonary edema . The results showed, however, that the Kf calculated for the dogs that received endotoxin was no different from that of control group (Kf = 0.005 ml/kg X min X mm Hg) . Instead, endotoxin constricted the pulmonary veins which led to a considerable rise in microvascular hydrostatic pressure above the level at which the lungs could not resist edema formation . We conclude that acute pulmonary edema that follows endotoxin insult and subsequent therapeutic volume replacement is due to an increased filtration force instead of an alteration in the microvascular permeability.

J Lab Clin Med, 1987 Sep, 110(3), 287 - 91
Kinetics of inhaled lipopolysaccharide in the guinea pig; Goto H et al.; The kinetics of endotoxin in the airway and the blood was studied after an inhalation exposure using guinea pigs . The amounts of endotoxin in the pulmonary lavage fluid, and in arterial and venous blood were determined . Forty minutes after the start of exposure to 400 micrograms/m3 of Escherichia coli 026:B6-LPS, 7.7 +/- 2.9 ng/ml of lipopolysaccharide (LPS) could be demonstrated in the pulmonary lavage fluid, and 60 minutes afterwards, the amount was 4.9 +/- 1.4 ng/ml . In the arterial blood, the amounts at 10 and 40 minutes of exposure, and 60 minutes afterwards were 21.2 +/- 23.8 pg/ml, 29.2 +/- 31.8 pg/ml, and 1.1 +/- 1.0 pg/ml, respectively . There was no significant difference of LPS content in the arterial and venous blood . The results demonstrate that the lung is a potent barrier for penetration of endotoxins into the body . The experimental model enables studies of how other exposures such as mineral dusts, tobacco smoke, or viral or bacterial infections might affect this defense.

Brain Behav Immun, 1987 Sep, 1(3), 216 - 30
Effects of Newcastle disease virus administration to mice on the metabolism of cerebral biogenic amines, plasma corticosterone, and lymphocyte proliferation; Dunn AJ et al.; Newcastle disease virus (NDV) administration to mice increased concentrations of plasma corticosterone, with a maximal effect at 8 h . This elevation of plasma corticosterone concentrations was not observed in hypophysectomized animals in which the completeness of the hypophysectomy was verified by functional tests . NDV administration consistently increased concentrations of free tryptophan in all brain regions examined (prefrontal cortex, hypothalamus, and brain stem) . It also caused an activation of cerebral catecholamine and indoleamine metabolism as determined by measurement of the amines and their catabolites . 3-Methoxy,4-hydroxyphenylethyleneglycol (MHPG), the major catabolite of norepinephrine (NE), homovanillic acid (HVA), a major catabolite of dopamine (DA), and 5-hydroxyindoleacetic acid (5-HIAA), the major catabolite of serotonin (5-HT), were all increased in both hypothalamus and brain stem . Ratios of catabolites to the parent amine, considered to be an index of utilization of the neurotransmitters, were increased for NE, DA, and 5-HT in the hypothalamus and for DA and 5-HT in the brain stem . This pattern of changes resembles that observed following stressors such as footshock or restraint . There were also significant increases of tryptophan, HVA, dihydroxyphenylacetic acid (DOPAC), and 5-HIAA in hypophysectomized relative to sham-operated mice . The NDV treatment also increased thymus weights and markedly decreased the proliferative responses of isolated spleen cells to phytohemagglutinin, concanavalin A, pokeweed mitogen, and Escherichia coli lipopolysaccharide . These changes were not caused by increased circulating corticosterone because they were present at equal magnitude in hypophysectomized mice . Thymosin alpha 1 concentrations in the plasma were not altered by NDV or hypophysectomy . These results indicate that administration of NDV to mice can initiate neurochemical and endocrine responses like those observed during stress and can also cause immunosuppression . They are thus consistent with the hypothesis that a virus can be a stressor.

Gene Anal Tech, 1987 Sep-Oct, 4(5), 105 - 10
A method for cloning mixtures of long, synthetic oligodeoxynucleotides; Carter-Muenchau P et al.; A method is described for cloning synthetic oligodeoxynucleotides, which can theoretically be of any length . The method requires only a single oligodeoxynucleotide strand and a vector with two unique restriction sites, one of which is for an enzyme that generates 3' protruding ends . A mixture of unpurified oligonucleotides containing a wild-type genetic regulatory sequence of the Escherichia coli gnd gene and two mutations of it was cloned into a plasmid carrying a gnd-lacZ protein fusion . Individual cloned oligonucleotides were readily identified by direct DNA sequencing of plasmid templates . The method is rapid, efficient, and has application to gene synthesis and site-directed mutagenesis.

Zhonghua Zhong Liu Za Zhi, 1987 Sep, 9(5), 379 - 81
{Pyometra and radiotherapy of cancer of the uterine cervix--analysis of 282 patients}; Yu GR et al.; From March 1958 to December 1980, 11284 patients with cancer of uterine cervix were treated with radiotherapy in our hospital . Among them, 282 were complicated with pyometra with an incidence of 2.5% . The incidence of pyometra was higher in patients treated by combined intracavitary with external irradiation than by other radiation methods . 74.1% was diagnosed during the manipulation of intracavitary therapy . Escherichia coli comprised 46.5% by culture . The 5 year survival rate was lower in patients with pyometra than without it . The patients who had a temperature over 38 degrees C and uterus enlarged to more than 6 weeks of pregnancy had a bad prognosis . It was even worse for patients whose pyometra did not heal or developed at the end of radiation . Endometrial biopsy was done in 36 patients with pyometra and cancer cells were seen in 55.6% of them . Endometrial biopsy positive rate reached to 77.8% in the patients whose pyometra was not cured or occurred after radiotherapy . This is the main reason of the poor prognosis . The authors believe that endometrial and cervical biopsy should be taken for cancer of uterine cervix complicated with pyometra; for biopsy positive patients during the treatment, the intracavitary dose should be increased; for positive patients after radiotherapy, operation is strongly indicated.

Acta Chir Scand, 1987 Sep, 153(9), 507 - 12
Intravenous versus intrapulmonary administration of corticosteroids in combination with fluid infusion in experimental septic shock; Ottosson J et al.; Three different routes of corticosteroid administration (intravenous (IV), intratracheal instillation (IT), and aerosol inhalation (AE)) were evaluated for the treatment of an experimental septic shock induced by E . coli, given intraperitoneally . The corticosteroids, dexamethasone and budesonide, were given alone and in combination with 3% albumin infusion . Shock induced a plasma volume loss to 69% of control levels . This was partially prevented by corticosteroids alone, and prevented completely by 3% albumin infusion . The two corticosteroids were identical with respect to prolongation of survival time and hematocrit changes, regardless of the route of administration . Plasma steroid concentrations of budesonide were similar after IT and IV administration . Increasing doses of corticosteroids up to 8 mg/kg significantly prolonged mean survival time from 9 to 13 hours . When antibiotics and 3% albumin infusions were combined, survival time increased to 15 hours . Corticosteroids added to antibiotics and 3% albumin increased 24-hour survival rate from 0 to 60% (p less than 0.001) . Intrapulmonary administration of corticosteroids is as effective as the intravenous route, and offers an important alternative, with obvious clinical implications . Further pharmacological modifications of the corticosteroids are necessary in order to evaluate local pulmonary versus the systemic effects.

Blood Rev, 1987 Sep, 1(3), 183 - 92
Role of chromosomal abnormalities in chronic lymphocytic leukemia; Gahrton G et al.; Chromosomal aberrations occur in both B-CLL and T-CLL . The polyclonal B-cell mitogens, in particular Epstein-Barr virus and lipopolysaccharide from E . coli, have been used successfully to reveal chromosomal abnormalities in 40-60% of patients with B-CLL, while T-cell mitogens have shown chromosomal aberrations in T-CLL . The most common clonal chromosomal aberration in B-CLL is an extra chromosome 12, alone or together with other abnormalities . Other common aberrations are 14q+, structural aberrations on 6, 11, 12 and 13 . Proto-oncogenes are frequently located close to breakpoints . The proto-oncogene c-K-ras is located on chromosome 12 and an abnormal transcript has recently been implicated in a subset of B-CLL-patients . An extra chromosome 12 as well as multiple chromosomal abnormalities in B-CLL appear to predict a less favourable prognosis . T-CLL is in most patients characterized by an inv(14), an extra 8q and structural abnormalities in chromosome 7 . The genes for the specific T-cell receptor as well as the immunoglobulin heavy chain are located on these chromosomes . Chromosomal aberrations appear to have pathogenetic importance in both B-CLL and T-CLL.

Comput Appl Biosci, 1987 Sep, 3(3), 223 - 7
Recognition of characteristic patterns in sets of functionally equivalent DNA sequences; Mengeritsky G et al.; An algorithm has been developed for the identification of unknown patterns which are distinctive for a set of short DNA sequences believed to be functionally equivalent . A pattern is defined as being a string, containing fully or partially specified nucleotides at each position of the string . The advantage of this 'vague' definition of the pattern is that it imposes minimum constraints on the characterization of patterns . A new feature of the approach developed here is that it allows a 'fair' simultaneous testing of patterns of all degrees of degeneracy . This analysis is based on an evaluation of inhomogeneity in the empirical occurrence distribution of any such pattern within a set of sequences . The use of the nonparametric kernel density estimation of Parzen allows one to assess small disturbances among the sequence alignments . The method also makes it possible to identify sequence subsets with different characteristic patterns . This algorithm was implemented in the analysis of patterns characteristic of sets of promoters, terminators and splice junction sequences . The results are compared with those obtained by other methods.

Mol Microbiol, 1987 Sep, 1(2), 143 - 50
Presence in the 'silent' terminus region of the Escherichia coli K12 chromosome of cryptic gene(s) encoding a new nitrate reductase; Bonnefoy V et al.; A cosmid complementing narG mutants defective in nitrate reductase activity was isolated from a genomic library of Escherichia coli . The restriction map of the insert differed from that of the narGHI operon . The new enzyme, termed NarZ, required molybdenum for activity . The expression of narZ was not affected by the factors controlling narGHI . Insertion mutations indicated that the narZ locus covered about 8 kb of DNA; narZ is located at 32.5 U on the chromosome, in the cotransduction gap near the replication terminus . Southern blot experiments under stringent conditions using narGHI or narZ DNA as probes revealed a large extent of homology, with a small area of very high homology . We propose that narZ and narGHI have descended from a common ancestor by gene duplication.

J Gen Microbiol, 1987 Sep, 133 ( Pt 9), 2531 - 8
Genetic analysis of conjugational recombination in Escherichia coli K12 strains deficient in RecBCD enzyme; Lloyd RG et al.; Conjugational recombination in Escherichia coli was investigated by comparing the effects of recN, recO, ruv and lexA mutations on the formation of recombinants in crosses with strains lacking RecBCD enzyme . The results presented reveal that recN and ruv mutations do not abolish residual recombination in a recB mutant, and have only a rather modest effect on recombination in recBC sbcA strains; in these respects they are quite different from recF, recJ and recO mutations . The differences between these two groups of genes are discussed in relation to the molecular exchanges needed to produce viable recombinants.

J Gen Microbiol, 1987 Sep, 133 ( Pt 9), 2411 - 20
An unmodified form of the ColE2 lysis protein, an envelope lipoprotein, retains reduced ability to promote colicin E2 release and lysis of producing cells; Pugsley AP et al.; Site-directed mutagenesis was used to replace the codon for the N-terminal cysteine residue of pColE2-P9-encoded mature lysis protein (CelB) by an arginine codon . In contrast to the wild-type CelB protein, the product of the mutated gene, which has an altered signal peptidase cleavage site, was neither processed nor acylated . However, the mutant protein retained sufficient residual activity to cause partial, Mg2+-suppressible lysis and could activate envelope phospholipase A1-A2 and promote colicin release, albeit with reduced efficiency compared to the wild-type protein . We propose that the uncleaved signal peptide of the mutant protein acts as the functional equivalent of the fatty acyl groups normally linked to the N-terminal cysteine residue of the wild-type protein, thereby anchoring the protein in the cell envelope where it exerts its various effects.

Postgrad Med J, 1987 Sep, 63(743), 767 - 75
Investigation of cereal toxicity in coeliac disease; Ciclitira PJ et al.; Coeliac disease is exacerbated by wheat gluten . A review of methods for the purification, identification and quantitation of wheat proteins to investigate coeliac disease is presented . Recent developments including amino acid sequencing and expression of wheat protein peptides in E . coli should permit characterization of the cereal peptide that exacerbates coeliac disease.

Antibiot Med Biotekhnol, 1987 Sep, 32(9), 683 - 5
{Chemotherapeutic effectiveness of mecillinam and dioxidine in an experiment using polyresistant Escherichia coli}; Antipov AV; Chemotherapeutic efficacy of mecillinam and dioxidine was studied on a model of Escherichia coli septicemia of albino mice caused by polyresistant variants of E . coli 675 . It was shown that the presence of RI plasmid in the bacterial cells markedly lowered the mecillinam chemotherapeutic efficacy whereas the presence of R64 plasmid did not change the drug efficacy as compared to the plasmid-free controls . It was noted that the presence of RI and R222 plasmids in the pathogen cells increased the dioxidine efficacy while pKM-101 plasmid had a contrary effect . Correlation between the level of dioxidine resistance of E . coli 675 in vitro and the drug chemotherapeutic efficacy in animals was observed.

J Biochem (Tokyo), 1987 Sep, 102(3), 607 - 12
Production of recombinant human pancreatic secretory trypsin inhibitor by Escherichia coli; Kikuchi N et al.; A synthetic gene for human pancreatic secretory trypsin inhibitor (PSTI) was fused to the coding sequence for the amino-terminal 135 amino acid residues of human interferon-gamma (IFN-gamma) by interposing a methionine codon sequence, and the resulting hybrid gene was efficiently expressed in Escherichia coli cells . Recombinant human PSTI (rHu-PSTI) was separated from the IFN-gamma/PSTI fused protein by cleavage at the methionine residue with cyanogen bromide . Finally, rHu-PSTI was purified by affinity chromatography on a bovine trypsin-CH-Sepharose 4B column . The amino acid composition, partial amino-terminal sequence, disulfide formation, human trypsin inhibitory activity, and immunoreactivity against rabbit anti-human PSTI serum of rHu-PSTI corresponded to those of the natural form.

J Biochem (Tokyo), 1987 Sep, 102(3), 503 - 11
Interaction of ras oncogene product p21 with guanine nucleotides; Hoshino M et al.; The nucleotide exchange reaction was observed with purified ras oncogene product p21 overproduced in Escherichia coli (Hattori, S . et al . (1985) Mol . Cell Biol . 5, 1449-1455) under various conditions . (NH4)2SO4 increased the rate of dissociation of bound GDP from c-rasH and v-rasH p21 . The dissociation kinetics were those of a first order reaction, and there was a linear relationship between the rate constant and the (NH4)2SO4 concentration . At any concentration of (NH4)2SO4, the exchange rate was faster with v-rasH p21 than that with c-rasH p21 . EDTA and (NH4)2SO4 synergetically stimulated the dissociation reaction . Nucleotide-free p21 was prepared by gel filtration on Sephadex G-25 in the presence of 5 mM EDTA and 200 mM (NH4)2SO4 at room temperature . The free p21 was quite thermolabile, but the addition of GDP or GTP completely protected p21 from thermal inactivation . The dissociation constants for GDP and GTP were determined with free p21 to be 8.9 and 8.2 nM, respectively, for v-rasH p21, and 1.0 and 2.6 nM for c-rasH p21 . In the presence of 200 mM (NH4)2SO4, these dissociation constants increased 3- to 12-fold.

Bioorg Khim, 1987 Sep, 13(9), 1186 - 93
{Duplication of a synthetic gene for human leukocyte interferon and its expression in polycistron mRNA with coupled translation system}; Kravchenko VV et al.; Using a chemically synthesised adapter, the coding part of an artificial gene for human leukocyte alpha 2 interferon (ifn-alpha 2) has been duplicated . The adapter contained a termination signal of the first gene (TAA) within the Shine-Dalgarno sequence of the second gene (TAAGGA), distance between the terminating codon and starting codon of the second gene being 11 nucleotides . In another case this distance was 69 nucleotides, with the same SD sequence . The expression of the tandems as a part of polycistrons has been studied under control of promoters Plac, (Ptrp)2 of E . coli, and PVIII of M13 phage . It was found that tandems of ifn-alpha 2 genes in polycistronic structures trp L-ifn-ifn and IX-VIII-ifn-ifn under control of promoters (Ptrp)2 and PVIII, respectively, provided high level of the interferon biosynthesis, thus differing from the tandem under Plac promoter control, which had only ifn-ifn translation coupling.

Biochem Int, 1987 Sep, 15(3), 645 - 52
Isolation of Escherichia coli release factor-2/ribosome complexes facilitated by efficient crosslinking and sensitive immunodetection of the ligand on the macromolecular particle; Kastner B et al.; Complexes between the protein ligand, release factor-2 (RF-2) and the 70S ribosome readily dissociate during purification . To stabilise the weak 70S.RF-2 complex the two components have been crosslinked using dimethyl suberimidate . A sensitive dot blot procedure employing RF-2 specific antibodies and detecting 0.1 ng of factor was developed to improve crosslinking yields . The proportion of the population of ribosomes carrying a release factor was increased several fold to at least 25% as estimated by immunoprecipitation . This combination of a sensitive immunoassay with an efficient crosslinking technique has general application in studies on the binding of a ligand with a macromolecular complex where an antibody against the ligand is available.

Acta Chir Scand, 1987 Sep, 153(9), 535 - 9
Treatment of experimental peritonitis in rats by transfer of peritoneal mononuclear cells from rats injected with semisoluble aminated glucan; Almdahl SM et al.; The efficacy of treatment with semisoluble aminated glucan (s.a.g.) and donor peritoneal mononuclear cells was investigated in two separate models of peritonitis (exogenous Escherichia coli challenge or caecal perforation) . Intraperitoneal administration of s.a.g . significantly protected against both forms of peritonitis . Our previous studies indicated this protective effect to be mediated by macrophage activation, and this was corroborated by the effect of injecting rats with s.a.g.-stimulated donor peritoneal cells (approximately 95% macrophages) immediately after induction of peritonitis . Increased bacterial clearance and survival time were achieved with this treatment as compared with rats injected with cells from saline-treated donors . Scanning electron microscopy demonstrated activation of macrophages from the s.a.g.-treated rats . The results provided further support for the concept that s.a.g . exerts its therapeutic effect by stimulation of macrophages.

Genetika, 1987 Sep, 23(9), 1705 - 7
{The ColIb-CA53 plasmid suppresses umuC- and umuD-mutations in Escherichia coli K-12}; Khmel' IA; The presence of the ColIa-CA53 plasmid in umuC and umuD mutant Escherichia coli K-12 cells restores their mutability under UV irradiation to a level that even exceeds that of the isogenic umuC+umuD+ strains, as well as increases their resistance to the lethal effects of UV irradiation . The ColIb-P9 plasmid which suppresses the umuC mutant phenotype, as we have shown earlier, acts in the same manner with respect to the umuD mutant cells . The results of the study demonstrate that both plasmids encode products that are functionally similar to those of the chromosomal genes umuC and umuD . The plasmids ColIa-CA53, ColIb-P9 and pKM101 are shown to have practically the same effect upon the mutagenesis and survival of the umuC, umuD mutant cells.

