Escherichia coli

J Neurochem, 1991 Nov, 57(5), 1615 - 22
Stress- and endotoxin-induced increases in brain tryptophan and serotonin metabolism depend on sympathetic nervous system activity; Dunn AJ et al.; Stressful treatments and immune challenges have been shown previously to elevate brain concentrations of tryptophan . The role of the autonomic nervous system in this neurochemical change was investigated using pharmacological treatments that inhibit autonomic effects . Pretreatment with the ganglionic blocker chlorisondamine did not alter the normal increases in catecholamine metabolites, but prevented the increase in brain tryptophan normally observed after footshock or restraint, except when the duration of the footshock period was extended to 60 min . The footshock- and restraint-related increases in 5-hydroxyindoleacetic acid (5-HIAA) were also prevented by chlorisondamine . The increases in brain tryptophan caused by intraperitoneal injection of endotoxin or interleukin-1 (IL-1) were also prevented by chlorisondamine pretreatment . The footshock-induced increases in brain tryptophan and 5-HIAA were attenuated by the beta-adrenergic antagonist propranolol but not by the alpha-adrenergic antagonist phenoxybenzamine or the muscarinic cholinergic antagonist atropine . Thus the autonomic nervous system appears to be involved in the stress-related changes in brain tryptophan, and this effect is due to the sympathetic rather than the parasympathetic limb of the system . Moreover, the main effect of the sympathetic nervous system is exerted on beta- as opposed to alpha-adrenergic receptors . We conclude that activation of the sympathetic nervous system is responsible for the stress-related increases in brain tryptophan, probably by enabling increased brain tryptophan uptake . Endotoxin and IL-1 also elevate brain tryptophan, presumably by a similar mechanism . The increase in brain tryptophan appears to be necessary to sustain the increased serotonin catabolism to 5-HIAA that occurs in stressed animals, and which may reflect increased serotonin release.

Microb Pathog, 1991 Nov, 11(5), 325 - 36
Localization and function of FanH and FanG, minor components of K99 fimbriae of enterotoxigenic Escherichia coli; Simons LH et al.; Specific antisera against FanG and against FanH were prepared by immunization with hybrid Cro-LacZ-FanG and Cro-LacZ-FanH proteins, respectively . Immunoblotting with these antisera revealed the presence of FanG and FanH as minor components in purified K99 fimbriae . Mutations were constructed in fanG and fanH and cells defective in FanG or FanH were characterized by ELISA, immunoblotting, adhesion assays and electron microscopy . A minicell experiment showed that the mutations in fanG or fanH had no effect on the expression of the other K99-specific proteins . Cells defective in FanG produced no fimbriae and did not agglutinate horse erythrocytes, but cell-free heat-shock preparations of these cells still bound the K99 glycolipid receptor . Cells defective in FanH produced 1-2% of the K99 fimbriae as compared with wild-type K99 producing cells . These mutant fimbriae appeared to be shorter but were still capable of binding the K99 glycolipid receptor . Apparently, FanG and FanH are not required for binding the K99 receptor . These results and analysis of K99 mutants by immunoblotting using a specific antiserum against another K99 minor component, FanF, indicated that the combinations FanF/FanG and FanF/FanH are required for the initiation and elongation (length determination) of K99 fimbriae formation, respectively.

Acta Virol, 1991 Nov, 35(6), 497 - 502
Analysis of strain-specific plasmid sequences from Coxiella burnetii; Minnick MF et al.; Acute isolates of Coxiella burnetii possess a 36-kbp plasmid termed QpH1 . DNA hybridizations show that QpH1 contains approximately 6-kbp region of DNA which is not present in the QpRS plasmid from chronic isolates . This QpH1-specific region of DNA contains the contiguous EcoRI fragments G, E, and D . The GED region was found to possess seven open reading frames (ORF's) coding for proteins ranging from 5.5 to 42.3 kDa in molecular mass when subcloned and expressed in vitro . Summing the predicted ORF's accounts for 95% of the GED coding potential . E . coli expression produced a stable 42.3-kDa protein from the pHIN19 subclone of GED . The ORF of the 42.3-kDa protein, termed cbhE', has been localized on GED by both in vitro transcription/translation and DNA sequencing . The cbhE' gene is estimated as 1142 bp in length with a putative promoter region of TCAACT (-35)-N16-TAAAAT (-10)-N14-AGAAGGA (Shine-Dalgarno)-N10-ATG.