Res Vet Sci, 1987 Sep, 43(2), 137 - 42
Effects of a specific thromboxane synthetase inhibitor in equine endotoxaemia; Semrad SD et al.; Thromboxane A2 may play a major role in circulatory shock . In some species, thromboxane synthetase inhibitors have a beneficial effect on shock induced by endotoxin, trauma, sepsis and administration of arachidonate . In some shock models, however, results with thromboxane synthetase inhibitors have been conflicting . The effect of UK-38,485, a selective thromboxane inhibitor, was evaluated in ponies injected with endotoxin intraperitoneally . Four groups of ponies were used to compare the effects of endotoxin alone, UK-38,485 alone, treatment with UK-38,485 before endotoxin challenge and treatment with UK-38,485 after endotoxin challenge . Haematological, metabolic, eicosanoid and clinical responses in each group were evaluated . The results indicated that UK-38,485 is an effective inhibitor of thromboxane A2 generation following endotoxin challenge . Prostacyclin values were elevated compared with baseline in ponies administered UK-38,485 and endotoxin . However, prostacyclin values were not significantly different from those of ponies receiving endotoxin alone . Furthermore, UK-38,485 failed to attenuate the haematological, metabolic and clinical manifestations commonly seen in the pony after endotoxin challenge.

Pflugers Arch, 1987 Sep, 410(1-2), 220 - 2
Thermoregulation of the rabbit during the late phase of endotoxin fever; Vybiral S et al.; In the late phase of the fever occurring 120 or more min after i.v . injection of endotoxin (1 microgram/kg) to female rabbits, marked shifts of thresholds for respiratory evaporative heat loss and for peripheral vasodilatation to higher body core temperatures were observed . In contrast, the threshold body core temperature for cold thermogenesis was shifted downwards . As a result, the interthreshold zone was widened . Within the body temperature range of 37.4 to 39.9 degrees C neither heat production or heat loss mechanisms were operant and the body temperature was determined mainly by passive heat transfer between the body and the environment . Outside this zone, the sensitivities of the heat and cold defence activities to changes in body core temperature appeared to be unchanged.

Mol Biol (Mosk), 1987 Sep-Oct, 21(5), 1297 - 309
{Creation of artificial hybrid operons with partially overlapping genes to achieve an expression of heterologous genes in Escherichia coli cells}; Mashko SV et al.; A new method of optimization of foreign gene expression in E . coli, based on the construction of hybrid operons with partially overlapping genes is described . The partial overlapping of the translation termination and initiation sites in the formed operon must provide translational coupling of appropriate gene product synthesis . Such an approach has provided the synthesis of human interferon alpha F in E . coli cells under the control of the lacUV5-promotor up to about (3-4).10(7) units per liter of bacterial culture . The reinitiation of the distal gene translation is shown to take place in the intercistronic region . Substitution of the lacUV5 promotor by the more efficient tac one allowed to increase the synthesis level of interferon alpha F to (1-2).10(8) units per liter . The conclusion is made about the equimolarity of distal and proximal to the promotor genes products syntheses when the intercistronic region of E . coli trpE-trpD genes are used for translational coupling.

EMBO J, 1987 Sep, 6(9), 2843 - 7
The pAR5 mutation and the allosteric mechanism of Escherichia coli aspartate carbamoyltransferase; Cherfils J et al.; Mutation pAR5 replaces residues 145'-153' at the C terminus of the regulatory (r) chains of Escherichia coli ATCase by a new sequence of six residues . The mutated enzyme has been shown to lack substrate cooperativity and inhibition by CTP . Solution X-ray scattering curves demonstrate that, in the absence of ligands, its structure is intermediate between the T form and the R form . In the presence of N-phosphonacetyl-L-aspartate, the mutant is similar to the wild type . An examination of the crystal structure of unligated ATCase reveals that the mutated site is at an interface between r and catalytic (c) chains, which exists only in the T allosteric form . A computer simulation by energy minimization suggests that the pAR5 mutation destabilizes this interface and induces minor changes in the tertiary structure of r chains . The resulting lower stability of the T form explains the loss of substrate cooperativity . The lack of allosteric inhibition may be related to a new electrostatic interaction made in mutant r chains between the C-terminal carboxylate and a lysine residue of the allosteric domain.

Biokhimiia, 1987 Sep, 52(9), 1411 - 6
{Preparation of Escherichia coli 70S ribosomes labeled with 35S}; Abdukaiumov M et al.; {35S}--70S ribosomes (150 Ci/mmol) were isolated from E . coli MRE-600 cells grown on glucose-mineral media in the presence of {35S} ammonium sulfate . The labeled 30S and 50S subunits were obtained from {35S} ribosomes by centrifugation in a sucrose density gradient of 10--30% under dissociating conditions (0.5 mM Mg2+) . The activity of {35S}--70S ribosomes obtained by reassociation of the labeled subunits during poly(U)-dependent diphenylalanine synthesis was not less than 70% . The activity of {35S}--70S ribosomes during poly(U)-directed polyphenylalanine synthesis was nearly the same as that of the standard preparation of unlabeled ribosomes . The 23S, 16S and 5S RNAs isolated from labeled ribosomes as total rRNA contained no detectable amounts of their fragments as revealed by polyacrylamide gel electrophoresis . The {35S} ribosomal proteins isolated from labeled ribosomes were analyzed by two-dimensional gel electrophoresis . The {35S} label was found in all proteins, with the exception of L20, L24 and L33 which did not contain methionine or cysteine residues.

Antimicrob Agents Chemother, 1987 Sep, 31(9), 1359 - 64
Autoradiographic study of tobramycin uptake by proximal and distal tubules of normal and pyelonephritic rats; Bergeron MG et al.; Multiple factors may modify the pharmacokinetics of aminoglycosides and affect their nephrotoxic potential . In the present study, the influence of Escherichia coli pyelonephritis on the renal handling of {3H}tobramycin was investigated . The accumulation of {3H}tobramycin in proximal and distal tubules in both normal and infected rats was compared . Following induction of pyelonephritis, disturbed intrarenal localization of the drug was noted . Grain counts were affected in both proximal and distal tubules . Decreased labeling was observed at all time intervals in the proximal tubules . Electron microscopy showed that radioactivity was associated mostly with lysosomes in both normal and infected rats 1 and 24 h following the injection of the drug . We could detect significantly higher amounts of drug in the distal tubules of the pyelonephritic kidney than the normal levels at 10 min and 24 h postinjection . The drug did not seem to be associated with any particular organelle and was evenly distributed within the distal tubular cells . The present study shows that the transport of tobramycin within the infected nephron is disturbed . These data might shed some light on the influence of infection on the intrarenal pharmacology of aminoglycosides.

Radiobiologiia, 1987 Sep-Oct, 27(5), 706 - 8
{Lethal and mutagenic effect of the XeCl laser on Escherichia coli}; Tiflova OA et al.; The survival rate and reversions to tryptophan-independence of Escherichia coli after XeCl laser irradiation (lambda = 308 nm) within the dose range from 10(3) to 10(5) J/m2 have been studied to show that LD37 is 10(4) J/m2, the survival rate at a maximum dose of 10(5)J/m2 is 1 per cent, and the number of mutants per 10(6) cells survived is 100.

Pediatr Infect Dis J, 1987 Sep, 6(9), 829 - 31
Patterns of adherence of diarrheagenic Escherichia coli to HEp-2 cells; Nataro JP et al.; A total of 516 Escherichia coli strains randomly isolated from coprocultures of 154 Chilean children with diarrhea and 66 controls were examined with DNA probes and tested for adherence to HEp-2 cells . Three adherence patterns were distinguished, localized, true diffuse and "aggregative." Enteropathogenic E . coli (EPEC) were detected by EPEC adherence factor probe among 86 of the 372 isolates (23%) from patients with diarrhea vs . 14 of 144 (10%) strains from controls (P less than 0.0002) . Of 95 strains that manifested localized adherence, 97% were EPEC adherence factor probe-positive; thus the HEp-2 assay may serve as an alternative to the probe in identifying EPEC adherence factor-positive EPEC . True diffuse adherence was not associated with diarrhea . In contrast the aggregative pattern appears to signify a new, distinct class of diarrheagenic E . coli (enteroadherent-aggregative E . coli) . The aggregative pattern was found in only 3 of 27 enterotoxigenic, 0 of 4 enteroinvasive, 0 of 2 enterohemorrhagic and 2 of 86 EPEC strains but in 84 of 253 probe-negative strains (P less than 0.00001) from patients with diarrhea; in comparison only 20 of 134 probe-negative strains from controls were aggregative E . coli (P less than 0.00001 vs . probe-negative strains from diarrhea patients).

Mol Gen Genet, 1987 Sep, 209(2), 408 - 10
Cloning and sequencing of the HU-2 gene of Escherichia coli; Kano Y et al.; The Escherichia coli HU-2 gene was cloned using a DNA fragment from the HU-1 gene as a probe . The amino acid sequence of the HU-2 protein deduced from the nucleotide sequence is in good agreement with the published sequence . The nucleotide sequence has a possible promoter and a typical ribosomal binding site upstream of the translation initiation codon (AUG) and a possible rho-independent terminater site downstream of the termination codon (UAA) of the gene.

Clin Nucl Med, 1987 Sep, 12(9), 713 - 6
Functional causes of the ductal obstructive pattern on hepatobiliary scans; Ford KB et al.; Hepatobiliary imaging with Tc-99m IDA derivatives has proven value for evaluation of biliary disease . Prompt hepatocellular uptake with persistent nonvisualization of the common bile duct and bowel is usually indicative of a high-grade common bile duct obstruction, but is not pathognomonic . A functional abnormality due to hepatocyte dysfunction resulting in intrahepatic cholestasis can also cause this pattern . Two cases of hepatocellular excretory dysfunction, one due to E . coli endotoxemia with intrahepatic cholestasis and the other due to acute hepatitis A that produces ductal obstructive patterns on Tc-99m disofenin scintigraphy in patients with documented patent biliary ducts, are reported . Transhepatic cholangiography or endoscopic retrograde cholangiography may be useful when the diagnosis of biliary ductal obstruction is in doubt.

Arch Biochem Biophys, 1987 Sep, 257(2), 485 - 7
A single-step large-scale purification of pyruvate oxidase; Zhang TF et al.; Pyruvate oxidase is an Escherichia coli peripheral membrane flavoprotein which catalyzes the oxidative decarboxylation of pyruvate to acetate and CO2 . Pyruvate oxidase, like several other peripheral membrane enzymes, can be activated either by binding to lipid amphiphiles or by limited protease digestion . This paper reports a rapid and convenient method for effecting the large-scale purification of pyruvate oxidase from crude enzyme preparations using a Triton X-114 phase separation technique . It appears likely that this purification procedure can be used successfully with the family of enzymes which respond to both lipid and protease activation.

Arch Biochem Biophys, 1987 Sep, 257(2), 464 - 71
Induction and inactivation of catalase and superoxide dismutase of Escherichia coli by ozone; Whiteside C et al.; Oxyradicals have been implicated in ozone (O3) toxicity and in other oxidant stress . In this study, we investigated the effects of O3 on the biosynthesis of the antioxidant enzymes catalase and superoxide dismutase in Escherichia coli to determine their role in the defense against ozone toxicity . Inhibition of growth and loss of viability were observed in cultures exposed to ozone . Results also showed an increase in the activities of catalase and superoxide dismutase in cultures exposed to ozone, which was shown to be due to true induction rather than activation of preexisting apoproteins . Cessation of O3 exposure resulted in 30 min of continual high rate of catalase biosynthesis followed by a gradual decrease in the level of the enzyme approaching that of control cultures . This decrease was attributed to a concomitant cessation of de novo enzyme synthesis and dilution of preexisting enzyme by cellular growth . Ozonation of cell-free extracts showed that superoxide dismutase and catalase are subject to oxidative inactivation by ozone . In vivo induction of these enzymes may represent an adaptive response evolved to protect cells against ozone toxicity.

Am J Vet Res, 1987 Sep, 48(9), 1363 - 6
Ultrastructure of equine endothelial cells exposed to endotoxin and flunixin meglumine and equine neutrophils; Turek JJ et al.; An in vitro system of cultured equine endothelial cells was evaluated as a model for endotoxin (ET) exposure in the horse . Primary cell lines from pulmonary vessels and aortas were cultured from tissues of 6 horses . Effects of ET alone with and without serum and in combination with the cyclo-oxygenase inhibitor flunixin meglumine and isolated equine neutrophils were evaluated by transmission electron microscopy . Cells plus serum were incubated with 10, 25, 50, or 100 micrograms of ET/ml of incubation medium for 1, 3, 8, or 24 hours . Cells without serum were cultured for 1 and 3 hours . Flunixin meglumine was used at a concentration of 20 micrograms/ml . Cells also were incubated in the presence of 1,000, 5,000, or 20,000 neutrophils/ml plus ET and in the presence of a combination of ET and flunixin meglumine for 1 or 3 hours . Endotoxin alone did not cause cell damage, and the only evidence of an effect was an increased number of secondary lysosomes at incubation hour 8 . At incubation hour 24, cells appeared normal . Endotoxin plus neutrophils caused cells to become round and detach from the growth substrate . Cell pathologic changes included swollen and distorted mitochondria and cytoplasmic vacuolization . Response to the ET plus neutrophil combination was variable and ranged from 5% to 50% of the cells being affected . The variability appeared to have some correlation with cell age, as well as individual preparation of neutrophils.

Somat Cell Mol Genet, 1987 Sep, 13(5), 563 - 8
DNA base sequence changes induced by ethyl methanesulfonate in a chromosomally integrated shuttle vector gene in mouse cells; Ashman CR et al.; We have analyzed the specificity of mutations induced by ethyl methanesulfonate (EtMes) in mouse cells carrying a selectable bacterial gene . The target gene was the Escherichia coli gpt gene contained within a retroviral shuttle vector integrated into mouse chromosomal DNA . Following mutagenesis by EtMes, cells with mutations in the gpt gene were selected as resistant to 6-thioguanine . Shuttle vector sequences were recovered from the mutant cell lines following fusion with monkey COS cells and introduced into bacteria as part of a bacterial plasmid . The DNA base sequences of the mutant genes were directly determined from plasmid DNA . All of the EtMes-induced mutations involving single base changes were found to be G:C to A:T transitions.

J Appl Physiol, 1987 Sep, 63(3), 1058 - 62
Effects of endotoxemia on pulmonary vascular resistances in unanesthetized sheep; Parker RE et al.; Ten experiments were conducted on nine sheep to determine the effects of endotoxemia (1.0 microgram/kg iv over 15 min) on the vascular resistances of two segments of the pulmonary circulation . The first segment (S1) was from the main pulmonary artery to the site in the pulmonary veins corresponding to the pressure measured with a deflated and wedged 7-Fr Swan-Ganz catheter . The second segment (S2) was from the wedge pressure measurement site to the left atrium . Endotoxemia caused both pulmonary arterial pressure and pulmonary arterial wedge pressure to increase significantly during early (phase 1) and late (phase 2) periods of response; left atrial pressure was significantly decreased during both phases . Normalized cardiac output decreased significantly at 35 and 180 min but not at 240 min after starting endotoxin infusion . The calculated resistance of S1 significantly increased from a base-line value of 3.03 +/- 0.31 (cmH2O.1-1.min) to 7.60 +/- 0.71, 6.34 +/- 1.22, and 6.66 +/- 1.35 at 35, 180, and 240 min, respectively . Calculated resistance of S2 was 1.32 +/- 0.14 at base line and increased significantly to 11.43 +/- 1.66 at 35 min, 4.45 +/- 0.47 at 180 min, and 3.32 +/- 0.61 at 240 min . The calculated percent of total pulmonary resistance in S2 increased significantly from approximately 31 to 59% during phase 1 and remained significantly increased at 41% from 90 to 180 min after endotoxin . Hematocrit increased by 40% at 35 min, whereas plasma total protein concentration increased by only 8% at 35 min.(ABSTRACT TRUNCATED AT 250 WORDS)

Biophys J, 1987 Sep, 52(3), 421 - 5
"Resonances" in the dielectric absorption of DNA?
Foster KR, Epstein BR, Gealt MA.
An attempt was made to confirm previous reports of resonant-like dielectric absorption of plasmid DNA in aqueous solutions at 1-10 GHz . The dielectric properties of the sample were measured using an automatic network analyzer with two different techniques . One technique used an open-ended coaxial probe immersed in the sample; the other employed a coaxial transmission line . No resonances were observed that could be attributed to the sample; however, resonance-type artifacts were prominent in the probe measurements . The coaxial line technique appears to be less susceptible to such artifacts . We note two important sources of error in the calibration of the automatic network analyzer using the probe technique.

Toxicology, 1987 Sep, 45(3), 257 - 68
Endotoxin inactivation by the humoral components in the tolerant rat serum; Yamaguchi Y et al.; A rapid inactivation of endotoxin has shown to occur following its incubation in serum obtained from endotoxin-tolerant rats with the aid of the limulus amebocyte lysate (LAL) assay . The tolerant rat had large quantities of lipopolysaccharide inhibitor (LPSI) activity, which does not appear to be complement . Heating tolerant rat serum for 60 min at 56 degrees C or the addition of lead acetate to the tolerant serum both resulted in the loss of LPSI activity . This paper focuses on the most unique properties of LPSI, namely it's alteration of activity after heating or the addition of lead acetate, compared with those properties of inhibitors for endotoxin which have been previously demonstrated by a number of investigators.

Proc Natl Acad Sci U S A, 1987 Sep, 84(18), 6496 - 500
Intramolecular recombination between partially homologous sequences in Escherichia coli and Xenopus laevis oocytes; Abastado JP et al.; We describe a system to analyze the individual contribution of a single physical DNA end on intramolecular recombination between partially homologous sequences . We took advantage of this partial sequence divergence to measure the distance separating the DNA end from the final recombination event . We show that a single physical DNA end stimulates recombination when located in a region of homology . Recombination frequency decreases gradually with the distance from the DNA end . A recombinational hot spot is found at the end of the region of homology . A large insertion of unrelated DNA interferes asymmetrically with this process, suggesting that a recombinogenic signal propagates along the region of homology.

Proc Natl Acad Sci U S A, 1987 Sep, 84(17), 6225 - 8
Confidence interval for the number of selectively neutral amino acid polymorphisms; Sawyer SA et al.; A statistical approach to the analysis of DNA sequences has been developed, which provides a confidence interval estimate for the proportion of naturally occurring amino acid polymorphisms that are selectively neutral . When applied to the gnd gene coding for 6-phosphogluconate dehydrogenase in a sample of seven natural isolates of Escherichia coli, the method indicates that the proportion of observed amino acid polymorphisms that are selectively neutral is unlikely to be greater than 49% (upper 95% confidence limit) . On the other hand, the observations are also consistent with a model in which all of the observed amino acid substitutions are mildly deleterious with an average selection coefficient approximating 1.6 X 10(-7) . Various models for the distribution of configurations at silent sites are also investigated.

Proc Natl Acad Sci U S A, 1987 Sep, 84(17), 6220 - 4
Spectra of spontaneous mutations in Escherichia coli strains defective in mismatch correction: the nature of in vivo DNA replication errors; Schaaper RM et al.; We have determined the DNA sequence changes in 487 spontaneous mutations in the N-terminal part of the lacI gene in mutH, mutL, and mutS strains of Escherichia coli . These strains display elevated spontaneous mutation rates because of a deficiency in the process of postreplicative mismatch correction . As a consequence the mutational spectra reveal the nature of spontaneous DNA replication errors . The spectra consist of base substitutions (75%) and single-base deletions (25%) . Among the base substitutions, transitions (both A.T----G.C and G.C----A.T) are strongly favored over transversions (96% versus 4%) . Large site-to-site differences are observed among identical base substitutions, presumably reflecting the modulating effects of neighboring bases . The single-base-deletion spectrum is dominated by a large hotspot at a run of adjacent identical base pairs, implying a Streisinger-slippage mechanism . The data, when compared to a previously determined wild-type spectrum, also provide information on the specificity of the mismatch repair system.