Zentralbl Veterinarmed B, 1991 Nov, 38(9), 689 - 700
Characterization of a new fimbrial antigen present in Escherichia coli strains isolated from calves; Varga J; Thirteen Escherichia coli strains isolated from calves with diarrhoea, supposed to carry a common antigen were examined for their hemagglutinating activity and compared by bacterial agglutination, double diffusion in two dimensions and by crossed immunoelectrophoresis (CIE) . Two of the strains were examined also in the electron microscope . Most of the strains agglutinated red blood cells of horse, ox, guinea pig and chicken, of which the agglutination of ox erythrocytes was mannose-resistant (MRHA) . None of the strains agglutinated human erythrocytes . All strains with MRHA of ox red blood cells, regardless to their O:K:H antigens could be agglutinated in unabsorbed or absorbed antisera produced against cultures C1209 (020:K-:H9) and C1213 (09:K36:H-) when live cells as antigens were used . None of these sera agglutinated reference strains carrying K88, K99, 987P, F41 or FY (Att25) antigen respectively . By the double gel diffusion test and by CIE in extracts (60 degrees C) of the strains a common heat labile antigen, responsible for the MRHA of ox red blood cells was identified . Electron microscopy revealed that this common antigen was represented by thin, long, hair-like fimbriae on cells of E . coli C1213, and that specific homologous antibodies attached to these fimbriae.

J Antimicrob Chemother, 1991 Nov, 28(5), 655 - 62
Inhibition of K88ab-mediated haemagglutination by polymyxin B nonapeptide; Matranga AM et al.; The ability of the cyclic peptide polymyxin B nonapeptide (PMBN) to inhibit haemagglutination of erythrocytes by Escherichia coli bearing K88ab, K99 or F41 fimbriae was examined . The agent strongly inhibited K88ab-mediated haemagglutination, but had little or no effect on haemagglutination mediated by K99 or F41 fimbriae . Inhibition of K88ab-mediated haemagglutination did not result from release of fimbrial adhesins from the bacterial cell surface, nor from solubilization of K88ab receptors in erythrocytes . Since PMBN also prevented haemagglutination mediated by partially-purified K88ab fimbriae, the agent may directly obstruct access of fimbriae to their mammalian receptor binding sites.

FEMS Microbiol Lett, 1991 Nov 1, 68(1), 57 - 62
Interaction of P-fimbriated Escherichia coli with human meconium; Adlerberth I et al.; The ability of Escherichia coli with different receptor specificities to interact with meconium was studied . E . coli strains expressing P-fimbriae, specific for Gal alpha 1-4Gal beta-containing receptors, were agglutinated by meconium at high titres . This reaction was inhibited by globotetraosylceramide . The attachment of P-fimbriated E . coli to human colonic epithelial cells of the HT-29 cell line was inhibited by meconium . Some type 1 fimbriated strains were agglutinated by meconium, but the agglutination was rarely blocked by methyl alpha-D-mannoside . The attachment by type 1 fimbriated strains to HT-29 cells was reduced by meconium only in some cases . These results suggest that meconium interacts with the P-fimbriae of E . coli, in a way that may influence bacterial colonization of the neonatal intestine.

J Cell Biol, 1991 Nov, 115(4), 1021 - 9
A sialoglycoprotein complex linked to the microvillus cytoskeleton acts as a receptor for pilus (AF/R1) mediated adhesion of enteropathogenic Escherichia coli (RDEC-1) in rabbit small intestine; Rafiee P et al.; Escherichia coli strain RDEC-1 is an enteroadherent, diarrheagenic pathogen in rabbits that utilizes AF/R1 pili for initial (stage 1) adherence, but the host receptors for this adhesion are unknown . Here we demonstrate that RDEC-1 binds, via AF/R1 pili, to a specific rabbit ileal microvillus membrane glycoprotein receptor complex of subunits 130 and 140 kD . The binding involves sialic acid present on oligosaccharide moieties of the glycoprotein receptor . Furthermore, the microvillus membrane glycoprotein receptor complex appears to be associated with cytoskeletal components via brush border myosin 1 . This newly described link between AF/R1 receptor and cytoskeletal components suggests that, in addition to this function in mucosal adherence, the pili may facilitate subsequent (second stage) close effacing attachment of RDEC-1 to the host epithelium by influencing cytoskeletal function.

Proc Natl Acad Sci U S A, 1991 Nov 1, 88(21), 9730 - 3
Acetyl-CoA carboxylase from Escherichia coli: gene organization and nucleotide sequence of the biotin carboxylase subunit; Kondo H et al.; Biotin carboxylase {biotin-carboxyl-carrier-protein:carbon-dioxide ligase (ADP-forming), EC 6.3.4.14} is the enzyme mediating the first step of the acetyl-CoA carboxylase {acetyl-CoA:carbon-dioxide ligase (ADP-forming), EC 6.4.1.2} reaction . We screened an Escherichia coli DNA library and a DNA fragment carrying the biotin carboxylase gene fabG, and its flanking regions were cloned . The gene for biotin carboxyl carrier protein was found 13 base pairs upstream of the fabG gene . Nucleotide sequencing of the recombinant plasmids revealed that the fabG codes for a 449-amino acid residue protein with a calculated molecular weight of 49,320, a value in good agreement with that of 51,000 determined by SDS/polyacrylamide gel electrophoresis of the purified enzyme . The deduced amino acid sequence of biotin carboxylase is also consistent with the partial amino acid sequence determined by Edman degradation . The primary structure of this enzyme exhibits a high homology with those of other biotin-dependent enzymes and carbamoyl-phosphate synthetase {carbon-dioxide:L-glutamine amino-ligase (ADP-forming, carbamate-phosphorylating), EC 6.3.5.5}; therefore, all these enzymes probably function through the same mechanism of reaction.

J Med Microbiol, 1991 Nov, 35(5), 270 - 7
Biochemical phenotypes of enteropathogenic Escherichia coli common to Iran and Sweden; Katouli M et al.; A collection of 143 strains of enteropathogenic Escherichia coli (EPEC) of 11 different serogroups isolated from children with diarrhoea, 71 in Sweden and 72 in Iran, was tested for similarity with a computerised biochemical fingerprinting method . From Sweden, there were 54 different phenotypes, 42 consisting of a single strain and 12 (common phenotypes) containing more than one isolate . From Iran, there were 48 different phenotypes, 38 with only one strain and 10 with more than one . Many of the strains which were biochemically similar, in both countries, also had similar virulence factors . Nine Swedish and 20 Iranian isolates showed biochemical identity to at least one of the strains of the other country, most of them belonging to serogroups O55, O119, O126, O127 and O128 . The value of the biochemical fingerprinting method as an epidemiological tool and its ability to evaluate clonal relations among E . coli strains in different geographical areas is discussed.