Proc Natl Acad Sci U S A, 1987 Sep, 84(17), 6152 - 6
Consequences of detailed balance in a model for sensory adaptation based on ligand-induced receptor modification; Walz D et al.; A model for exact sensory adaptation has been published by Segel and co-workers in several papers {e.g., Knox, B . E., Devreotes, P . N., Goldbeter, A . & Segel, L . A . (1986) Proc . Natl . Acad . Sci . USA 83, 2345-2349} . The model comprises a pair of states whose relative probabilities are determined by the binding of a ligand . A second pair of states related by the same ligand binding is accessible as a consequence of either a conformational change or a "covalent modification." By taking proper account of detailed balance, we show that the notion of covalent modification in this context includes three cases, two of which involve dissipation of metabolic energy . The condition for exact adaptation is dependent on metabolite concentrations in all cases of covalent modification . The performance of the model is critically examined on thermodynamic and kinetic grounds.

Mutat Res, 1987 Sep, 184(2), 99 - 105
Sodium arsenite inhibits spontaneous and induced mutations in Escherichia coli; Nunoshiba T et al.; Sodium arsenite at a non-toxic concentration was found to inhibit strongly mutagenesis induced by ultraviolet light (UV), 4-nitroquinoline-1-oxide (4NQO), furylfuramide (AF-2) and methyl methane-sulfonate (MMS) as well as spontaneous mutation in the reversion assay of E . coli WP2uvrA/pKM101 . The effect was not, however, seen in the case of the mutagenesis induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) . In order to elucidate the mechanism of the mutation-inhibitory effect of sodium arsenite, its action on umuC gene expression and DNA-repair systems was investigated . It was found that sodium arsenite depressed beta-galactosidase induction, corresponding to the umuC gene expression . For UV-irradiated E . coli strains possessing different DNA-repair capacities, sodium arsenite decreased the UV survival rates of WP2, WP2uvrA{uvrA} and WP67{uvrA polA}, increased those of SOS-uninducible strains having either the recA+ or uvrA+ such as CM571 {recA}, CM561 {lexA(Ind-)} and CM611{uvrA lexA (Ind-)}, and did not affect that of the uvrA recA double mutant, WP100 . From these results, we assume that sodium arsenite may have at least two roles in its antimutagenesis: as an inhibitor of umuC gene expression, and as an enhancer of the error-free repairs depending on the uvrA and recA genes.

J Bacteriol, 1987 Sep, 169(9), 4327 - 34
Export of protein in Escherichia coli: a novel mutation in ompC affects expression of other major outer membrane proteins; Catron KM et al.; A mutation within the ompC structural gene of Escherichia coli K-12 which affects expression of outer membrane proteins was characterized . The mutation consisted of a 6-base-pair deletion near the 3' end of the gene which removed the amino acids Val-300 and Gly-301 of the mature coding sequence but otherwise left the reading frame intact . The deletion occurred within a region highly conserved among the porins . No protein product was detected from a single copy of the mutant gene . The mutation caused a trans-dominant decrease in the expression of a wild-type ompC allele . The mutation caused a similar decrease in the amounts of OmpA, OmpF, LamB, and Lc proteins, yet it did not appear to affect the minor outer membrane proteins . It had no significant effect on transcription from either ompF or ompC promoters as measured with lacZ operon fusions . The effects of the mutation on other proteins were completely eliminated when the signal sequence was disrupted so that the mutant protein no longer interacted with the secretion machinery of the cell but instead accumulated as precursor in the cytoplasm . A model is proposed involving the translocation of proteins to the outer membrane and the importance of protein conformation in this process.

J Bacteriol, 1987 Sep, 169(9), 4128 - 34
Suppression of rpoH (htpR) mutations of Escherichia coli: heat shock response in suhA revertants; Tobe T et al.; Temperature-resistant pseudorevertants were isolated from rpoH (htpR) mutants of Escherichia coli K-12 that cannot grow at a high temperature owing to a deficiency in sigma 32 required for the induction of heat shock proteins . Among them was a class of revertants carrying a suppressor mutation, designated suhA, that suppressed all the nonsense and missense rpoH mutations tested . suhA is located at 77 min, about 1 min away from rpoH, on the genetic map . In contrast to the rpoH mutants, the suhA revertants that contained both rpoH (nonsense) and suhA mutations were fully or partially proficient in the induction of heat shock proteins upon exposure to a high temperature . Under these conditions, transcription from two heat shock promoters as determined by operon fusion was transiently activated . In one of the rpoH(Am) suhA revertants studied in detail, an increase in temperature caused the synthesis of significant amounts of sigma 32, accompanied by increased stability and accumulation of rpoH mRNAs . On the other hand, the same mutation (suhA6) only weakly suppressed the rpoH deletion mutant; however, two of the major heat shock genes, dnaK and groE, were apparently induced in the absence of sigma 32 . Thus, suhA6 seems to bring about the induction of heat shock genes by at least two mechanisms, one increasing the level of sigma 32 synthesis, and the other activating some transcription factor other than sigma 32.

Infect Immun, 1987 Sep, 55(9), 2293 - 5
Release of lactoferrin by polymorphonuclear leukocytes after aerosol challenge with Escherichia coli; LaForce FM et al.; Mice with cyclophosphamide-induced granulocytopenia were challenged with aerosolized Escherichia coli, their lungs were lavaged at 1 and 4 h, and total cell counts, differential counts, and levels of lactoferrin, transferrin, and albumin were measured in the lung lavage fluid . Lung lavage fluid from cyclophosphamide-treated mice had few neutrophils and no increase in lactoferrin levels, whereas control mice had significant increases in both . Transferrin levels did not change in either group . Neutrophils are the source of increased lactoferrin levels in lung lavage fluid after aerosol challenge.

Infect Immun, 1987 Sep, 55(9), 2121 - 5
Substitutions of cysteine residues of Escherichia coli heat-stable enterotoxin by oligonucleotide-directed mutagenesis; Okamoto K et al.; The Escherichia coli 18-amino-acid, heat-stable enterotoxin STp has six cysteine residues linked intramolecularly by three disulfide bonds . These disulfide bonds are important for toxic activity, but the precise role of each bond is not clear . We substituted cysteine residues of STp in vivo by oligonucleotide-directed site-specific mutagenesis to dissociate each disulfide bond and examined the biological activities of the resulting mutants . The Cys-6----Ala and Cys-17----Ala mutations caused a complete loss of toxic activity . The Cys-5----Ala, Cys-10----Ser, and Gly-16, Cys-17----Cys-16, Gly-17 mutations caused a large decrease in toxic activity . These results mean that all three disulfide bonds formed at fixed positions are required for full expression of the biological activity of STp . However, a weak but significant toxicity still remained after three mutations, Cys-5----Ala, Cys-10----Ser, and Gly-16, Cys-17----Cys-16, Gly-17 . This indicates that STp has some flexibilities in its conformation to exert toxic activity and that the role of each disulfide bond exerting toxic activity is not quite the same.

Infect Immun, 1987 Sep, 55(9), 2103 - 9
Effects of fibronectin and other salivary macromolecules on the adherence of Escherichia coli to buccal epithelial cells; Hasty DL et al.; The effect of saliva and fibronectin (Fn) on the adherence of a type 1 fimbriated strain of Escherichia coli to human buccal epithelial cells was studied . Saliva pretreatment of epithelial cells led to a dose-dependent increase in adherence that was inhibited by alpha-methyl mannoside, which is typical of a type 1 fimbria-mediated event . The molecules responsible for affecting this increased adherence were nondialyzable and were recovered after lyophilization . E . coli adherence was stimulated by individual saliva samples from each of 11 volunteers . Fn inhibited E . coli adherence to saliva-treated buccal cells by more than 60% . Biotinylated E . coli and Fn were reacted with Western blots of whole saliva to identify the receptors that might explain the phenomenon described above . Both E . coli and Fn bound to 57- and 62-kilodalton (kDa) protein bands in Western blots of sodium dodecyl sulfate gels of whole saliva . The binding of E . coli to these bands was inhibited by pretreatment with unlabeled Fn . To study these salivary components, samples of saliva were electrophoresed on sodium dodecyl sulfate gels, strips corresponding to the appropriate molecular weights were cut out, and the proteins were eluted electrophoretically . Material that eluted from strips at 57 and 62 kDa, but not that from a control strip, stimulated E . coli adherence to buccal cells . Alternatively, saliva was fractionated over 100- and 50-kDa cutoff filters . Of the three fractions obtained, only the fraction passing through the 100-kDa filter and retained by the 50-kDa filter stimulated E . coli adherence to buccal cells . This fraction also increased the binding of Fn to buccal cells . These observations suggest the possibility that one or more salivary components bind to the surface of buccal cells and serve as receptors for type 1 fimbriated E . coli . Fn also binds to this isolated material; and it is apparently by these interactions, at least in part, that saliva stimulates and Fn inhibits E . coli adherence . The way in which these interactions may affect bacterial adherence in vivo remains to be elucidated.

Infect Immun, 1987 Sep, 55(9), 2057 - 60
Protease degradation of Escherichia coli heat-stable, mouse-negative, pig-positive enterotoxin; Whipp SC; Enterotoxigenic Escherichia coli produces three enterotoxins: heat-labile toxin, a mouse-positive heat-stable toxin, and a mouse-negative heat-stable toxin (STb) . The only species in which a response to STb has been documented is the pig, and this response is inconsistent . When STb was placed in 60 ligated jejunal segments (loops) in six pigs, a positive response (net secretion) was observed in only 40 loops . In contrast, when the jejunal lumen was pre-rinsed with 50 ml of saline, the same STb preparation induced net secretion in 60 of 60 loops . STb did not induce secretion in rinsed loops when jejunal luminal washings were collected and mixed with STb in vitro . The anti-STb activity of jejunal luminal washings was filterable through 0.45-micron-pore-size filters, was destroyed by heating at 100 degrees C for 15 min, and was blocked by soybean trypsin inhibitor . STb was inactivated when incubated with trypsin in vitro for 60 min at 37 degrees C . It is concluded that STb is susceptible to trypsin degradation and that variable amounts of trypsin-like activity in swine jejuna are responsible for inconsistent responses to STb in jejunal loops of swine . These results also suggest that the concept of species specificity of the STb response should be reexamined.

J Korean Med Sci, 1987 Sep, 2(3), 189 - 94
Liver cell adenoma in a neonate--report of an autopsy case; Suh YL et al.; A case of liver cell adenoma that was incidentally found at postmortem examination of a neonate who died of E . coli sepsis is described . The adenoma was a sharply demarcated, not encapsulated mass located subcapsularly in the right lobe, and was pale tan to light yellowish round nodule of 0.9 cm in diameter . Microscopically, the tumor was composed of sheets and cords of uniform and slightly enlarged hepatocytes separated by dilated sinusoids . There were no portal zones or central veins to suggest the normal lobular architecture . The nuclei were bland and the cytoplasm varied from clear to acidophilic, containing lipid vacuoles . Ultrastructural examination showed that the hepatocytes of the tumor had highly differentiated organelles, reminiscent of normal hepatocytes.

J Photochem Photobiol B, 1987 Sep, 1(1), 13 - 31
Partial tRNA deacylation specifically triggers Escherichia coli cell volume reduction; Caldeira de Araujo A et al.; Limitation of Escherichia coli cell growth rate either by means of continuous 366 nm illumination, which is known to decrease the in vivo acylation level of some tRNA species, or by means of specific inhibitors of tRNA acylation allows the division rate to remain unchanged for a few generations, resulting in cell volume reduction . In contrast the cell volume remains stable or increases after treatment with inhibitors of DNA replication and transcription, or with drugs acting at any other step of protein synthesis . The conclusion that limiting acylation of some tRNA species is the triggering event is confirmed by the use of thermosensitive mutants of aminoacyl-tRNA synthetases or of tRNA (the divE strain mutated in the tRNA1Ser gene) . Other cellular responses modulate the expression of cell volume reduction . The relA+ stringent response helps expression of the effect but does not appear to be strictly required . However, cell volume reduction may be masked under conditions triggering the SOS response . The data suggest that tRNA acylation is one of the major steps where cells sense change in their nutrient environment.

J Photochem Photobiol B, 1987 Sep, 1(1), 1 - 11
recA-dependent DNA repair in UV-irradiated Escherichia coli; Smith KC et al.; UV-radiation-induced lesions in DNA result in the formation of excision gaps, daughter-strand gaps (DSG) and double-strand breaks (DSB), which are repaired by several different mechanisms . Postreplication repair . The recA gene is a master gene that controls all of the pathways of postreplication repair . The repair of DSG proceeds by one pathway that is also recF dependent, and one pathway that is constitutive and independent of the recF and recBC genes . A small fraction of the recF recB-independent repair of DSG is dependent upon the umuC gene, and may define an error-prone pathway of postreplication repair . Unrepaired DSG can be converted to DSB, which are normally repaired by the RecBCD pathway . However, in the recBC sbcB background, these DSB are repaired by a recF-dependent process . The RecF pathways of postreplication repair appear to utilize DNA containing a single-stranded region (either a gap or a DSB with a single-stranded end), while the RecBCD pathway appears to utilize the blunt ends of duplex DNA to promote the recombinational repair of DSB . The polA gene (especially the 5'----3' exonuclease activity of DNA polymerase I) functions in pathways of postreplication repair (both for the repair of DSG and DSB) that are largely independent of the recF gene . Nucleotide excision repair . The repair of excision gaps is independent of the recA gene in cells with unreplicated chromosomes, but is recA dependent in cells with partially replicated chromosomes at the time of UV irradiation . This recA-dependent repair of excision gaps appears to be analogous to the recF- and recB-dependent pathways of postreplication repair, i.e . the RecF pathway repairs DNA gaps, and the RecBCD pathway repairs the DSB that arise at unrepaired gaps.

Biochimie, 1987 Sep, 69(9), 957 - 63
Relationship between size of mRNA ribosomal binding site and initiation factor function; Canonaco MA et al.; The rate and the extent of the binding of initiator fMet-tRNA(fMet) to 30S ribosomal subunits in the presence of IF1, IF2 and GTP is either inhibited or slightly stimulated by the presence of IF3 depending on whether the initiation triplet AUG or the polynucleotide poly(AUG) is used as template . To determine the length of the template required for the transition from the AUG- to the poly(AUG)-type of behavior in the presence of IF3, the ribosomal binding of fMet-tRNA was studied in response to AUG triplets extended on either the 5'- or the 3'-side by stretches of homo-oligonucleotides of different lengths . When the binding of fMet-tRNA was studied at equilibrium it was found that IF3 no longer inhibits the amount of ternary complex formed if AUG is extended either 10 nucleotides on the 5'- or 35-40 nucleotides on the 3'-side . When the initial rate of ternary complex formation is considered, shorter extensions (4 nucleotides on the 5'-side or 20-30 nucleotides on the 3'-side) are sufficient to elicit a substantial stimulation by IF3 . These results are discussed in relation to the mechanism of action of the initiation factors in the selection of the initiation region of the mRNA by ribosomes.

Biochimie, 1987 Sep, 69(9), 949 - 56
Structural dynamic aspects of protein synthesis on ribosomes; Spirin A; Three types of conformational changes in the translating ribosome are considered: (1) intersubunit movement (ribosome unlocking) during translocation; (2) L7/L12 stalk mobility affected by elongation factors; (3) change of tRNA residue during its transition from the A-site to the P-site . Relevant experimental data are reviewed.

Biochimie, 1987 Sep, 69(9), 925 - 38
An apparent conformational change in tRNA(Phe) that is associated with the peptidyl transferase reaction; Odom OW et al.; Fluorescence techniques were used to detect changes in the conformation of tRNA(Phe) that may occur during the peptidyl transferase reaction in which the tRNA appears to move between binding sites on ribosomes . Such a conformational change may be a fundamental part of the translocation mechanism by which tRNA and mRNA are moved through ribosomes . E . coli tRNA(Phe) was specifically labeled on acp3U47 and s4U8 or at the D positions 16 and 20 . The labeled tRNAs were bound to ribosomes as deacylated tRNA(Phe) or AcPhe-tRNA . Changes in fluorescence quantum yield and anisotropy were measured upon binding to the ribosomes and during the peptidyl transferase reaction . In one set of experiments non-radiative energy transfer was measured between a coumarin probe at position 16 or 20 and a fluorescein attached to acp3U47 on the same tRNA(Phe) molecule . The results indicate that the apparent distance between the probes increases during deacylation of AcPhe-tRNA as a result of peptide bond formation . All of the results are consistent with but in themselves do not conclusively establish that tRNA undergoes a conformational change as well as movement during the peptidyl transferase reaction.

Biochimie, 1987 Sep, 69(9), 911 - 23
Evidence that the G2661 region of 23S rRNA is located at the ribosomal binding sites of both elongation factors; Hausner TP et al.; Alpha-sarcin cleaves one phosphodiester bond of 23S rRNA within 70S ribosomes or 50S subunits derived from E . coli . The resulting fragment was isolated and sequenced . The cleavage site was identified as being after G2661 and is located within a universally conserved dodecamer . Cleavage after G2661 specifically blocked the binding of both elongation factors, i.e . that of the ternary complex Phe-tRNA*EF-Tu*GMPPNP and of EF-G*GMPPNP, whereas all elongation-factor independent functions of the ribosome, such as association of the ribosomal subunits, tRNA binding to A and P sites, the accuracy of tRNA selection at both sites, the peptidyl transferase activity, and the EF-G independent, spontaneous translocation, were not affected at all . Control experiments with wheat germ ribosomes yielded an equivalent inhibition pattern . The data suggest that the universally conserved dodecamer containing the cleavage site G2661 is located at the presumably overlapping region of the binding sites of both elongation factors.

Prostaglandins, 1987 Sep, 34(3), 385 - 400
The effect of endotoxin on the lipoxygenase-mediated conversion of exogenous and endogenous arachidonic acid in mouse peritoneal macrophages; Schade UF; The influence of lipopolysaccharide (LPS, endotoxin) or its lipid A component (bacterial and synthetic) on the synthesis of zymosan induced leukotriene C4, prostaglandin E2 and prostacyclin and on the conversion of exogenous arachidonic acid was studied in mouse peritoneal macrophages . It was found that following preincubation with LPS the amount of leukotriene C4 released during phagocytosis of zymosan was substantially decreased . The levels of prostaglandin E2 and prostacyclin, however, were the same in LPS-treated cells and controls . Likewise, pretreatment with LPS impaired the capacity to convert exogenously added arachidonic acid to mono- and di-HETE's . Lipid A (bacterial and synthetic) exhibited the same activity as LPS . LPS had no effect on macrophages of the endotoxin low responder mouse strain (C3H/HeJ) . Several explanations could be possible for the observed LPS effect . The finding that low doses of alpha-tocopheryl acetate prevented the LPS-induced decrease of LTC4 synthesis indicates a protective role of this agent . We would, therefore, favour the idea that lipoxygenases undergo oxidative selfinactivation during LPS action.

EMBO J, 1987 Sep, 6(9), 2835 - 41
Release of a chimeric protein into the medium from Escherichia coli using the C-terminal secretion signal of haemolysin; Mackman N et al.; Recently, we have identified a novel topogenic sequence at the C terminus of Escherichia coli haemolysin (HlyA) which is essential for its efficient secretion into the medium . This discovery has introduced the possibility of using this secretion system for the release of chimeric proteins from E . coli directly into the medium . We have now successfully fused this C-terminal signal to a hybrid protein containing a few residues of beta-galactosidase and the majority of the E . coli outer membrane porin OmpF lacking its own N-terminal signal sequence . We find that this chimeric protein is specifically translocated across the inner and outer membranes and is released into the medium . In addition, we have further localized the HlyA secretion signal to the final 113 amino acids of the C terminus . In fact, a specific secretion signal appears to reside at least in part within the last 27 amino acids of HlyA.

EMBO J, 1987 Sep, 6(9), 2617 - 25
The nuclear migration signal of Xenopus laevis nucleoplasmin; Burglin TR et al.; Nucleoplasmin is the most abundant protein in the nucleus of Xenopus laevis oocytes . Its ability to target to the nucleus when microinjected into the cytoplasm has been the subject of many studies central to our understanding of how proteins segregate into nuclei . Using a cDNA clone we constructed beta-galactosidase-nucleoplasmin hybrids in modified bacterial expression vectors . The fusion proteins were expressed in Escherichia coli, purified and injected into the cytoplasm of X . laevis oocytes . The distribution of the fusion proteins between the cytoplasmic and nuclear compartments were analysed after incubation of various lengths of time . The results show that the signal sequence for nuclear transport is located close to the carboxy terminus of the protein . The signal sequence has been mapped to a small stretch of amino acids, containing a stretch of four lysines analogous to the SV40 large-T antigen signal.

J Appl Physiol, 1987 Sep, 63(3), 1008 - 11
Lowered pulmonary arterial pressure prevents edema after endotoxin in sheep; Allen SJ et al.; Escherichia coli endotoxin causes increased capillary membrane permeability and increased pulmonary arterial pressure (PAP) in sheep . If the pulmonary hypertension extends to the level of the microvasculature, then the increased microvascular pressure may contribute to the pulmonary edema caused by endotoxin . We tested the hypothesis that reducing the pulmonary hypertension would reduce the amount of edema caused by endotoxin . Twelve sheep were chronically instrumented with catheters to measure PAP, left atrial pressure, and central venous pressure . The sheep were divided into two groups . One group (E) of six sheep received an intravenous infusion of 4 micrograms/kg of E . coli endotoxin . The second group (E + SNP) received the same dose of endotoxin as well as a continuous infusion of sodium nitroprusside (SNP) to reduce PAP . Three hours after the endotoxin infusions, the sheep were terminated and the extravascular fluid-to-blood-free dry weight ratios of the lungs were determined (EVF) . The base-line PAP was 17.5 +/- 2.7 mmHg . A two-way analysis of variance demonstrated a significant difference (P less than 0.01) in PAP between the E and E + SNP groups . Although PAP in each group varied as a function of time, the difference between the two groups did not . The mean PAP for the E + SNP group (20.9 +/- 1.5 mmHg) was lower than the E group PAP of 27.3 +/- 2.1 mmHg after the endotoxin spike . Furthermore, the E + SNP group EVF (3.9 +/- 0.2) was significantly less than the EVF of the E group (4.7 +/- 0.5).(ABSTRACT TRUNCATED AT 250 WORDS)

Infect Immun, 1987 Sep, 55(9), 2164 - 70
Enhancement of resistance to Escherichia coli infection in mice by dihydroheptaprenol, a synthetic polyprenol derivative; Araki S et al.; The effect of a chemically synthesized polyprenol derivative, dihydroheptaprenol (DHP), on the nonspecific resistance of mice to infection with Escherichia coli was investigated . Mice that had been injected intramuscularly with 100 mg of DHP per kg of body weight, prepared as a microemulsion with lecithin, 1 to 4 days before infection showed enhanced resistance to subcutaneous (s.c.) infection with E . coli . When DHP-injected mice were inoculated s.c . with 3 X 10(8) E . coli, which induces fatal acute systemic infection in normal mice, propagation of bacteria in the blood, liver, and spleen was significantly inhibited . Enhanced resistance of athymic (nude) mice to E . coli infection was also induced by DHP . DHP markedly stimulated the generation of peripheral blood neutrophils, significantly enhanced clearance of E . coli from the bloodstream, and activated neutrophils and peritoneal macrophages for H2O2 generation . DHP restored the resistance to E . coli infection in cyclophosphamide-treated mice over the normal level . Furthermore, DHP shortened the period of the recovery of neutrophils and also enhanced clearance of E . coli from the bloodstream in cyclophosphamide-treated mice . DHP was nontoxic for mice and rats (400 mg/kg intramuscularly and 800 mg/kg s.c.) and nonpyrogenic at a dose of 30 mg/kg when administered intravenously to rabbits . These results suggest that the mechanism of action of DHP for enhancing resistance in mice may be, at least in part, its ability to stimulate the generation of potent neutrophils and to activate macrophages in the reticuloendothelial system.

Mutat Res, 1987 Sep, 180(1), 67 - 73
RecA-independent mutagenesis in Escherichia coli may be subject to glucose repression; Thomas SM et al.; The frameshift mutagen 9-aminoacridine (9AA) causes DNA damage via a recA+-independent mechanism in Escherichia coli . In this study we have exposed E . coli cells carrying the lacZ19124 frameshift marker to 9AA in defined minimal media, washed them, and plated to score for Lac+ revertants . Our results show that 9AA-induced reversion to Lac+ occurs in the absence of any exogenous carbon source and when cells are plated on media which do not allow much, if any, cell replication prior to expression of the revertant phenotype . When glycerol (1% w/v) was added to the liquid treatment medium, the number of Lac+ E . coli revertants was similar to that obtained when no carbon source was present . By contrast the addition of glucose (1% w/v) during the mutagenesis treatment caused a significant decrease in the number of revertants . Further experiments indicate that the repressing effects of glucose may be due to a reduction in cAMP concentration, since 9AA mutagenesis was abolished in a cya strain in which no adenylate cyclase is produced . These results are consistent with (but do not prove) the notion that at least one part of the process leading to 9AA mutagenesis is subject to catabolite repression.

J Bacteriol, 1987 Sep, 169(9), 4228 - 34
Cloning and characterization of the Escherichia coli K-12 alanine-valine transaminase (avtA) gene; Wang MD et al.; avtA, which encodes the alanine-valine transaminase, transaminase C, was cloned in vivo with high- and low-copy-number mini-Mu cloning vectors . The phenotype conferred by the cloned avtA+ gene usually depended upon the plasmid copy number; most high-copy-number avtA+ plasmids permitted isoleucine-requiring ilvE strains to grow in the absence of isoleucine (multicopy suppression), while low-copy-number avtA+ plasmids did not . avtA was mapped to a 1.25-kilobase segment by comparison of the restriction maps of 24 independent mini-Mu plasmids and then by gamma-delta (Tn1000) mutagenesis of a pBR322-avtA+ plasmid . The direction of transcription of avtA on the cloned fragment was determined with fusions to a promoterless lac gene.

J Bacteriol, 1987 Sep, 169(9), 4177 - 83
Plasmid transfer in Streptomyces lividans: identification of a kil-kor system associated with the transfer region of pIJ101; Kendall KJ et al.; The 8.9-kilobase Streptomyces plasmid pIJ101 is self-transmissible at high frequency into recipient strains . By genetic analysis of the transfer region of the plasmid, we identified six plasmid-encoded loci involved in gene transfer and the associated pocking phenomenon . Two loci, kilA and kilB, could not be cloned into Streptomyces lividans on a minimal pIJ101-based replicon unless suitable kil-override (kor) genes were present, either in cis or in trans . korA could control the lethal effects of both kilA and kilB, whereas korB could control only the effects of kilB . KilB mutants were defective in their pocking reaction; kilA mutants produced no visible pocks whatsoever . Mutations in two other loci, tra and spd, produced no pocks and defective pocks, respectively . These results suggest that kilA, kilB, tra, and spd are intimately involved in plasmid transfer and that the actions of kilA and kilB are regulated by the products of the korA and korB genes.

J Bacteriol, 1987 Sep, 169(9), 4163 - 70
Cluster of genes controlling synthesis and activation of 2,3-dihydroxybenzoic acid in production of enterobactin in Escherichia coli; Nahlik MS et al.; The Escherichia coli gene cluster encoding enzymatic activities responsible for the synthesis and activation of 2,3-dihydroxybenzoic acid in the formation of the catechol siderophore enterobactin was localized to a 4.2-kilobase chromosomal DNA fragment . Analysis of various subclones and transposon insertion mutations confirmed the previously suggested gene order as entEBG(AC) and provided evidence to suggest that these genes are organized as three independent transcriptional units, composed of entE, entBG, and entAC, with the entBG mRNA transcribed in a clockwise direction . Plasmid-specific protein expression in E . coli minicells identified EntE and EntB as 58,000- and 32,500-dalton proteins, respectively, while no protein corresponding to EntG was detected . The EntA and EntC enzymatic activities could not be separated by genetic or molecular studies . A small DNA fragment encoding both activities expressed a single 26,000-dalton polypeptide, suggesting that this protein is a multifunctional enzyme catalyzing two nonsequential reactions in the biosynthetic pathway . A protein of approximately 15,000 daltons appears to be encoded by the chromosomal region adjacent to the entAC gene, but no known function in enterobactin biosynthesis or transport can yet be ascribed to this polypeptide.

J Bacteriol, 1987 Sep, 169(9), 4154 - 62
Molecular characterization of the Escherichia coli enterobactin cistron entF and coupled expression of entF and the fes gene; Pettis GS et al.; The Escherichia coli entF gene, which encodes the serine-activating enzyme involved in enterobactin synthesis, has been localized to a 4.7-kilobase-pair DNA fragment inserted in the vector pBR328 . This recombinant molecule, pITS32, restored the ability of an entF mutant to grow on low-iron medium and to produce enterobactin . Examination of its translation products by minicell and electrophoretic analyses revealed a protein of approximately 160,000 daltons, which we identified as the EntF protein . A small DNA segment from pITS32 containing the translational start site for entF allowed the low constitutive expression of beta-galactosidase when cloned (pITS301) upstream of the lacZ structural gene in the vector pMC1403 . In contrast, a clone (pITS312) containing the identical entF-lacZ fusion and a larger region upstream of entF including the entire fes gene and extending into the fepA gene (whose transcription is in the opposite direction relative to entF) expressed beta-galactosidase in high yet inducible amounts in response to fluctuations in the metabolic iron concentration . Transposon insertion mutations in the fes gene but not an insertion near the 5' region of fepA in pITS312 reduced this high inducible expression to the low constitutive level seen for pITS301 . These observations are most readily explained by the presence of a regulatory region located upstream of fes which mediates the iron-regulated expression of a transcript that includes the fes and entF genes.

J Bacteriol, 1987 Sep, 169(9), 3994 - 4002
Analysis of transfer genes and gene products within the traB-traC region of the Escherichia coli fertility factor, F; Moore D et al.; A series of plasmids that carry overlapping segments of F DNA encoding the genes in the traB-traC interval was constructed, and a restriction enzyme map of the region was derived . Plasmids carrying deletions that had been introduced at an HpaI site within this interval were also isolated . The ability of these plasmids to complement transfer of F lac plasmids carrying mutations in traB, traV, and traW, and traC was analyzed . The protein products of the plasmids were labeled in UV-irradiated cells and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography . These analyses showed that the product of traV is a polypeptide that migrates with an apparent molecular weight of 21,000 . It was not detected when {35S}methionine was used to label plasmid products, but was readily detected in 14C-amino acid labeling experiments . A 21,500-dalton product appeared to stem from the region assigned to traP . A 9,000-dalton product was found to stem from a locus, named traR, that is located between traV and traC . No traW activity could be detected from the region of tra DNA examined . Our data also indicated that traC is located in a more promoter-proximal position than suggested on earlier maps . The plasmids constructed are expected to be useful in studies designed to identify the specific functions of the traB, -P, -V, -R, and -C products.

J Bacteriol, 1987 Sep, 169(9), 3898 - 903
Cloning and characterization of dnaA(Cs), a mutation which leads to overinitiation of DNA replication in Escherichia coli K-12; Braun RE et al.; The product of the dnaA gene is essential for the initiation of chromosomal DNA replication in Escherichia coli K-12 . A cold-sensitive mutation, dnaA(Cs), was originally isolated as a putative intragenic suppressor of the temperature sensitivity of a dnaA46 mutant (G . Kellenberger-Gujer, A . J . Podhajska, and L . Caro, Mol . Gen . Genet . 162:9-16, 1978) . The cold sensitivity of the dnaA(Cs) mutant was attributed to a loss of replication control resulting in overinitiation of DNA replication . We cloned and sequenced the dnaA gene from the dnaA(Cs) mutant and showed that it contains three point mutations in addition to the original dnaA46(Ts) mutation . The dnaA(Cs) mutation was dominant to the wild-type allele . Overproduction of the DnaA(Cs) protein blocked cell growth . In contrast, overproduction of wild-type DnaA protein reduced the growth rate of cells but did not stop cell growth . Thus, the effect of elevated levels of the DnaA(Cs) protein was quite different from that of the wild-type protein under the same conditions.

J Virol, 1987 Sep, 61(9), 2691 - 701
The pseudorabies virus gII gene is closely related to the gB glycoprotein gene of herpes simplex virus; Robbins AK et al.; We have looked for conserved DNA sequences between four herpes simplex virus type 1 (HSV-1) glycoprotein genes encoding gB, gC, gD, and gE and pseudorabies virus (PRV) DNA, HSV-1 DNA fragments representing these four glycoprotein-coding sequences were hybridized to restriction enzyme fragments of PRV DNA by the Southern blot procedure . Specific hybridization was observed only when HSV-1 gB DNA was used as probe . This region of hybridization was localized to a 5.2-kilobase (kb) region mapping at approximately 0.15 map units on the PRV genome . Northern blot (RNA blot) analysis, with a 1.2-kb probe derived from this segment, revealed a predominant hybridizing RNA species of approximately 3 kb in PRV-infected PK15 cells . DNA sequence analysis of the region corresponding to this RNA revealed a single large open reading frame with significant nucleotide homology with the gB gene of HSV-1 KOS 321 . In addition, the beginning of the sequenced PRV region also contained the end of an open reading frame with amino acid homology to HSV-1 ICP 18.5, a protein that may be involved in viral glycoprotein transport . This sequence partially overlaps the PRV gB homolog coding sequence . We have shown that the PRV gene with homology to HSV-1 gB encoded the gII glycoprotein gene by expressing a 765-base-pair segment of the PRV open reading frame in Escherichia coli as a protein fused to beta-galactosidase . Antiserum, raised in rabbits, against this fusion protein immunoprecipitated a specific family of PRV glycoproteins of apparent molecular mass 110, 68, and 55 kilodaltons that have been identified as the gII family of glycoproteins . Analysis of the predicted amino acid sequence indicated that the PRV gII protein shares 50% amino acid homology with the aligned HSV-1 gB protein . All 10 cysteine residues located outside of the signal sequence, as well as 4 of 6 potential N-linked glycosylation sites, were conserved between the two proteins . The primary protein sequence for HSV-1 gB regions known to be involved in the rate of virus entry into the cells and cell-cell fusion, as well as regions known to be associated with monoclonal antibody resistance, were highly homologous with the PRV protein sequence . Furthermore, monospecific antibody made against PRV gII immunoprecipitated HSV-1 gB from infected cells . Taken together, these findings suggest significant conservation of structure and function between the two proteins and may indicate a common evolutionary history.

Mol Biol (Mosk), 1987 Sep-Oct, 21(5), 1310 - 21
{Effectiveness of translation coupling in hybrid operons}; Mashko SV et al.; The possibility of creating artificial overlappons was studied on the model of two genes, that coding for the N-terminal part of lambda cro protein and the cat of E . coli . To test the dependence of translational coupling efficiency on the intercistronic region a series of recombinant DNA molecules carrying different hybrid operons with partially overlapping genes was constructed . The translational efficiency of the distal to the promoter gene was shown to depend on the intercistronic region structure: overlapping of the AUG codon with the terminating one of the proximal gene in the UGAUG manner is optimal for the translational coupling, and the displacement of AUG at several nucleotides in both directions decreases the translational reinitiation efficiency for the ribosomes, that have synthesised the first gene product.

Mol Cell Biol, 1987 Sep, 7(9), 3049 - 56
Adenovirus E1A requires synthesis of a cellular protein to establish a stable transcription complex in injected Xenopus laevis oocytes; Richter JD et al.; The Escherichia coli-expressed adenovirus E1A 13S mRNA product injected into Xenopus oocytes was active, as assessed by its ability to stimulate the transcription of an injected gene which is normally responsive to E1A in mammalian cells . In the presence of the protein synthesis inhibitors pactamycin or cycloheximide, E1A was correctly posttranslationally modified (phosphorylated) and transported to the nucleus; but it failed to stimulate the transcription of an injected gene containing the human heat shock protein 70 promoter . The basal (unstimulated) level of transcription of the gene was unaffected by these inhibitors . If oocytes were cultured in the presence of cycloheximide after E1A stimulated transcription, however, the high level of transcription was maintained for several hours without new protein synthesis . Results of competition studies with the same promoter (the heat shock protein 70 promoter) linked to two marked genes demonstrated that once the induction of transcription by E1A took place, the stimulated levels of transcription were maintained, even when they were challenged with excess competitor DNA . Results of these studies suggest that E1A requires the synthesis of a cellular protein to form a stable transcription complex.

Mol Gen Genet, 1987 Sep, 209(2), 399 - 402
Molecular genetic analysis of the pyr-4 gene of Neurospora crassa; Glazebrook JA et al.; By means of S1 nuclease mapping, one transcription origin and three termini are identified for the pyr-4 gene of Neurospora crassa, the same origin being used also by Escherichia coli on the cloned gene . Translation of the clone in mini-cells gives a 50,000 dalton gene product, the same size as that determined for the Neurospora native enzyme . Putative CAAT and TATA boxes, and upstream and downstream potential secondary structures, are identified.

Mol Gen Genet, 1987 Sep, 209(2), 382 - 90
The Escherichia coli rep mutation . X . Consequences of increased and decreased Rep protein levels; Colasanti J et al.; Recombinant DNA techniques were used to study various aspects of rep gene function in Escherichia coli . In order to enhance expression of the Rep protein, the rep gene was cloned into the vector pKC30 under the control of the lambda pL promoter . By trimming away a portion of the DNA sequence immediately upstream of the translational start site of rep, we were able to obtain very high levels of Rep protein upon induction . Cells carrying such plasmids showed no ill effects from the high concentration of the protein . To ascertain the consequence of the absence of Rep protein on the cell, the chromosomal copy of the gene was deleted using a homologous recombination technique . The viability of E . coli strains completely lacking the rep gene proves that the Rep function is not essential, at least in wild-type cells under laboratory conditions . We confirmed that in the absence of Rep function there is an increase in the average number of growing forks in exponentially growing cells; augmentation of Rep protein levels above normal, however, did not detectably decrease the number of growing forks.

Virology, 1987 Sep, 160(1), 292 - 6
Binding of cellular polypeptides to human adenovirus type 5 E1A proteins produced in Escherichia coli; Egan C et al.; Cellular proteins of 300, 107, 105, 68 and 65 kDa have previously been shown to associate specifically with the early region 1A (E1A) proteins of human adenovirus type 5 . In the present study we report that, to varying degrees, these proteins also were capable of binding to E1A products produced in Escherichia coli from plasmids carrying cDNAs corresponding to the 1.1- and 0.9-kb E1A mRNAs . When these purified E1A proteins were mixed in solution with extracts from mock-infected human cells, the 68- and 65-kDa species bound very efficiently to the 1.1-kb mRNA product and somewhat less so with that of the 0.9-kb mRNA . The 107-, 105-, and 300-kDa species bound poorly, if at all, to both E1A products . Using the E1A 1.1-kb mRNA product which had been covalently attached to Sepharose beads, the 68-, 65-, and 300-kDa species bound efficiently, and binding of protein which migrated in SDS gels in the region of the 107- and 105-kDa species was also observed . In addition to these proteins, several other cellular polypeptides of 30, 33, 75, 95, 150, 180, and greater than 300 kDa were shown to bind to E1A-Sepharose and thus may also be E1A-binding proteins . The present data confirm the specificity of the previously identified cellular proteins for E1A products and show that binding of the 300-, 65-, and 68-kDa species does not require the presence of any other viral polypeptide . In contrast, the inefficient binding of the 107- and 105-kDa species to Escherichia coli-expressed E1A protein may suggest that these interactions require either eukaryotic-specific post-translational modifications of the E1A protein, or the presence of additional Ad5 gene products.