J Exp Med, 1991 Nov 1, 174(5), 1167 - 77
Localized adherence by enteropathogenic Escherichia coli is an inducible phenotype associated with the expression of new outer membrane proteins; Vuopio-Varkila J et al.; Enteropathogenic Escherichia coli grow as discrete colonies on the mucous membranes of the small intestine . A similar pattern can be demonstrated in vitro; termed localized adherence (LA), it is characterized by the presence of circumscribed clusters of bacteria attached to the surfaces of cultured epithelial cells . The LA phenotype was studied using B171, an O111:NM enteropathogenic E . coli (EPEC) strain, and HEp-2 cell monolayers . LA could be detected 30-60 min after exposure of HEp-2 cells to B171 . However, bacteria transferred from infected HEp-2 cells to fresh monolayers exhibited LA within 15 min, indicating that LA is an inducible phenotype . Induction of the LA phenotype was found to be associated with de novo protein synthesis and changes in the outer membrane proteins, including the production of a new 18.5-kD polypeptide . A partial NH2-terminal amino acid sequence of this polypeptide was obtained and showed it to be identical through residue 12 to the recently described bundle-forming pilus subunit of EPEC . Expression of the 18.5-kD polypeptide required the 57-megadalton enteropathogenic E . coli adherence plasmid previously shown to be required for the LA phenotype in vitro and full virulence in vivo . This observation, the correspondence of the 18.5-kD polypeptide to an EPEC-specific pilus protein, and the temporal correlation of its expression with the development of the LA phenotype suggest that it may contribute to the EPEC colonial mode of growth.

J Bacteriol, 1991 Nov, 173(22), 7240 - 8
Targeted mutagenesis of the b subunit of F1F0 ATP synthase in Escherichia coli: Glu-77 through Gln-85; McCormick KA et al.; Subunit b of Escherichia coli F1F0 ATP synthase contains a large hydrophilic region thought to be involved in the interaction between F1 and F0 . Oligonucleotide-directed mutagenesis was used to evaluate the functional importance of a segment of this region from Glu-77 through Gln-85 . The mutagenesis procedure employed a phagemid DNA template and a doped oligonucleotide primer designed to generate a predetermined collection of missense mutations in the target segment . Sixty-one mutant phagemids were identified and shown to contain nucleotide substitutions encoding 37 novel missense mutations . Mutations were isolated singly or in combinations of up to four mutations per recombinant phagemid . F1F0 ATP synthase function was studied by mutant phagemid complementation of a novel E . coli strain in which the uncF (b) gene was deleted . Complementation was assessed by observing growth on solid succinate minimal medium . Many phagemid-encoded uncF (b) gene mutations in the targeted segment resulted in growth phenotypes indistinguishable from those of strains expressing the native b subunit, suggesting abundant F1F0 ATP synthase activity . In contrast, several specific mutations were associated with a loss of enzyme function . Phagemids specifying the Ala-79----Pro, Arg-82----Pro, Arg-83----Pro, or Gln-85----Pro mutation failed to complement uncF (b) gene-deficient E . coli . F1F0 ATP synthase displayed the greatest sensitivity to mutations altering a single site in the target segment, Ala-79 . The evidence suggests that Ala-79 occupies a restricted position in the enzyme complex.

Infect Immun, 1991 Nov, 59(11), 4013 - 8
Use of purified F1845 fimbrial adhesin to study localization and expression of receptors for diffusely adhering Escherichia coli during enterocytic differentiation of human colon carcinoma cell lines HT-29 and Caco-2 in culture; Kerneis S et al.; Whole diffusely adhering Escherichia coli (DAEC) C1845 cells bearing the F1845 adhesive factor bind diffusely to differentiated human colon carcinoma cell lines HT-29 and Caco-2 . By using antibodies directed against the purified fimbrial adhesin F1845 factor, the expression of the DAEC F1845-specific brush border receptors in the polarized human intestinal HT-29 and Caco-2 epithelial cells was studied by indirect immunofluorescence . A low level of DAEC F1845 receptors in undifferentiated intestinal cells was detected; they were localized in a cluster of cells . DAEC F1845 receptors were expressed at a high level in differentiated HT-29 and Caco-2 cells . DAEC F1845 receptors were expressed at a strikingly high level in the apical domains of the cells and developed during enterocytic differentiation in culture, in parallel with the apical expression of the intestinal brush border hydrolase, sucrase-isomaltase.

Infect Immun, 1991 Nov, 59(11), 3924 - 9
HeLa cell adherence, actin aggregation, and invasion by nonenteropathogenic Escherichia coli possessing the eae gene; Cantey JR et al.; Enteropathogenic Escherichia coli (EPEC) produce diarrhea in humans by a mechanism that involves close adherence to epithelial cells in the intestine and colon . Close adherence is associated with effacement of microvilli and condensation of actin beneath the bacteria, a process termed attaching/effacing adherence . Attaching/effacing adherence of EPEC occurs in vitro in tissue culture, simplifying the study of the molecular genetics of this process . An EPEC gene (eae) necessary for attaching/effacing adherence was recently characterized . Enterohemorrhagic E . coli and the rabbit-specific RDEC-1 strain adhere in a like fashion in vivo and hybridize with eae . However, these strains adhere poorly to tissue culture cells, complicating the in vitro study of attaching/effacing adherence . In order to develop an in vitro model for the study of attaching/effacing activity of non-EPEC bacteria, a plasmid encoding the F1845 adhesin of an E . coli strain (C1845) isolated from a patient with diarrhea was transformed into RDEC-1 and enterohemorrhagic E . coli . The transformed strains adhered in a diffuse pattern to HeLa cells, and they aggregated HeLa cell actin at points of adherence in the fluorescein-isothiocyanate-labeled phalloidin assay . They also invaded HeLa cells in a gentamicin invasion assay, although not to the extent seen with EPEC . The construction of adherent non-EPEC strains facilitates the molecular study of the attaching/effacing properties and invasiveness of these strains in tissue culture models.

Carcinogenesis, 1991 Nov, 12(11), 2089 - 92
A new technique for determining the distribution of N7-methylguanine using an automated DNA sequencer; Shoukry S et al.; We have developed a method to determine rapidly the sequence specificity of DNA alkylation resulting from chemical treatment . The utility of this approach is demonstrated here in a study of the sequence specificity of alkylation by dimethylsulphate (DMS) . The method is independent of the sequence chosen and makes use of the polymerase chain reaction (PCR) to generate a fluorescently labelled DNA target . In this study, a 302 bp segment of the Escherichia coli lacI gene was amplified and the product purified by liquid chromatography on a Mono Q column . This DNA was alkylated with DMS and treated with hot piperidine to produce single-strand breaks at sites of N7 alkylation . The distribution of the break points, and hence the position and extent of alkylation, were determined on an Applied Biosystems 370A automated DNA sequencer.

Arch Biochem Biophys, 1991 Nov 1, 290(2), 397 - 406
Solubilization and reprecipitation from intestinal brush border membranes of a complex containing guanylate cyclase activatable by the heat-stable enterotoxin; Katwa LC et al.; Extraction of pig intestinal brush border membranes with the zwitterionic detergent 3-{(3-cholamidopropyl)-dimethylammonio}-1-propanesulfonate (Chaps) in the presence of 0.5 M KCl yielded a solution which contained 60-70% of the receptor for the Escherichia coli heat-stable enterotoxin (STa) and of the Lubrol PX-activated guanylate cyclase activity present in the membrane . When the supernatant solution was diluted fivefold with 10 mM Hepes buffer (pH 7.4) and kept at 4 degrees C overnight, a precipitate formed . Centrifugation yielded a pellet (P2) which contained 25-30% of both the cyclase and the receptor in the original membranes, with a 2.5- to 3-fold enrichment of both . The process could be repeated for further enrichment (P4) . The addition of MgCl2 to the diluted extract affected both basal and STa-stimulated activity of P2; 1 mM was optimal . P2 resembled membranes with respect to competitive inhibition of 125I-STa binding by STa, and the concentration-dependent activation of cyclase by STa . Guanylate cyclase in resolubilized P2 was also activated by STa . Most of the enzymes interfering with guanylate cyclase determinations were removed, as were the brush border marker enzymes sucrase and gamma-glutamyltransferase, and a GTP-binding protein that is a pertussis toxin substrate . Specific cross-linking of 125I-STa to receptors in the membrane was preserved in P2 and P4, the three proteins showing the strongest radioactivity having relative molecular masses of 55,000-60,000, 70,000-80,000, and 135,000-140,000 . P2 and P4 appear to contain a complex of membrane proteins with certain functional properties intact.