J Bacteriol, 1987 Sep, 169(9), 3921 - 5
Identification of a vanadate-sensitive, membrane-bound ATPase in the archaebacterium Methanococcus voltae; Dharmavaram RM et al.; Membrane-bound ATPase activity was detected in the methanogen Methanococcus voltae . The ATPase was inhibited by vanadate, a characteristic inhibitor of E1E2 ATPases . The enzyme activity was also inhibited by diethylstilbestrol . However, it was insensitive to N,N'-dicyclohexylcarbodiimide, ouabain, and oligomycin . The enzyme displayed a high preference for ATP as substrate, was dependent on Mg2+, and had a pH optimum of approximately 7.5 . The enzyme was completely solubilized with 2% Triton X-100 . The enzyme was insensitive to oxygen and was stabilized by ATP . There was no homology with the Escherichia coli F0F1 ATPase at the level of DNA and protein . The membrane-bound M . voltae ATPase showed properties similar to those of E1E2 ATPases.

Mol Microbiol, 1987 Sep, 1(2), 211 - 7
Primary structure and subcellular localization of two fimbrial subunit-like proteins involved in the biosynthesis of K99 fibrillae; Roosendaal E et al.; Analysis of the nucleotide sequence of the distal part of the fan gene cluster encoding the proteins involved in the biosynthesis of the fibrillar adhesin, K99, revealed the presence of two structural genes, fanG and fanH . The amino acid sequence of the gene products (FanG and FanH) showed significant homology to the amino acid sequence of the fibrillar subunit protein (FanC) . Introduction of a site-specific frameshift mutation in fanG or fanH resulted in a simultaneous decrease in fibrillae production and adhesive capacity . Analysis of subcellular fractions showed that, in contrast to the K99 fibrillar subunit (FanC), both the FanH and the FanG protein were loosely associated with the outer membrane, possibly on the periplasmic side, but were not components of the fimbriae themselves.

Mol Microbiol, 1987 Sep, 1(2), 169 - 78
Nucleotide sequence, regulation and functional analysis of the papC gene required for cell surface localization of Pap pili of uropathogenic Escherichia coli; Norgren M et al.; The papC gene of uropathogenic Escherichia coli is required for the formation of digalactoside-binding Pap pili . papC forms part of an operon wherein the regulatory gene papB, the major pilin gene papA, a minor pilin-like gene papH, and papC are co-transcribed . Furthermore, the extent of PapC synthesis was found to affect the number of pili expressed on the cell surface . The DNA sequence of the papC gene is presented and its deduced amino acid sequence is compared to that of the FaeD protein encoded by the K88 pili gene cluster . The PapC protein was localized to the E . coli outer membrane where it may form a trans-membrane channel through which pilin subunits are surface localized.

Res Vet Sci, 1987 Sep, 43(2), 233 - 8
Production and evaluation of monoclonal antibodies directed against the K88 fimbrial adhesin produced by Escherichia coli enterotoxigenic for piglets; Thorns CJ et al.; Fifty murine monoclonal antibodies were produced against a purified preparation of K88ab antigen . Two monoclonal antibodies were selected for further studies together with a monoclonal antibody directed against the c region of K88 (5CA3) . Monoclonal antibody K88-13 bound to all K88+ Escherichia coli examined, whereas K88-18 and 5CA3 bound to K88ab+ and K88ac+ strains, respectively . The monoclonal antibodies failed to react with K88+ E coli grown at 18 degrees C or with K88- E coli grown at 37 degrees C or 18 degrees C . Binding of monoclonal antibody K88-13 to K88 antigen was blocked by OK antisera to G7 (O8:K87 K88ab) and Abbotstown (O149:K91 K88ac), whereas absorbed antisera to K88b, c and d had no effect . Monoclonal K88-18 was inhibited by OK G7 antiserum and absorbed antiserum to K88b . One hundred and forty-nine strains of K88+ E coli representing seven somatic O groups were agglutinated by antibody K88-13; 142 of these, from at least six somatic O groups were agglutinated by 5CA3 and the remainder by K88-18 . Monoclonal antibody K88-13 bound to cryostat sections of ileum from piglets infected with E coli strains Abbotstown and G7 . K88-18 and 5CA3 bound only to sections from piglets infected with G7 and Abbotstown strains, respectively . It is concluded that monoclonal antibody K88-13 recognises an epitope on the a region of K88 while monoclonal antibody K88-18 is directed towards the b region . These monoclonal antibodies together with 5CA3 can be used to routinely identify K88+ E coli isolates.

Proc Natl Acad Sci U S A, 1987 Sep, 84(18), 6506 - 10
Integration host factor is required for the DNA inversion that controls phase variation in Escherichia coli; Eisenstein BI et al.; The on-and-off expression (phase variation) of type 1 fimbriae, encoded by fimA, in Escherichia coli is controlled by the inversion of a promoter-containing 314-base-pair DNA element . This element is flanked on each side by a 9-base-pair inverted, repeat sequence and requires closely linked genes for inversion . Homology analysis of the products of these genes, fimB and fimE, reveals a strong similarity with the proposed DNA binding domain of lambda integrase, which mediates site-specific recombination in the presence of integration host factor . Integration host factor, encoded by himA and hip/himD, binds to the sequence 5' TNYAANNNRTTGAT 3', where Y = pyrimidine and R = purine, in mediating integration-excision . In analyzing the DNA flanking the fim 314-base-pair inversion sequence, we found the adjacent sequence 5' TTTAACTTATTGAT 3', which corresponds perfectly with the consensus integration host factor binding site . To characterize the role of himA in phase variation, we transduced either a deletion of himA or an insertionally inactivated hip/himD gene into an E . coli strain with a fimA-lacZ operon fusion . We found the rate of phase variation decreases sharply from 10(-3) to less than 10(-5) per cell per generation . Southern hybridization analysis demonstrates that the himA mutation results in a failure of the switch-generated genetic rearrangement . When the transductant was transformed with a himA+ plasmid, normal switching returned . Thus integration host factor is required for normal type 1 fimbriae phase variation in E . coli.

Proc Natl Acad Sci U S A, 1987 Sep, 84(17), 6083 - 7
Role of glutamic acid-181 in DNA-sequence recognition by the catabolite gene activator protein (CAP) of Escherichia coli: altered DNA-sequence-recognition properties of {Val181}CAP and {Leu181}CAP; Ebright RH et al.; It has been proposed that Glu-181 of the catabolite gene activator protein (CAP) makes direct contact with certain base pairs of the specific DNA site . We have purified wild-type CAP and two substituted CAP variants, {Val181}CAP and {Leu181}CAP, and have assessed the DNA-sequence-recognition properties in vitro with respect to positions 5, 6, 7, 8, and 16 of the DNA site . The data indicate that {Val181}CAP and {Leu181}CAP fail to discriminate between the consensus DNA base pair and the three non-consensus-DNA base pairs at 2-fold-related positions 7 and 16 of the DNA site . In contrast, {Val181}CAP and {Leu181}CAP retain the ability to discriminate between different base pairs at positions 5 and 8 of the DNA site . We conclude that Glu-181 of CAP makes a direct contact with 2-fold-related positions 7 and 16 of the DNA site, as proposed previously based on in vivo results . We propose that upon replacement of Glu-181 by valine or leucine, this contact is eliminated and is replaced by no other functional contact . We estimate that the contact by Glu-181 with each position contributes -0.7 kcal/mol to the total CAP-DNA binding free energy.

J Bacteriol, 1987 Sep, 169(9), 4279 - 84
Mutations that create new promoters suppress the sigma 54 dependence of glnA transcription in Escherichia coli; Reitzer LJ et al.; Escherichia coli rpoN mutants lack sigma 54 and are therefore unable to initiate the transcription of glnA at glnAp2, which is required for the production of a high intracellular concentration of glutamine synthetase . We have found that the dependence on sigma 54 can be overcome by mutations that have apparently created a new sigma 70-dependent promoter . The position -35 RNA polymerase contact site of this new promoter overlaps glnAp2 . The initiation of transcription at the new promoter is inhibited by sigma 54-RNA polymerase even in the absence of nitrogen regulator I-phosphate, the activator required for the initiation of transcription at glnAp2 . The results suggest that in cells growing with an excess of nitrogen and therefore lacking nitrogen regulator I-phosphate, sigma 54-RNA polymerase is bound at glnAp2.

J Bacteriol, 1987 Sep, 169(9), 3926 - 31
Isolation, genetic mapping, and characterization of Escherichia coli K-12 mutants lacking gamma-glutamyltranspeptidase; Suzuki H et al.; Escherichia coli K-12 mutants lacking gamma-glutamyltranspeptidase (EC 2.3.2.2) were isolated after mutagenesis of cells with ethyl methanesulfonate . They lost the enzyme activity to different extents . The mutations of two mutants that had lost the enzyme activity completely were mapped at 76 min of the E . coli K-12 linkage map . These mutations made the cells neither nutrient requiring nor cold sensitive . The mutants leaked much more glutathione into the medium than the wild type . We propose the symbol ggt for these mutations.

Infect Immun, 1987 Sep, 55(9), 2204 - 7
Serological response to the P fimbriae of uropathogenic Escherichia coli in pyelonephritis; de Ree JM et al.; Uropathogenic Escherichia coli strains isolated from four patients with pyelonephritis were characterized by their O:K serotype, hemolysin production, mannose-resistant hemagglutination, and the serotype of the P fimbriae . These P fimbriae were serotyped with specific monoclonal antibodies . Serum samples from the patients were analyzed for the presence of specific antibodies to the P fimbriae . In all cases antifimbrial antibodies were found, strongly suggesting that these P fimbriae are expressed in vivo . However, the antibodies in the patient sera were not able to inhibit the mannose-resistant hemagglutination . This finding suggests that these antibodies react with the fimbrial components and not with the minor components which are responsible for adhesion.

J Gen Microbiol, 1987 Sep, 133 ( Pt 9), 2631 - 8
The isolation of fumB mutants of Escherichia coli; Henson JM et al.; Escherichia coli strains lacking the terminus region of the chromosome (min 29-36) due to an IS10-promoted deletion did not grow well in rich medium; they also did not grow on fumarate minimal medium because fumAC (min 35.7) is deleted . Strains with secondary mutations that partially suppress the deletion phenotype displayed healthier growth on rich medium and grew on minimal fumarate medium . These suppressor mutants had an IS10 insertion just upstream of the fumB structural gene (min 93.4) . A strain with a Tn10 insertion at this location was constructed and used to delete nonessential fumB; fumB deletion mutants grew well on both rich and minimal fumarate media.

Plasmid, 1987 Sep, 18(2), 135 - 41
Enhanced transforming activity of ultraviolet-irradiated pSV2-gpt is due to damage outside the gpt transcription unit; Leadon SA et al.; We have shown that when pSV2-gpt is introduced into human cells by calcium phosphate coprecipitation, the yield of Gpt+ transformants is increased by irradiating the plasmid with 254 nm uv . To elucidate the mechanism underlying this response, we constructed pSV2-gpt molecules in which the uv damage was confined to a particular region: a 3.0-kb region containing the pBR322 sequences and simian virus 40 (SV40) sequences not required for expression of the gpt gene, or a 2.3-kb fragment containing the Escherichia coli gpt gene together with the SV40 early promoter and sequences needed for splicing and polyadenylation . The transforming activity of the plasmid was greatly enhanced by uv damage confined to the 3.0-kb pBR322 region and increased linearly with uv dose up to 1 kJ/m2, but remained relatively constant at doses between 2 and 8 kJ/m2 . Positioning the damaged region upstream, or both upstream and downstream, from the gpt transcription unit increased the uv enhancement slightly compared to positioning the damaged region only downstream . In contrast, transforming activity was significantly decreased by damage in the 2.3-kb gpt transcription unit . These results suggest that uv damage outside a selectable marker gene in a plasmid can increase the probability of stable integration of the plasmid into the genome of recipient cells without inhibiting expression of of the gene.

Plasmid, 1987 Sep, 18(2), 111 - 9
Mode of insertion of the broad-host-range plasmid RP4 and its derivatives into the chromosome of Myxococcus xanthus; Jaoua S et al.; The mode of insertion of the broad-host-range plasmid RP4 into the chromosome of Myxococcus xanthus strain DZ1 has been analyzed . The plasmid integrated in numerous sites of the chromosome and generated insertional mutations . There is a hot spot of integration located between 31.5 and 34.5 kb clockwise from the EcoRI site of the plasmid . In the absence of this segment the insertion can, however, take place, but much less efficiently . The presence of transposable elements on the plasmid decreases severely the insertion frequency . Once integrated, RP4 could be transferred back to Escherichia coli, either by precise excision or with a segment of the Myxococcus chromosome . The role of site-specific recombination in RP4 integration is discussed.

Genetika, 1987 Sep, 23(9), 1702 - 4
{Site of integration of IS1-element responsible for expression of the yeast ADE1 gene in Escherichia coli}; Gedminene GK et al.; Cloned yeast ADE1 gene is expressed in Escherichia coli, due to integration of the IS1 bacterial element into the non-coding 5' region . Primary structure analysis of the integration site of IS1 element demonstrated that this site is situated in a region flanked by two inverted repeats of yeast DNA which are homologous to the right end of the IS1 element . The region up-stream (5') to the left inverted repeat is AT-rich.

Mol Biol (Mosk), 1987 Sep-Oct, 21(5), 1201 - 19
{Dissociation of unstable cointegrate plasmids formed during transposition of IS1 element: the role of IS1 encoded recombinase}; Danilevich VN; The properties of IS1/Tn9'-mediated cointegrates between plasmids pDK57 (pBR322:: :: Tn9') and pRP3.1--the deletion derivative of RP1 were investigated . It was found that IS1/Tn9'-mediated integration of pDK57 into the active transcribed regions of pRP3.1 (in particular kan and tet genes) leads to formation of unstable cointegrates capable of resolving in E . coli K-12 rec+ and recA cells . The structure of dissociation products of unstable cointegrates was studied . According to the data received in rec+ cells, the unstable cointegrates mainly produced plasmids pDK57 and pBR322::IS1--Cms-derivative of pDK57 as resolution products . In recA cells the cointegrates dissociate in different ways, and this process leads to the formation of not only pDK57 and pBR322::IS1, but also to the production of the deletion derivatives of these plasmids as well as to the derivatives of pDK57 and pBR322::IS1, containing duplications of IS1 or separate parts of Tn9' . It was concluded that the IS1-specific recombinase is involved in the dissociation (resolution) of unstable IS1/Tn9'-mediated cointegrates . This recombinase recognizes the sites localized in both inverted termini of IS1 as well as in the adjacent DNA segments . Hence, it is possible, that the IS1 recombinase is involved also in the generation of IS1-adjacent delations.

J Antimicrob Chemother, 1987 Sep, 20(3), 303 - 12
Influence of specific growth rate and nutrient-limitation upon the sensitivity of Escherichia coli towards polymyxin B; Wright NE et al.; The sensitivity of Escherichia coli to the lytic action of polymyxin B was assessed for cells grown in a chemostat at a variety of specific growth rates and under conditions of carbon, nitrogen, phosphorus and magnesium limitation . Magnesium and phosphorus limited cells demonstrated a trend of increased resistance with increasing growth-rate, whereas carbon and nitrogen limited cells increased their sensitivity as the growth rate increased . Divergent patterns of sensitivity, such as these, allowed a number of models for resistance towards polymyxin to be assessed . It was not possible to attribute polymyxin sensitivity to any single envelope component; rather the patterns of sensitivity reflect, in a complex manner, presence of envelope proteins and acidic phospholipids.

EMBO J, 1987 Sep, 6(9), 2719 - 25
The highly conserved amino-terminal region of the protein encoded by the v-myb oncogene functions as a DNA-binding domain; Klempnauer KH et al.; The retroviral oncogene v-myb encodes a 45,000 Mr nuclear protein (p45v-myb) that is predominantly associated with the chromatin of transformed cells . It has previously been shown that p45v-myb, when released from chromatin by salt-treatment, binds to DNA . To analyse the biochemical properties of p45v-myb in more detail we have expressed the v-myb coding region in Escherichia coli . Our results demonstrate that bacterially expressed myb protein has an intrinsic DNA-binding activity . Using two alternative strategies, (i) inhibition of DNA-binding by monoclonal antibodies and (ii) analysis of DNA-binding activities of partially deleted forms of the bacterial myb protein, we show that the DNA-binding domain is located in the amino-terminal region of the v-myb protein . This region has been highly conserved between myb genes of different species . Our results are therefore consistent with the hypothesis that DNA-binding is an important aspect of myb protein function.

Resuscitation, 1987 Sep, 15(3), 187 - 99
Prevention by granulocyte depletion of endotoxin-induced adult respiratory distress syndrome . An experimental study in pigs; Modig J et al.; To see whether circulating granulocytes are of importance in mediating endotoxin-induced pulmonary failure with similar characteristics to the human adult respiratory distress syndrome (ARDS), we made pigs granulocytopenic by injecting hydroxyurea via a central venous line for 10 days . The circulating granulocyte count decreased by 95% prior to the start of the continuous (6 h) endotoxin infusion (n = 8), whereas pigs sham-infused for 10 days had a normal granulocyte count before starting the continuous endotoxin infusion (n = 8) . Four granulocyte depleted pigs served as controls and received physiological saline instead of endotoxin . No physiological changes occurred in controls during the observation period of 6 h and all animals survived . Granulocyte depletion in response to endotoxin was associated with significantly attenuated pulmonary hypertension, significantly better preservation of cardiac output, significantly better maintained arterial oxygenation and a significantly smaller increase in extravascular lung water throughout the observation period as compared with non-granulocytopenic animals . Seven out of 8 granulocytopenic animals survived the observation period as against 4 out of 8 in non-granulocytopenic animals . Although one should be extremely careful in applying experimental results to the clinical situation, our findings might support the potential use of agents which interfere with granulocyte activation in septic-induced ARDS.

Mol Gen Genet, 1987 Sep, 209(2), 257 - 64
Site-specific integration and excision of pMEA100 in Nocardia mediterranei; Madon J et al.; Nocardia mediterranei strain LBG A3136 contains the 23.7 kb element pMEA100 in a chromosomally integrated form as well as in the free state (Moretti et al . 1985) . The integrated form of this element can be excised precisely from the Nocardia chromosome without any accompanying rearrangements in flanking chromosomal DNA . After transfer into plasmid-free mutant strains, pMEA100 reintegrates site specifically into its original chromosomal locus . The exact mapping of the pMEA100 integration site was accomplished by restriction analysis and DNA sequencing . The attachment site of pMEA100, the junctions of its integrated form and plasmid-free chromosomal DNA of N . mediterranei contain an identical 47 bp long sequence which is probably required for site-specific recombination connected with integration and excision of pMEA100 . Only one such sequence was found in the chromosome of pMEA100-free N . mediterranei derivatives as suggested by the single integration locus.

Arch Biochem Biophys, 1987 Sep, 257(2), 352 - 6
Contrasting fates of the paraquat monocation radical in Escherichia coli B and in Dunaliella salina; Rabinowitch HD et al.; Anaerobic cultures of Escherichia coli exposed to paraquat (PQ2+) accumulated the corresponding monocation radical PQ+., both within the cells and in the suspending medium . The green alga, Dunaliella salina, which is susceptible to a light- and O2-dependent toxicity of PQ2+, was nevertheless unable to cause accumulation of PQ+ . when illuminated anaerobically and could, moreover, discharge the ESR signal and the blue color of PQ+ . accumulated by E . coli . Spin trapping allowed demonstration of the photoproduction of O2- within D . salina and of the augmentation of that O-2 production by PQ2+ . D . salina appears to contain an electron sink and a heat-labile mechanism for transferring electrons from PQ+ . to that sink . This mechanism was demonstrable anaerobically but did not prevent PQ+.-mediated O2- production under aerobic conditions.