J Virol, 1991 Nov, 65(11), 6084 - 93
Helix-loop-helix transcriptional activators bind to a sequence in glucocorticoid response elements of retrovirus enhancers; Corneliussen B et al.; A family of nuclear proteins, designated SL3-3 enhancer factors 2 (SEF2), were found to interact with an Ephrussi box-like motif within the glucocorticoid response element in the enhancer of the murine leukemia virus SL3-3 . Mutation of the DNA sequence decreased the basal enhancer activity in various cell lines . The important nucleotides for binding of SEF2 are conserved in most type C retroviruses . Various cell types displayed differences both in the sets of SEF2-DNA complexes formed and in their amounts . A cDNA which encoded a protein that interacted specifically with the SEF2-binding sequence was isolated from human thymocytes . The nucleotide sequence specificity of the recombinant protein, expressed in Escherichia coli, corresponded to that of at least one of the nuclear SEF2 proteins . Sequence analysis of the cDNA revealed that it belongs to the basic helix-loop-helix class of DNA-binding proteins . Several mRNA transcripts of different sizes were identified . Molecular analysis of cDNA clones revealed multiple related mRNA species containing alternative coding regions, which are most probably a result of differential splicing.

Glycobiology, 1991 Nov, 1(5), 441 - 7
Sialic acid activation; Kean EL; Cytidine 5'-monophosphosialic acid (CMP-sialic acid) is the activated form of sialic acid which is required for the biosynthesis of sialic acid-containing complex carbohydrates . Its discovery over 30 years ago by the laboratory of Dr Saul Roseman was a landmark in research dealing with the biosynthesis of these compounds . A review is presented of the salient features concerning this molecule: its discovery, chemistry, biosynthesis, subcellular location, enzymatic cleavage, transport and molecular biology . This report does not deal with its utilization by the sialyltransferases.

Development, 1991 Nov, 113(3), 723 - 34
Developmental analysis of the retinoic acid-inducible RAR-beta 2 promoter in transgenic animals; Mendelsohn C et al.; Retinoic acid (RA) is a signalling molecule important for pattern formation during development . There are three known types of nuclear receptors for RA in mammals, RAR-alpha, RAR-beta and RAR-gamma, which transduce the RA signal by inducing or repressing the transcription of target genes . Here we describe the developmental expression pattern of the mouse RAR-beta 2 promoter . Independent lines of transgenic animals expressing RAR-beta 2 promoter sequences fused to the E . coli beta-galactosidase gene were examined throughout the course of embryogenesis and found to exhibit reproducible and specific patterns of beta-galactosidase expression in a majority of sites that have been shown previously to contain mRAR-beta transcripts . In the limbs, mRAR-beta 2 promoter activity and mRAR-beta transcripts were both excluded from precartilagenous condensations; interestingly, mRAR-beta 2 promoter activity was observed in the apical ectodermal ridge (AER) where mRAR-beta transcripts could not be detected, while no mRAR-beta 2 promoter activity or mRAR-beta transcripts were associated with the limb region that contains the zone of polarizing activity (ZPA) . Analysis of the lacZ expression pattern in embryos from mothers treated with teratogenic doses of RA, indicated that mRAR-beta 2 promoter is selectively induced in a manner suggesting that overexpression of the mRAR-beta 2 isoform is involved in RA-generated malformations . The normal and induced expression pattern of the mRAR-beta 2 promoter suggests several possible roles for mRAR-beta 2 in development of the limbs, as an inhibitor of cartilage formation, in programmed cell death and in the formation of loose connective tissue.