Mutat Res, 1987 Sep, 192(1), 1 - 6
Mutagenicity studies with N6, O2'-dibutyryl cyclic adenosine 3',5'-monophosphate (DBcAMP); Shimada H et al.; N6,O2'-Dibutyryl cyclic adenosine 3,5-monophosphate (DBcAMP) was studied for mutagenicity using the rec assay, the Ames method, and in vitro cytogenetics . DBcAMP had no mutagenic effect on B . subtilis in the rec assay, or on S . typhimurium (TA1535, TA1537, TA1538, TA98 and TA100) or E . coli (WP2 uvrA) . In the cytogenetic study, a significant increase in chromosomal aberrations was observed at a concentration of 50,000 micrograms/ml, but it was considered that this effect could be attributed to the secondary effect of the high osmotic pressure in the culture medium . These results suggest that DBcAMP has no mutagenic potential.

Virology, 1987 Sep, 160(1), 176 - 82
Intertypic recombinants of herpes simplex virus types 1 and 2 infected-cell polypeptide 4; Smith CA et al.; The wild-type ICP4s (infected-cell polypeptide 4) encoded by herpes simplex virus types 1 and 2 (HSV-1 and HSV-2) are functionally interchangeable . In order to test the functional interchangeability of their intramolecular domains, a series of intertypic ICP4 genes was constructed and characterized to determine if any of the encoded chimeric proteins were functionally impaired . We generated the recombinants in Escherichia coli using cloned ICP4 genes and the lambda recombination vectors developed by D . Carroll and R . S . Ajioka (1980, Gene 10, 273-281) and D . Carroll, R . S . Ajioka, and C . Georgopoulos (1980, Gene 10, 261-271) . We chose to generate the recombinants in E . coli in order to avoid imposing any restrictions with respect to the biological activities of their chimeric protein products . Six different recombinants encoding chimeric ICP4s were studied . As determined by restriction enzyme analysis, one of the six encodes an ICP4 protein whose amino-terminus is type 1 and whose carboxy-terminus is type 2 . Five recombinants encode ICP4 proteins whose amino-termini are type 2 and carboxy-termini, type 1 . The recombinant ICP4 proteins were assessed for their ability to stimulate transcription driven by the HSV-1 thymidine kinase promoter and for their ability to complement the growth of d120 and hr259, deletion mutants in HSV-1 and HSV-2 ICP4, respectively . All six recombinants exhibited wild-type levels of functional activity in both assay systems, demonstrating the colinearity of sequences specifying the intramolecular domains of HSV-1 and HSV-2 ICP4 and their functional interchangeability.

Proc Natl Acad Sci U S A, 1987 Sep, 84(18), 6434 - 7
Three-dimensional structure of a genetically engineered variant of porcine growth hormone; Abdel-Meguid SS et al.; The three-dimensional structure of a genetically engineered variant of porcine growth hormone, methionyl porcine somatotropin (MPS), has been determined at 2.8-A resolution, using single crystal x-ray diffraction techniques . Phases were obtained by use of a single isomorphous K2OsCl6 derivative and were improved by use of the density modification procedure . The MPS structure is predominantly helical . It consists mainly of four antiparallel alpha-helices arranged in a left twisted helical bundle, a structural motif observed in a number of other unrelated proteins . However, the way the four helices are connected in the bundle is unusual and, to our knowledge, has never been reported before . Alignment of the amino acid sequence of MPS with that of other growth hormones reveals that residues within the alpha-helices are predominantly invariant and thus these invariant residues are necessary to maintain the structural integrity of these proteins.

Proc Natl Acad Sci U S A, 1987 Sep, 84(18), 6369 - 73
Macrophage synthesis of nitrite, nitrate, and N-nitrosamines: precursors and role of the respiratory burst; Iyengar R et al.; The macrophage cell line RAW 264.7 when activated with Escherichia coli lipopolysaccharide and interferon-gamma synthesized nitrite (NO3-) and nitrate (NO3-) . Medium change after the activation showed that L-arginine was the only amino acid essential for this synthesis . D-Arginine would not substitute for L-arginine . Other analogues that could replace L-arginine were L-homoarginine, L-arginine methyl ester, L-arginamide, and the peptide L-arginyl-L-aspartate . L-Argininic acid, L-agmatine, L-ornithine, urea, L-citrulline, and ammonia were among the nonprecursors, while L-canavanine inhibited this L-arginine-derived NO2-/NO3- synthesis . When morpholine was added to the culture medium of the activated RAW 264.7 macrophages, N-nitrosation took place, generating N-nitrosomorpholine . GC/MS experiments using L-{guanido-15N2}arginine established that the NO2-/NO3- and the nitrosyl group of N-nitrosomorpholine were derived exclusively from one or both of the terminal guanido nitrogens of arginine . Chromatographic analysis showed that the other product of the L-arginine synthesis of NO2-/NO3- was L-citrulline . The role of the respiratory burst in NO2-/NO3- synthesis was examined using the macrophage cell lines J774.16 and J774 C3C . Both cell lines synthesized similar amounts of NO2-/NO3- . However, J774 C3C cells do not produce superoxide and hence do not exhibit the respiratory burst . Additional experiments also ruled out the involvement of the respiratory burst in NO2-/NO3- synthesis.

Proc Natl Acad Sci U S A, 1987 Sep, 84(17), 6050 - 4
Expression of enzymatically active poliovirus RNA-dependent RNA polymerase in Escherichia coli; Morrow CD et al.; The poliovirus genome is replicated by a virus-encoded RNA-dependent RNA polymerase (RNA polymerase) . The RNA polymerase is first synthesized as a larger precursor polypeptide, which is subsequently processed by a viral proteinase, 3Cpro, to give the mature polymerase molecule, 3Dpol . To further characterize the poliovirus RNA polymerase, we have constructed plasmids that expressed this protein in Escherichia coli . The plasmids consisted of fusions between the E . coli DNA encoding the first 13 amino acids of the trp operon leader protein and viral genes encoding the 3Cpro and 3Dpol polypeptides . E . coli harboring such plasmids gave significant, inducible levels of enzymatically active RNA polymerase as determined by the poly(A).oligo(U) poly(U) polymerase assay . The purified RNA polymerase activity from E . coli corresponded to a protein with the approximate molecular weight of the mature 3Dpol protein . The availability of a recombinant, enzymatically active poliovirus RNA polymerase provides a system in which we can precisely delineate the role this enzyme plays in the regulation of poliovirus replication.

Biochimie, 1987 Sep, 69(9), 991 - 9
A three-dimensional model of domain III of the Escherichia coli small ribosomal subunit; Elson D et al.; A three-dimensional model of domain III (nucleotides 920 to 1395) of the 30S ribosomal subunit of E . coli is proposed . The data used as a guide in folding the secondary structure of the RNA into a tertiary structure are four long range RNA-RNA interactions proposed by us on the basis of experiments performed in this laboratory plus two sets of data from other laboratories: protein-RNA cross-linking sites for proteins S1, S3, S7, S10 and S12, and the interprotein distances determined by neutron scattering . The model is consistent with nearly all of the published experimental findings on the structure of domain III.

J Biochem (Tokyo), 1987 Sep, 102(3), 455 - 64
Use of a series of ompF-ompC chimeric proteins for locating antigenic determinants recognized by monoclonal antibodies against the ompC and ompF proteins of the Escherichia coli outer membrane; Yamada H et al.; A method is presented for the efficient location of antigenic determinants using a series of chimeric proteins . By means of in vivo homologous recombination between the ompC and ompF genes coding for OmpC and OmpF, homologous proteins of the Escherichia coli outer membrane, a series of ompF-ompC chimeric genes was constructed (Nogami, T., Mizuno, T., & Mizushima, S . (1985) J . Bacteriol . 164, 797-801, and this work) . The OmpF-OmpC chimeric proteins expressed by these genes were successfully used to locate antigenic determinants recognized by monoclonal antibodies, which specifically react with either the OmpC or OmpF protein . Interaction between monoclonal antibodies and the chimeric proteins was examined by means of either enzyme-linked immunosorbent assay or immunoblot analysis . The antigenic determinants recognized by three anti-OmpC antibodies and one anti-OmpF antibody were thus located . Finally, the polypeptides covering these regions were chemically synthesized for two of them and then tested as to their reactivity with the antibodies . The peptides reacted with the corresponding antibodies when the former were chemically coupled with bovine serum albumin . Most of the monoclonal antibodies isolated in this work were highly specific to the unfolded monomer of the protein against which the antibody was raised . But they did not react with the trimer, the native form . These results are discussed in relation to the structures and functions of the OmpC and OmpF proteins . The use of a series of monoclonal antibodies for studying the mechanism of protein translocation across the cytoplasmic membrane is also discussed.

EMBO J, 1987 Sep, 6(9), 2607 - 15
Analysis of cytokeratin domains by cloning and expression of intact and deleted polypeptides in Escherichia coli; Magin TM et al.; Using recombination of an appropriate expression vector system (pINDU) with a complete cDNA encoding a basic (type II) cytokeratin, i.e . cytokeratin 8 (1) of Xenopus laevis, we transformed Escherichia coli cells to synthesize considerable amounts of an insoluble eukaryotic cytoskeletal protein . The cytokeratin was deposited in large 'inclusion bodies' in the bacterial cytoplasm but did not form detectable filamentous structures . However, when the E . coli-expressed cytokeratin was purified and combined in vitro with an authentic cytokeratin of the complementary, i.e . acidic (type I) subfamily, it formed typical intermediate-sized filaments (IFs) . Using Bal31 deletion from either the 5' or the 3' end of the cDNA, series of polypeptides progressively deleted from the amino or the carboxy terminus were produced in E . coli and identified by monoclonal antibodies . These assays allowed the mapping of epitopes . The deletion polypeptides of cytokeratin 8 were further examined to localize the region(s) involved in the heterotypic binding of alpha-helices of type I cytokeratins, using an in vitro nitrocellulose blot binding assay . We show that a region of 37 amino acids located in the central portion of coil 2 of the alpha-helical rod domain is sufficient for the specific recognition of a radiolabelled type I cytokeratin, i.e . cytokeratin 18 (D) from rat liver . In addition, deletion polypeptides containing only coil 1 of the alpha-helical rod also bind strongly the complementary cytokeratin . This indicates that the capability of heterotypic recognition and complex formation is not restricted to a single signal sequence but is located in distant and independent alpha-helical domains.(ABSTRACT TRUNCATED AT 250 WORDS)

J Nutr, 1987 Sep, 117(9), 1629 - 37
Immunologically mediated growth depression in chicks: influence of feed intake, corticosterone and interleukin-1; Klasing KC et al.; The effects of an immune response on growth and feed efficiency in chicks and the role of interleukin-1 (IL-1) and corticosterone (Cort) as mediators of the response were investigated . Daily injections of either sheep red blood cells or the inflammatory agent Sephadex resulted in significantly (P less than 0.05) lower rates of weight gain, feed intake and efficiency of feed utilization than controls fed ad libitum, indicating an immunologically mediated stress . Feeding control chicks the same amount of diet as that consumed by immunologically challenged chicks did not completely equalize rates of weight gain . Injections of a crude preparation of IL-1, but not Cort, resulted in weight gain, feed intake and efficiency of feed utilization that were similar to those of immunologically challenged chicks . The concentrations of IL-1 and Cort, measured by bioassay and radioimmunoassay, respectively, in serum from immunologically challenged chicks were significantly higher than in nonchallenged chicks . To determine the influence of IL-1 and Cort on protein accretion in skeletal muscles, the extensor digiti communus and ulnaris lateralis were incubated in the presence of these two hormones at concentrations similar to that seen in serum after an immunologic challenge . Cort did not affect the rate of protein degradation but resulted in rates of protein synthesis that were significantly lower than controls . IL-1 did not affect the rate of protein synthesis but resulted in rates of protein degradation that were about 24% greater than controls.

Proc Natl Acad Sci U S A, 1987 Sep, 84(18), 6516 - 20
Autogenous control of the S10 ribosomal protein operon of Escherichia coli: genetic dissection of transcriptional and posttranscriptional regulation; Freedman LP et al.; The S10 ribosomal protein operon is regulated autogenously by the product of one of the genes of the operon, the gene encoding ribosomal protein L4 . We have used site-directed mutagenesis to isolate leader mutations affecting L4 control . The phenotypes of these mutants demonstrate that L4 regulates both transcription and translation of the S10 operon . Several mutations abolish both levels of L4 control; others eliminate either transcriptional or translational control with little or no effect on the other mode of regulation . We conclude that L4-mediated transcriptional and translational control share some sequence requirements, but the two regulatory processes recognize somewhat different features of the S10 leader . Primary as well as secondary structures within the S10 leader appear to be involved.

Mutat Res, 1987 Sep, 180(1), 1 - 9
Bleomycin-induced mutagenesis in repackaged lambda phage: base substitution hotspots at the sequence C-G-C-C; Povirk LF; DNA isolated from lambda phage was treated with bleomycin A2 plus Fe2+ . The bleomycin-damaged DNA was added to lambda packaging extracts and the resulting phage were grown in SOS-induced E . coli . Under these conditions, treatment of the DNA with 0.8 microM bleomycin reduced the viability of the repackaged phage to 3% and increased the frequency of clear-plaque mutants in the progeny by a factor of 16 . Bleomycin-induced mutations which mapped to the DNA-binding domain of the cI gene were subjected to DNA-sequence analysis . The most frequent events were single-base substitutions at G:C base pairs, nearly all of which occurred at cytosines in the sequence Py-G-C . Cytosines in the third position of the sequence C-G-C-C were particularly susceptible to mutation . At A:T base pairs, mutations were less frequent and were a mixture of single-base substitutions and -1 frameshifts, occurring primarily at G-T and A-T sequences . Thus, the overall specificity of bleomycin-induced mutations matches that of bleomycin-induced DNA lesions (strand breaks and apyrimidinic sites), which are formed at G-C (particularly Py-G-C), G-T and, to a lesser extent, A-T sequences . Furthermore, the frequency of various types of substitutions was consistent with selective incorporation of A and T residues opposite apyrimidinic sites at these sequences . The highly selective nature of bleomycin-induced mutations may explain the lack of mutagenesis by this compound in a number of reversion assays.

J Bacteriol, 1987 Sep, 169(9), 4203 - 10
Escherichia coli K-12 mutants in which viability is dependent on recA function; Clyman J et al.; A gene required for growth and viability in recA mutants of Escherichia coli K-12 was identified . This gene, rdgB (for Rec-dependent growth), mapped near 64 min on the E . coli genetic map . In a strain carrying a temperature-sensitive recA allele, recA200, and an rdgB mutation, DNA synthesis but not protein synthesis ceased after 80 min of incubation at 42 degrees C, and there was extensive DNA degradation . The rdgB mutation alone had no apparent effect on DNA synthesis or growth; however, mutant strains did show enhanced intrachromosomal recombination and induction of the SOS regulon . The rdgB gene was cloned and its-gene product identified through the construction and analysis of deletion and insertion mutations of rdgB-containing plasmids . The ability of a plasmid to complement an rdgB recA mutant was correlated with its ability to produce a 25-kilodalton polypeptide as detected by the maxicell technique.

J Bacteriol, 1987 Sep, 169(9), 4003 - 10
Lipopolysaccharide tightly bound to porin monomers and trimers from Escherichia coli K-12; Rocque WJ et al.; Lipopolysaccharide (LPS) bound to isolated porin was detected on polyacrylamide gels by using a carbohydrate-specific silver stain and on Western blots by using anti-lipid A monoclonal antibodies . Porin was isolated from Escherichia coli JF733 (Ra chemotype) and D21f2 (Re chemotype) . Isolated porin was separated from loosely associated LPS by polyacrylamide gel electrophoresis (PAGE) in sodium dodecyl sulfate (SDS) . Unheated porin traveled on gels as aggregates, presumably trimers, with an apparent molecular weight of 78,000 to 83,000 . After heating to 100 degrees C for 2 min in SDS, the porin traveled as a monomer with a molecular weight of 36,000 . The unheated, high-molecular-weight trimer band reacted in the gel with the carbohydrate-specific silver stain, while the heated monomer band showed no staining . In contrast, lipid A-specific monoclonal antibodies showed reactivity on Western blots to the 36,000-molecular-weight band but not to the trimer . Finally, both monomer and trimer bands were isolated from gels and rerun by SDS-PAGE . LPS was released from the trimer preparation when the sample was heated, but the monomer band that was formed by heating the trimer isolate still reacted with anti-lipid A antibodies . Quantitative Limulus amebocyte lysate analysis revealed an approximately equal molar ratio of LPS to protein in the electroeluted porin monomer . Thus, some but not all of the LPS could be released from trimer complexes by boiling in SDS . The isolated monomer did not release more LPS on boiling in SDS a second time but still had LPS tightly bound, as detected by lipid A-specific monoclonal antibodies.

J Neurochem, 1987 Sep, 49(3), 756 - 63
Isolation of NILE glycoprotein-related cDNA probes; Sajovic P et al.; The nerve growth factor (NGF)-induced large external (NILE) glycoprotein is an NGF-inducible surface component of PC12 cells that is also widely distributed in the nervous system . It has recently been shown to be indistinguishable from the high-molecular-weight species of the brain antigens L1 and neuron-glia cell adhesion molecule (Ng-CAM) and may have a function in regulating cell adhesion in the developing nervous system . We have used polyclonal anti-NILE antisera to screen a lambda gt11 cDNA library made from NGF-treated PC12 cells . Four molecular probes have been isolated that encode parts of the apoprotein, related proteins, or both . These probes are 1,500, 800, 330, and 300 base pairs long, respectively, and in Northern blots they recognize a family of messages having lengths of approximately 5.9, 3.4, 2.4, and 1.9 kilobases . The two smaller messages are modestly but reproducibly up-regulated by NGF in PC12 cells, as is the NILE glycoprotein; however, only the two larger species would appear to be large enough to encode it . These messages are prominent in brain but not in nonneural tissues, in accordance with the observed levels of the protein . The recombinant phages produce fusion proteins that share specific epitopes with the NILE glycoprotein but not with other proteins . In these experiments, filters coated with recombinant fusion protein were prepared . Antibodies bound to and eluted from these filters specifically immunoprecipitate NILE glycoprotein, but not other proteins, from PC12 cell extracts . Other activities present in the original sera are not specifically retained by recombinant fusion proteins, and Escherichia coli lysates made with nonrecombinant lambda gt11 do not absorb the anti-NILE activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochem Biophys Res Commun, 1987 Aug 31, 147(1), 315 - 21
Purification and characterization of recombinant human interleukin-1 beta produced in Escherichia coli; Kikumoto Y et al.; Recombinant human interleukin-1 beta (rIL-1 beta) produced in Escherichia coli was purified to homogeneity by a combination of mass ion exchange column chromatography, ion exchange and gel filtration high performance liquid chromatography . The purified rIL-1 beta had a molecular weight of 18 kD on SDS-polyacrylamide gel electrophoresis and an isoelectric point of 6.9 on analytical isoelectric focusing . These values were almost same as those of natural interleukin-1 beta . The amino acid composition and amino acid sequence of the amino terminal region were consistent with those deduced from the cDNA sequence . In addition, the primary structure was confirmed by peptide mapping with lysyl-endopeptidase on reverse phase HPLC . Besides rIL-1 beta with amino terminal Ala, two molecular species, {Met0} rIL-1 beta and {desAla1} rIL-1 beta, were also obtained . Biological and physicochemical properties of the three species of rIL-1 beta were compared.

FEBS Lett, 1987 Aug 31, 221(1), 87 - 90
The primary structure of iron superoxide dismutase from Escherichia coli; Schinina ME et al.; The complete amino acid sequence of iron superoxide from Escherichia coli has been determined . The sequence was deduced from analysis of peptides obtained after cleavage of the carboxymethylated apoenzyme with trypsin . Stapholococcus aureus protease or CNBr . The polypeptide chain is made up of 192 residues and is easily aligned with the other known amino acid sequences of iron and manganese superoxide dismutases from various sources . The iron superoxide dismutase from E . coli shows a significantly higher homology with the iron enzyme from a different organism than with the manganese isoenzyme from E . coli.

Biochem Biophys Res Commun, 1987 Aug 31, 147(1), 501 - 5
Cyclic AMP-induced expression of the mouse lactate dehydrogenase-A promoter-cat fusion gene in Chinese hamster ovary wild-type cells, but not in cAMP-dependent protein kinase mutant cells; Hou EW et al.; The promoter region of mouse lactate dehydrogenase-A gene was fused with the chloramphenicol acetyltransferase gene of Escherichia coli, and the expression of this fusion gene was induced by cyclic AMP in Chinese hamster ovary wild-type cells, but not in mutants affecting the regulatory or catalytic subunit of cAMP-dependent protein kinase.

Cell, 1987 Aug 28, 50(5), 681 - 91
MAT alpha 1 protein, a yeast transcription activator, binds synergistically with a second protein to a set of cell-type-specific genes; Bender A et al.; We show by electrophoresis mobility shift and by DNAase I footprinting assays that the alpha 1 product of the yeast alpha mating-type locus binds to homologous sequences within the control regions of the three known alpha-specific genes . Binding requires both alpha 1 and a second yeast protein(s) (called PRTF) that is present in all three cell types (a, alpha, and a/alpha); neither protein binds alone . Binding and competition experiments using synthetic oligonucleotides indicate that PRTF binds to only part of the homology found at alpha-specific genes and imply that alpha 1 binds to the remainder . Our results suggest that alpha 1 renders gene expression alpha-specific by creating a binding site for PRTF . Similar experiments lead to the idea that PRTF also plays a role in transcription of a-specific genes . Perhaps a-specificity is achieved through the occlusion of the PRTF binding site by alpha 2, the negative regulator encoded by the alpha mating-type locus.

Science, 1987 Aug 28, 237(4818), 992 - 8
Characterization by tandem mass spectrometry of structural modifications in proteins; Biemann K et al.; Tandem mass spectrometry can be used to solve a number of protein structural problems that are not amenable to conventional methods for amino acid sequencing . Typical problems that use this approach involve characterization of peptides with blocked amino termini or peptides that have been otherwise posttranslationally processed, such as, by phosphorylation or sulfation . The structure and homogeneity of synthetic peptides can also be evaluated . Since peptides can be selectively characterized in the presence of other peptides or contaminants, the need for extensive purification is reduced or eliminated.

Science, 1987 Aug 28, 237(4818), 1044 - 6
Activation of adenovirus promoters by the adenovirus E1A protein in cell-free extracts; Spangler R et al.; The primary product of the adenovirus E1A gene is a protein that is sufficient for controlling host-cell proliferation and immortalizing primary rodent cells . The mechanism by which the protein induces these cellular effects is poorly understood, but might be linked to its ability to regulate RNA transcription from a number of viral and cellular genes . The mechanism of E1A's transcriptional-activation (trans-activation) was studied here by monitoring the protein's effect on specific adenovirus promoters in two types of transcriptional systems in vitro . One of these systems consisted of extracts from transformed cells constitutively expressing E1A, and the other consisted of extracts of HeLa cells supplemented with a plasmid-encoded E1A protein purified from Escherichia coli . The results show that the E1A protein specifically stimulates transcription from adenovirus promoters; thus, the induction of cellular transcription factors is not necessary to explain the stimulation of transcription by E1A.

Biochemistry, 1987 Aug 25, 26(17), 5439 - 47
The elasticity of uniform, unilamellar vesicles of acidic phospholipids during osmotic swelling is dominated by the ionic strength of the media; Haines TH et al.; Uniform, unilamellar vesicles have been prepared by the pH-modification technique . The initial sizes of the vesicles were from 200 to 700 nm and were measured to within 1-3% by photo correlation spectroscopy . Vesicles were made of the dioleoyl esters of phosphatidic acid, phosphatidylcholine, phosphatidylglycerol, phosphatidylethanolamine, the diphytanyl ethers of phosphatidylglycerol, Escherichia coli lipids, and lac permease reconstituted into E . coli lipids . The vesicle suspensions were prepared and then diluted with electrolyte (KCl) and/or nonelectrolyte (sucrose, trehalose, pentaerythritol) impermeants . The amplitude of the swelling is linearly proportional to the osmotic pressure difference across the bilayer . We have determined the elastic modulus, the elastic limit (percent surface expansion at bursting), and the transbilayer pressure difference at bursting for each of these vesicles at constant osmolarity but at different ionic strengths . We find that the elastic properties of the bilayer vary by a factor of 10 in electrolyte media as compared to isosmolal nonelectrolyte media and that this variation appears to be related to both the charge density at the surface and the ionic strength of the media . Anionic lipid vesicles in 150 mM KCl have a significantly higher modulus (50 X 10(7) dyn/cm2) and transbilayer pressure difference (40 mosM) at bursting with a small capacity to stretch (3-4% surface expansion) compared to the same vesicles suspended in nonelectrolyte impermeants . The latter vesicles undergo a significant surface expansion (8-10%), display a low modulus (3 X 10(7) dyn/cm2), and burst at 3-4 mosM bilayer pressure difference . Vesicles suspended in media of constant osmolarity at various ionic strengths display properties with proportional values.(ABSTRACT TRUNCATED AT 250 WORDS)

Nucleic Acids Res, 1987 Aug 25, 15(16), 6539 - 52
Visualization of an AAF induced frameshift mutation: molecular views of base displacement in B-DNA from minimized potential energy calculations; Broyde S et al.; Energy minimized structures of base displacement in an AAF modified B-DNA dodecamer are presented . A rational search strategy, beginning with a global search of the conformation space of the modified deoxydinucleoside monophosphate, together with model building by computer graphics, has been employed . A number of different minimum energy conformations have been located which reveal base displaced structures . These show fluorene interstrand stacking, fluorene inter- and intrastrand stacking, and non-stacked fluorene situated in the denatured bulge . The local helix axis is bent to various extents in the different forms, and one or two base pairs are fully denatured . One structure of special interest offers a molecular view that suggests how AAF can induce the -2 deletion mutation observed in AAF modified E . coli.

Biochemistry, 1987 Aug 25, 26(17), 5548 - 55
Reversible unfolding of ribosomal protein E-L30: an NMR study; van de Ven FJ et al.; Ribosomal protein E-L30 unfolds reversibly at pH values between 7.0 and 4.5 . Unfolding of the protein involves a fast and a slow equilibrium, which depend on the degree of protonation of His19 and His33 . Both the fast equilibrium between protonated and deprotonated histidines and the slow equilibrium between folded and unfolded protein could be monitored by means of 500-MHz 1H NMR spectroscopy . The degree of protonation of His19 and His33 appears to be determinant in the unfolding process of the protein . It is shown however that even when the histidines are uncharged, the protein has only limited stability, probably as a result of the presence of all four Glu's of E-L30 in its triple-stranded beta-sheet . At equimolar concentrations of the folded and unfolded form, the rate constant characterizing the transition between these forms is approximately 0.14 s-1 . Making use of sequential resonance assignments of the 1H NMR spectrum {van de Ven, F.J.M., & Hilbers, C.W . (1986) J . Mol . Biol . 192, 419-441}, the fast equilibrium could be interpreted in terms of alterations in the spatial structure of E-L30 in a specific domain of the molecule . This domain is also affected by temperature although not in exactly the same manner as by pH.

Nucleic Acids Res, 1987 Aug 25, 15(16), 6469 - 88
Purification of the FLP site-specific recombinase by affinity chromatography and re-examination of basic properties of the system; Meyer-Leon L et al.; The FLP protein, a site-specific recombinase encoded by the 2 micron plasmid of yeast, has been purified to near homogeneity from extracts of E . coli cells in which the protein has been expressed . The purification is a three column procedure, the final step employing affinity chromatography . The affinity ligand consists of a DNA polymer with multiple FLP protein binding sites arranged in tandem repeats . This protocol yields 2 mg of FLP protein which is 85% pure . The purified protein is highly active, stable for several months at -70 degrees C and free of detectable nucleases . The molecular weight and N-terminal sequence are identical to that predicted for the FLP protein by the DNA sequence of the gene . Purified FLP protein primarily, but not exclusively, promotes intramolecular recombination . Intermolecular recombination becomes the dominant reaction when E . coli extracts containing no FLP protein are added to the reaction mixture . These extracts are not specifically required for recombination, but demonstrate that some properties previously attributed to FLP protein can be assigned to contaminating proteins present in E . coli.

J Biol Chem, 1987 Aug 25, 262(24), 11841 - 6
Purification and characterization of a glycine betaine binding protein from Escherichia coli; Barron A et al.; A major component of the Escherichia coli response to elevated medium osmolarity is the synthesis of a periplasmic protein with an Mr of 31,000 . The protein was absent in mutants with lambda placMu insertions in the proU region, a locus involved in transport of the osmoprotectant glycine betaine . This periplasmic protein has now been purified to homogeneity . Antibody directed against the purified periplasmic protein crossreacts with the fusion protein produced as a result of the lambda placMu insertion, indicating that proU is the structural gene specifying the 31-kDa protein . The purified protein binds glycine betaine with high affinity but has no affinity for either proline or choline, clarifying the role of proU in osmoprotectant transport . The amino-terminal sequence of the mature glycine betaine binding protein is Ala-Asp-Leu-Pro-Gly-Lys-Gly-Ile-Thr-Val-Asn-Pro.

Biochemistry, 1987 Aug 25, 26(17), 5428 - 32
Acid unfolding and self-association of recombinant Escherichia coli derived human interferon gamma; Arakawa T et al.; The secondary and tertiary structure of recombinant human interferon gamma, determined by far- and near-UV circular dichroism, showed a transition from the native state to an unfolded state below pH 4.5 . The acid unfolding was extensively studied at pH 3.5 as a function of NaCl concentration . Addition of 0.05-0.2 M NaCl to a pH 3.5 sample increased the amount of beta-sheet structure with no change in the amount of alpha-helix and also induced reversible self-association of interferon gamma to form large aggregates from the monomer . When samples at pH 3.5 were dialyzed against 0.1 M ammonium acetate (pH 6.9) to refold interferon gamma, the samples that contained NaCl in acid formed aggregates upon dialysis while those without NaCl formed a dimer apparently identical with the starting protein (i.e., before acid treatment) . Thus, the self-association of interferon gamma in acid is closely correlated with its aggregation behavior in 0.1 M ammonium acetate after removal of acid.

Biochemistry, 1987 Aug 25, 26(17), 5422 - 7
Sedimentation equilibrium measurements of recombinant DNA derived human interferon gamma; Yphantis DA et al.; Recombinant DNA derived human interferon gamma (IFN-gamma) from Escherichia coli was examined by equilibrium ultracentrifugation . Short-column equilibrium experiments at pH 6.9 in 0.1 M ammonium acetate buffer gave a z-average molecular weight of 33,500 +/- 1400 at infinite dilution, corresponding to 1.98 +/- 0.08 times the formula weight . Long- (2.6 mm) column experiments at pH 7.5 in 0.04 M imidazole buffer gave a molecular weight of 33,400 +/- 500 . Under the latter conditions IFN-gamma behaves somewhat nonideally, with the departure from ideality accounted for by an effective (Donnan) charge of about 6+ . No association of this dimer to form tetramer or higher polymers was observed, with the association constant for formation of tetramer from dimer K24 found to be less than 34 L mol-1 . Similarly, no dissociation to monomers was observable, with the dissociation constant to monomer K21 being less than 5 X 10(-8) mol L-1 . At pH 3.55 in 0.02 M buffer (acetate plus acetic acid), there was virtually complete dissociation of the dimer to monomer . Extreme nonideality was seen in this low ionic strength system, and the effective charge on the protein was estimated to be about 11+ . The reduced molecular weight M(1 -upsilon rho) of the monomer was found to be about 4.09 +/- 0.20 kg mol-1; this corresponds to a molecular weight of 16,410 +/- 820, with the Scatchard definition of components . A small amount of a polymer with a molecular weight of about 0.5 X 10(6) was detected under these conditions.

Biochemistry, 1987 Aug 25, 26(17), 5416 - 22
tRNA recognition site of Escherichia coli methionyl-tRNA synthetase; Leon O et al.; We have previously shown that anticodon bases are essential for specific recognition of tRNA substrates by Escherichia coli methionyl-tRNA synthetase (MetRS) {Schulman, L . H., & Pelka, H . (1983) Proc . Natl . Acad . Sci . U.S.A . 80, 6755-6759} and that the enzyme tightly binds to C34 at the wobble position of E . coli initiator methionine tRNA (tRNAfMet) {Pelka, H., & Schulman, L . H . (1986) Biochemistry 25, 4450-4456} . We have also previously demonstrated that an affinity labeling derivative of tRNAfMet can be quantitatively cross-linked to the tRNA binding site of MetRS {Valenzuela, D., & Schulman, L . H . (1986) Biochemistry 25, 4555-4561} . Here, we have determined the site in MetRS which is cross-linked to the anticodon of tRNAfMet, as well as the location of four additional cross-links . Only a single peptide, containing Lys465, is covalently coupled to C34, indicating that the recognition site for the anticodon is close to this sequence in the three-dimensional structure of MetRS . The D loop at one corner of the tRNA molecule is cross-linked to three peptides, containing Lys402, Lys439, and Lys596 . The 5' terminus of the tRNA is cross-linked to Lys640, near the carboxy terminus of the enzyme . Since the 3' end of tRNAfMet is positioned close to the active site in the N-terminal domain {Hountondji, C., Blanquet, S., & Lederer, F . (1985) Biochemistry 24, 1175-1180}, this result indicates that the carboxy ends of the two polypeptide chains of native dimeric MetRS are folded back toward the N-terminal domain of each subunit.

J Biol Chem, 1987 Aug 25, 262(24), 11446 - 8
The stereospecific labilization of the C-4' pro-S hydrogen of pyridoxamine 5'-phosphate is abolished in (Lys258----Ala) aspartate aminotransferase; Kochhar S et al.; Reconstitution of wild-type apoaspartate aminotransferase from Escherichia coli with {4'-3H}pyridoxamine 5'-phosphate results in stereospecific release of the pro-S C-4' 3H to the solvent . The reaction follows first-order kinetics (t1/2 = 15 min at pH 7.5 and 25 degrees C), its rate constant being similar to that found previously with mitochondrial aspartate aminotransferase from chicken (Tobler, H.P., Christen, P., and Gehring, H . (1986) J . Biol . Chem . 261, 7105-7108) . Substituting the active site residue Lys258 by alanine via site-directed mutagenesis yields a catalytically inactive enzyme (Malcolm, B . A., and Kirsch, J . F . (1985) Biochem . Biophys . Res . Commun . 132, 915-921) . This mutant enzyme fails to release any measurable 3H from bound {4'-3H}pyridoxamine 5'-phosphate . The data are consistent with earlier proposals that Lys258 is indispensable for the ketimine/aldimine tautomerization, and corroborate the previous conclusion that 3H exchange from enzyme-bound pyridoxamine 5'-phosphate mechanistically corresponds to the deprotonation at C-4' of the ketimine intermediate during the transamination reaction.

J Biol Chem, 1987 Aug 25, 262(24), 11813 - 6
Mutagenesis affecting the carboxyl terminus of the biotinyl subunit of transcarboxylase . Effects on biotination; Murtif VL et al.; Biotin is added to biotin-containing enzymes as a post-translational modification catalyzed by holoenzyme synthetase . This reaction is fairly general in that synthetase from one organism will modify enzymes from heterologous sources . This suggests that the polypeptides share some structural characteristic(s) that define(s) them as biotin enzymes . We have reported previously that when the gene coding for the 1.3 S biotinyl subunit of transcarboxylase is expressed in Escherichia coli, the polypeptide produced is biotinated by the cellular synthetase . Using in vitro mutagenesis of this gene, we have begun to define the primary structure involved in the enzymatic addition of biotin to a lysine residue . We show here that the carboxyl terminus of the 1.3 S subunit is critical in biotination . Mutations affecting the COOH-terminal residue do not influence the modification, but elimination of the hydrophobic side chain of the penultimate residue abolishes biotin addition.

J Biol Chem, 1987 Aug 25, 262(24), 11785 - 9
Isolation and characterization of two types of cDNA for mitochondrial adenylate kinase and their expression in Escherichia coli; Kishi F et al.; Two cDNA clones for mitochondrial adenylate kinase were isolated from a cDNA library of bovine liver poly(A)+ RNA by using synthetic oligodeoxynucleotides as probes . The clone containing a 0.9-kilobase insert had the reading frame for a 241-residue protein (AK2A), while the other clone containing a 1.6-kilobase insert had the frame for a 234-residue protein (AK2B) . Nucleotide sequences of these two clones were the same in the 5' portion up to the coding sequence for the 233rd residue, but different in the remaining 3' portions . The reported amino acid sequence of mitochondrial adenylate kinase from bovine heart corresponded to AK2A . Neither AK2A nor AK2B had a cleavable NH2-terminal presequence as that found in other imported mitochondrial proteins . RNA blot analysis of poly(A)+ RNAs from bovine liver and heart revealed three species of mRNA with approximate sizes of 0.9, 1.4, and 1.7 kilobases . The 1.7- and 1.4-kilobase species were specific for AK2B, whereas the 0.9-kilobase species was specific for AK2A . In the liver, the 1.7-kilobase mRNA was more abundant, whereas in the heart the 0.9-kilobase mRNA was predominant . The 1.4-kilobase mRNA was present only in the heart . The AK2A- and AK2B-coding sequences were expressed in Escherichia coli cells under the control of trc promoter . Both the products reverted the temperature-sensitive phenotype of the adenylate kinase mutant of E . coli.

J Biol Chem, 1987 Aug 25, 262(24), 11422 - 7
Interactions of the catabolite activator protein (CAP) at the galactose and lactose promoters of Escherichia coli probed by hydroxyl radical footprinting . The second CAP molecule which binds at gal and the one CAP at lac may act to stimulate transcription in the same way; Shanblatt SH et al.; The catabolite activator protein (CAP) binding sites of the Escherichia coli galactose and lactose operons were probed by hydroxyl radical footprinting . This method reveals each base that is protected by the bound protein . The patterns of protection seen for the primary CAP sites at gal and lac were virtually identical . In the presence of RNA polymerase the footprint of the second CAP molecule at gal was found to be very similar to those at the other two sites . This upstream site in gal align's perfectly with the lac CAP site with respect to the start of P1 transcription . Replacing most of the gal second CAP site DNA with heterologous sequences did not abolish binding although it became noticeably weaker . In vitro transcription studies of this hybrid gal promoter DNA further demonstrated the reduced affinity of the second CAP . These results are consistent with molecular models proposed for specific CAP binding and suggest that the second CAP at gal may be responsible for overall stimulation of transcription at this operon . Thus, in spite of differences in stoichiometry, the mechanisms of activation by CAP at gal and lac may be quite similar.

J Biol Chem, 1987 Aug 25, 262(24), 11584 - 90
Fusion of trpB and trpA of Escherichia coli yields a partially active tryptophan synthetase polypeptide; Yanofsky C et al.; The separate alpha and beta polypeptides of the tryptophan synthetase of bacteria are represented in fungi by a fusion polypeptide in which the first third is homologous to bacterial alpha chains and the remainder is homologous to bacterial beta chains . In the yeast polypeptide, a short nonhomologous "connector" joins the two homologous segments . The chromosomal order of all bacterial genes that specify tryptophan synthetase beta and alpha chains, respectively, is trpB-trpA . Fusion of these genes in their present arrangement would result in the synthesis of a polypeptide with a segmental order, N-beta-alpha-C, opposite that observed in fungi . To investigate possible explanations for the apparent transposition that occurred in the evolution of the fungal gene we have made two fusions of trpB and trpA of Escherichia coli in their natural orientation . We find that the fusion proteins are synthesized but both are less active catalytically than the wild type bacterial protein . In addition, the fusion proteins associate abnormally, they are activated only slightly by wild type alpha or beta 2, and they are less sensitive than the wild type protein to inhibition by antibodies to alpha or beta 2 . The fusion proteins have normal substrate affinities . Our findings suggest that the altered structures of the fusion proteins affect catalytic ability and the locations of the alpha and/or beta chain combining sites . This structural distortion may have prevented the natural selection of direct gene fusions during the course of the fungal gene's evolution.

Biochemistry, 1987 Aug 25, 26(17), 5486 - 92
F0 portion of Escherichia coli ATP synthase: orientation of subunit c in the membrane; Deckers-Hebestreit G et al.; Incubation of right-side-out oriented membrane vesicles of Escherichia coli with tetranitromethane resulted in the nitration of tyrosine residues (Tyr-10 and Tyr-73) of subunit c from the ATP synthase . Cleavage of the protein with cyanogen bromide and separation of the resulting fragments, especially of the tyrosine-containing peptides, clearly demonstrated that the distribution of the nitro groups is similar at any time and at any pH value chosen for the analysis . Furthermore, the percentage of 3-nitrotyrosine present in the two peptide fragments was in good agreement with that obtained for the intact polypeptide chain . While the modification of the tyrosine residues in subunit c with the lipophilic tetranitromethane is independent of the orientation of the membrane vesicles, the subsequent partial conversion of the 3-nitrotyrosine to the amino form only occurred when membrane vesicles with right-side-out orientation were treated with the ionic, water-soluble sodium dithionite, which at certain concentrations cannot penetrate biological membranes . Cleavage of subunit c isolated from nitrated and subsequently reduced membrane vesicles and separation of the resulting fragments by high-pressure liquid chromatography showed that the 3-nitrotyrosine in the Tyr-73-containing peptides has been completely reduced, while the nitro group in peptides containing Tyr-10 remained nearly unaffected.

Biochemistry, 1987 Aug 25, 26(17), 5541 - 8
Magnetic interaction between the tyrosyl free radical and the antiferromagnetically coupled iron center in ribonucleotide reductase; Sahlin M et al.; Ribonucleotide reductases from Escherichia coli and from mammalian cells are heterodimeric enzymes . One of the subunits, in the bacterial enzyme protein B2 and in the mammalian enzyme protein M2, contains iron and a tyrosyl free radical that both are essential for enzyme activity . The iron center in protein B2 is an antiferromagnetically coupled pair of high-spin ferric ions . This study concerns magnetic interaction between the tyrosyl radical and the iron center in the two proteins . Studies of the temperature dependence of electron paramagnetic resonance (EPR) relaxation and line shape reveal significant differences between the free radicals in proteins B2 and M2 . The observed temperature-dependent enhanced EPR relaxation and line broadening of the enzyme radicals are furthermore completely different from those of a model UV-induced free radical in tyrosine . The results are discussed in terms of magnetic dipolar as well as exchange interactions between the free radical and the iron center in both proteins . The free radical and the iron center are thus close enough in space to exhibit magnetic interaction . For protein M2 the effects are more pronounced than for protein B2, indicating a stronger magnetic interaction.

Biochemistry, 1987 Aug 25, 26(17), 5433 - 9
Affinity labeling of Escherichia coli phenylalanyl-tRNA synthetase at the binding site for tRNAPhe; Hountondji C et al.; Periodate-oxidized tRNA(Phe) (tRNA(oxPhe)) behaves as a specific affinity label of tetrameric Escherichia coli phenylalanyl-tRNA synthetase (PheRS) . Reaction of the alpha 2 beta 2 enzyme with tRNA(oxPhe) results in the loss of tRNAPhe aminoacylation activity with covalent attachment of 2 mol of tRNA dialdehyde/mol of enzyme, in agreement with the stoichiometry of tRNA binding . Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the PheRS-{14C}tRNA(oxPhe) covalent complex indicates that the large (alpha, Mr 87K) subunit of the enzyme interacts with the 3'-adenosine of tRNA(oxPhe) . The {14C}tRNA-labeled chymotryptic peptides of PheRS were purified by both gel filtration and reverse-phase high-performance liquid chromatography . The radioactivity was almost equally distributed among three peptides: Met-Lys{Ado}-Phe, Ala-Asp-Lys{Ado}-Leu, and Lys-Ile-Lys{Ado}-Ala . These sequences correspond to residues 1-3, 59-62, and 104-107, respectively, in the N-terminal region of the 795 amino acid sequence of the alpha subunit . It is noticeable that the labeled peptide Ala-Asp-Lys-Leu is adjacent to residues 63-66 (Arg-Val-Thr-Lys) . The latter sequence was just predicted to resemble the proposed consensus tRNA CCA binding region Lys-Met-Ser-Lys-Ser, as deduced from previous affinity labeling studies on E . coli methionyl- and tyrosyl-tRNA synthetases {Hountondji, C., Dessen, P., & Blanquet, S . (1986) Biochimie 68, 1071-1078}.

J Mol Biol, 1987 Aug 20, 196(4), 853 - 75
Structural asymmetry in the CTP-liganded form of aspartate carbamoyltransferase from Escherichia coli; Kim KH et al.; The protein and solvent structure of the CTP-liganded form of aspartate carbamoyltransferase from Escherichia coli yields an R-factor of 0.155 for data to a resolution of 2.6 A . The model has 7353 protein atoms, 945 sites for solvent, and two molecules of CTP . A total of 25 of the 912 residues of the model exist in more than one conformation . The root-mean-square deviation of bond lengths and angles from their ideal values is 0.013 A and 2.1 degrees, respectively . The model reported here reflects a correction in the trace of the regulatory chain . One molecule of CTP binds to each of the two regulatory chains of the asymmetric unit of the crystal . The interactions between the pyrimidine of each CTP molecule and the protein are similar . The 4-amino group of CTP binds to the carbonyl groups of residues 89 (tyrosine) and 12 (isoleucine) of the regulatory chain . The nitrogen of position 3 of the pyrimidine binds to the amide group of residue 12; the 2-keto group binds to lysine 60 . The 2'-OH group of the ribose forms hydrogen bonds with lysine 60 and the carbonyl group of residue 9 (valine) . The binding of the phosphate groups of CTP to the regulatory chain probably reflects an incomplete association of CTP with the enzyme at pH 5.8 . A lattice contact influences the interaction between the triphosphate group of one CTP molecule and the protein . For the other CTP molecule, only lysine 94 binds to the phosphate groups of CTP . Of the two regulatory and two catalytic chains of the asymmetric unit of the crystal, there are only two significant violations of non-crystallographic symmetry . The active site in the vicinity of arginine 54 of one catalytic chain is larger than the active site of its non-crystallographic mate . The "expanded" cavity accommodates four solvent molecules in the vicinity of arginine 54 as opposed to two molecules of water for the "contracted" cavity . Furthermore, arginine 54 in the "expanded" pocket adopts two conformations, either hydrogen-bonding to glutamate 86 or to the phenolic oxygen atom of tyrosine 98; residues 86 and 98 are in a catalytic chain related by 3-fold symmetry to the catalytic chain of arginine 54 . In the "contracted" pocket, arginine 54 binds only to glutamate 86.(ABSTRACT TRUNCATED AT 400 WORDS)

J Mol Biol, 1987 Aug 20, 196(4), 789 - 99
Positive regulation of the Escherichia coli L-rhamnose operon is mediated by the products of tandemly repeated regulatory genes; Tobin JF et al.; The rhaC gene, whose product is the positive activator of the genes required for L-rhamnose utilization, has been cloned along with the rhamnose structural genes . The rhaC sequence shows two partially overlapping reading frames, encoding two proteins of molecular weight 32,000 and 35,000 RhaS and RhaR . Both proteins show significant homology to AraC, the positive activator of the arabinose operon . S1 mapping located transcriptional start points and showed that RhaR, and possibly RhaS, positively regulate transcription from the structural gene promoters as well as transcription from their own promoter . In-vivo dimethyl sulfate footprinting and DNase I footprinting indicate that the RhaR protein may bind to DNA elements upstream from its RNA polymerase binding site.

J Mol Biol, 1987 Aug 20, 196(4), 781 - 8
Location and sequence of the promoter of the gene for the NADH-dependent nitrite reductase of Escherichia coli and its regulation by oxygen, the Fnr protein and nitrite; Jayaraman PS et al.; The DNA sequence containing the start of the Escherichia coli nirB gene is reported . The N-terminal amino acid sequence of purified NADH-dependent nitrite reductase coincided with that predicted from the DNA sequence, confirming that nirB is the structural gene for nitrite reductase apoprotein and identifying the translation start point . Using nuclease S1 mapping, the sole transcription startpoint for the nirB gene was found 23 or 24 base-pairs upstream from the ATG initiation codon . By subcloning successively smaller DNA fragments into a beta-galactosidase expression vector plasmid, we located the promoter within a sequence bounded by a TaqI site at +14 with respect to the transcription startpoint and a HpaII site at -208 . Measurements in vivo of beta-galactosidase expression and RNA levels due to nirB promoter activity showed that this promoter was activated during anaerobic growth . Optimal activity was found only after anaerobic growth in the presence of nitrite . The sequence of the nirB promoter is compared with sequences found at other anaerobically activated promoters.

Anal Biochem, 1987 Aug 15, 165(1), 137 - 41
Determination of plasmid copy number by the "boiling" method; Ivanov IG et al.; A fast and reliable approach for determination of plasmid copy number in Escherichia coli is proposed, based on the "boiling" method (5) for separation of plasmid and chromosomal DNA . The method includes in vivo uniform labeling of total bacterial DNA, separation of DNA into plasmid and chromosomal DNA fractions, and quantitation of DNA in the two fractions by radioactivity measurement . No isolation and purification of native DNA are necessary.

J Biol Chem, 1987 Aug 15, 262(23), 11382 - 8
Studies on the role of actin's N tau-methylhistidine using oligodeoxynucleotide-directed site-specific mutagenesis; Solomon LR et al.; The primary structure of all actins except that isolated from Naegleria gruberi contains a unique N tau-methylhistidine (MeHis) at position 73 . This modified residue has been implicated as possibly being important for the post-translational processing of actin's amino terminus, the binding of actin to DNase I, and in the polymerization of G-actin . We have investigated the potential role of MeHis in each of these processes by utilizing site-directed mutagenesis to change His-73 of skeletal muscle actin to Arg and Tyr . Wild type and mutant actins were synthesized in vivo, using non-muscle cells transfected with mutant cDNAs, and in vitro by translating mutant RNAs synthesized using SP6 RNA polymerase in a rabbit reticulocyte lysate . We have found that actins containing Arg or Tyr at position 73 undergo amino-terminal processing, bind to DNase I-agarose, and become incorporated into the cytoskeleton of a nonmuscle cell as efficiently as wild type actin . Furthermore, using an in vitro copolymerization assay we have found that although there is no difference between the Arg mutant and the wild type actins, the Tyr mutant has a slightly greater critical concentration for polymerization . These results show that MeHis is not absolutely required for any of these processes.

J Biol Chem, 1987 Aug 15, 262(23), 11176 - 81
Purification and characterization of human recombinant interleukin-1 beta; Meyers CA et al.; A human interleukin-1 (IL-1) beta cDNA was cloned, and the region coding for the mature protein was expressed in Escherichia coli . The 17-kDa biologically active product was purified in 40% yield to apparent homogeneity, without chaotropes, from the soluble fraction of sonicated cell lysates . The recombinant IL-1 beta was characterized by amino acid analysis, NH2- and COOH-terminal sequence analysis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, spectroscopy, and biological assay . Specific biological activity was 4.6 X 10(8) units/mg in a co-mitogenic IL-2 induction assay using cultured EL-4 T-lymphocytes . The molar extinction coefficient was determined to be 10,300 cm-1 M-1 at 280 nm . NH2-terminal sequence analysis revealed that 70% of the product begins with the Ala corresponding to the NH2 terminus of the natural protein, while 30% begins with the following Pro . No initiator Met was observed . Both of the sulfhydryl groups are reactive to Ellman's reagent and to iodoacetamide under nonreducing conditions, indicating that the Cys residues do not form disulfide bonds . S-Carboxamidomethyl-Cys-rIL-1 beta retained biological activity in the IL-2 induction assay . Circular dichroism suggested an extensive beta sheet structure for rIL-1 beta.

J Biol Chem, 1987 Aug 15, 262(23), 10965 - 70
Contribution of different organs to increased glucose consumption after endotoxin administration; Meszaros K et al.; Glucose utilization of different organs (spleen, liver, ileum, kidney, skin, lung, and testis) was investigated in vivo in conscious rats 3, 24, or 48 h after treatment with 100 micrograms of endotoxin/100 g of body weight . Glucose uptake was determined by the 2-deoxyglucose technique, which was validated by demonstrating that endotoxin treatment did not alter either the intracellular retention of the phosphorylated metabolites (P-2-dGlc) of the tracer or the discrimination against 2-deoxyglucose in pathways of glucose metabolism . At 3 h after endotoxin the accumulation of P-2-dGlc was markedly increased in the liver (4.8-fold), spleen and skin (2.9-fold), lung (2.4-fold), and ileum and kidney (2.1-fold), as compared to time-matched controls . This effect was sustained in the liver at 24 and 48 h, was diminishing but still significant in spleen, ileum, and kidney, and absent in skin and lung . Accumulation of P-2-dGlc in the testis remained unchanged after endotoxin . Glucose uptake by individual organs and their contribution to whole body glucose utilization in control and endotoxin-treated rats were compared based on P-2-dGlc accumulation data . Organs rich in mononuclear phagocytes (liver and spleen) exhibited a marked and prolonged increase in glucose uptake after endotoxin . Yet the bulk of the increment in the whole body glucose disappearance rate (Rd) was due to three large tissues (skin, intestine, and muscle, accounting for more than 80% of the total P-2-dGlc accumulation in soft tissues), which showed a more moderate and transient increase in glucose utilization.

J Biol Chem, 1987 Aug 15, 262(23), 10938 - 45
Investigation of the role of individual tryptophan residues in the binding of Escherichia coli single-stranded DNA binding protein to single-stranded polynucleotides . A study by optical detection of magnetic resonance and site-selected mutagenesis; Khamis MI et al.; Fluorescence and optical detection of triplet state magnetic resonance (ODMR) spectroscopy have been employed to study the complexes formed between single-stranded polynucleotides and Escherichia coli ssb gene products (SSB) in which tryptophans 40, 54, and 88 are selectively, one residue at a time, replaced by phenylalanine using site-specific oligonucleotide mutagenesis . Fluorescence titrations and ODMR results indicate that tryptophans 40 and 54 are the only tryptophan residues in E . coli single-stranded DNA binding protein that are involved in stabilizing the protein-nucleic acid complexes via stacking interactions . Wavelength-selected ODMR measurements on E . coli SSB reveal the presence of two spectrally distinct tryptophan sites (Khamis, M . I., Casas-Finet, J . R., and Maki, A . H . (1987) J . Biol . Chem . 262, 1725-1733) . Our present results indicate that tryptophan 54 belongs to the blue-shifted site, while tryptophan 40 belongs to the red-shifted site of the protein.

Arch Biochem Biophys, 1987 Aug 15, 257(1), 194 - 9
Importance of disulfide linkage for constructing the biologically active human interleukin-2; Yamada T et al.; Recombinant human interleukin-2 (rIL-2) produced in Escherichia coli possesses a free thiol group at Cys-125 and a disulfide linkage between Cys-58 and Cys-105, as in the case for natural human interleukin-2 . Treatment of rIL-2 with 200 mM dithiothreitol resulted in the cleavage of the Cys-58-Cys-105 disulfide bond . The reduced form of rIL-2 thus obtained retained only 10% of the in vitro biological activity of the native form, as measured by the ability to stimulate the growth of an IL-2-dependent mouse natural killer cell line, NKC3 . Far-uv circular dichroism studies indicated that the cleavage of the disulfide bond results in a decrease of alpha-helix content . Near-uv circular dichroism studies suggested that the native molecule is folded into a rigid tertiary structure, while the reduced form showed a spectrum similar to that of rIL-2 denatured in the presence of 6 M guanidine.HCl . The once-reduced molecule was readily reoxidized in the presence of 10 microM Cu2+ to form the native molecule with full biological activity . These results strongly demonstrate that the Cys-58-Cys-105 disulfide linkage in the IL-2 molecule is essential for constructing a rigid and biologically active form of IL-2.

J Biol Chem, 1987 Aug 15, 262(23), 11375 - 81
Expression of cDNAs for G proteins in Escherichia coli . Two forms of Gs alpha stimulate adenylate cyclase; Graziano MP et al.; Complementary DNAs that encode two forms of the alpha subunit (Gs alpha) of the guanine nucleotide-binding protein responsible for stimulation of adenylate cyclase (Gs) have been inserted into plasmid vectors for expression in Escherichia coli . Following transformation of either of these plasmids into E . coli K38, Gs alpha accumulates to 0.4-0.8 mg/liter (approximately 0.1% of total protein), as judged by immunoblot analysis with specific antisera . Based on deduced amino acid sequence, the two cDNAs should encode proteins with molecular weights of 44,500 and 46,000, respectively (Robishaw, J.D., Smigel, M . D., and Gilman, A . G . (1986) J . Biol . Chem . 261, 9587-9590) . Expression of these cDNAs in E . coli yields proteins that co-migrate on sodium dodecyl sulfate-polyacrylamide gels with the Gs alpha subunits from S49 lymphoma cell membranes, with apparent molecular weights of 45,000 and 52,000, respectively . Low levels of activity are detected in the 100,000 X g supernatant after lysis and fractionation of E . coli expressing either form of Gs alpha . Partial purification of Gs alpha from E . coli lysates yields preparations in which significant and stable activity can be assayed . Both forms of Gs alpha migrate through sucrose gradients as soluble, monodisperse species in the absence of detergent . As expressed in E . coli, both forms of Gs alpha can reconstitute isoproterenol-, guanine nucleotide-, and fluoride-stimulated adenylate cyclase activity in S49 cyc-cell membranes to approximately the same degree and can be ADP-ribosylated with {32P}NAD+ and cholera toxin . However, based on the specific activity of purified rabbit liver Gs, only 1-2% of the Gs alpha expressed in E . coli appears to be active . Incubation of partially purified fractions of recombinant Gs alpha with guanosine 5'-(3-O-thio)triphosphate and resolved beta gamma subunits isolated from purified bovine brain G proteins results in a 7-10-fold increase in Gs activity . Incubation of bovine brain beta gamma with recombinant Gs alpha also leads to a dramatic increase in observed levels of cholera toxin-catalyzed {32P}ADP-ribosylation.

J Biol Chem, 1987 Aug 15, 262(23), 11280 - 3
Interaction with RNA polymerase of nucleosomal cores lacking one H2A.H2B dimer; Gonzalez PJ et al.; Nucleosomal particles lacking one H2A.H2B dimer interact with RNA polymerase from Escherichia coli more strongly than the complete nucleosomal core particles . Moreover, the in vitro transcription of the H2A.H2B-deficient particles is much more efficient than that of the whole nucleosomal cores, both in the presence and absence of rifampicin . Although a substantial fraction of particles in the preparation of whole nucleosomal cores binds to RNA polymerase, the efficiency of these particles as transcription templates is very small . This block to transcription is partially eliminated when one H2A.H2B dimer is released from the core particle . Our results suggest that the lack of one H2A.H2B dimer from nucleosomal particles might be required for the formation of complexes with RNA polymerase active in transcription.

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