J Protozool, 1991 Nov-Dec, 38(6), 25S - 28S
Pathobiology of cryptosporidiosis (C . baileyi) in broiler chickens; Blagburn BL et al.; Pathologic and clinicopathologic changes were examined in broiler chickens inoculated with Cryptosporidium baileyi (Cb) alone or in combination with infectious bronchitis virus (IBV), infectious bursal disease virus (IBDV) or Escherichia coli (Ec) . Concurrent infections with Cb and either IBV or Ec resulted in a greater respiratory inflammatory response than either agent given alone . Concurrent Cb, IBV or Ec infections resulted in a decreased density of respiratory cryptosporidial stages . No interactions between Cb and IBDV were observed . Clinicopathologic results in broiler chicks exhibiting signs of respiratory cryptosporidiosis indicated that pO2 decreased, pCO2 increased, HCO3 increased and CO2 increased . Changes in blood gases and serum electrolyte values correlated with signs of acute respiratory disease . Blood gases and serum electrolyte values were unchanged in birds with bursal and cloacal infections only . Results of these studies clarified pathogenetic events associated with avian respiratory cryptosporidiosis, and demonstrated that cryptosporidiosis may enhance the severity of respiratory disease caused by other avian pathogens.

Biull Eksp Biol Med, 1991 Nov, 112(11), 532 - 4
{Mutations of the genetic region Fin of the F-like plasmid pAP18-1}; Buianova NI et al.; With help of nitrosoguanidine 60 mutants of F-like plasmids pAP18-1 drd::Tn 5 and pAP18-1::Tn 9 were induced which determined resistance of E . coli cells of specific phage MS2 . Mutational changes in fin-locus of those plasmids were accompanied by phenotypic reversion Fin(-)-Fin+.

Res Microbiol, 1991 Nov-Dec, 142(9), 937 - 42
Genetic studies on the promoter of malT, the gene that encodes the activator of the Escherichia coli maltose regulon; Raibaud O et al.; We report the construction of a chromosomal malT-lacZ gene fusion that is expressed under the control of the malT promoter in Escherichia coli K12 . The resulting hybrid protein is soluble and stable in crude cellular extracts, which allowed us to measure very low levels of malTp activity . In this note, we confirm and extend previous observations on the regulation of malTp . We show that the promoter is 40-times less active in the absence of cAMP receptor protein (CRP) than in its presence, that CRP works by binding to the site centred at position -70.5, and that all of the elements necessary and sufficient for the regulation by CRP are located downstream from position -122.

Genetika, 1991 Nov, 27(11), 1912 - 9
{Uncoupling expression of genes coding for the synthesis of proteins involved in transport and utilization of fructose in Escherichia coli K-12}; Bol'shakova TN et al.; A novel mutation fruS localised in the fru operon has been obtained . The mutation uncouples expression of genes determining fructose specific uptake and utilization . In the fruS bacteria fruA and fruF genes (coding for enzyme II and FPr, respectively) become constitutive, while the fruK gene (responsible for fructose-1-phosphate kinase synthesis) remains inducible . In contrast to the already known mutations making the whole fru operon constitutive, the fruS mutation: 1) does not lead to xylitol sensitivity; 2) does not depress growth on lactate, pyruvate and alanine; 3) does not decrease PEP-synthase activity.

Biochimie, 1991 Nov, 73(11), 1361 - 74
Multiple IS insertion sequences near the replication terminus in Escherichia coli K-12; Moszer I et al.; In order to assess the feasibility of semi-automatic procedures for large genome sequencing, a fragment of 9.4 kb of Escherichia coli chromosomal DNA isolated at random was sequenced . It was found to map at 30 min on the chromosome map and to harbour two insertion sequences (IS2 and IS30) as well as several putative coding sequences which had no feature in common with known proteins.

Biol Chem Hoppe Seyler, 1991 Nov, 372(11), 991 - 7
Biosynthesis of tetrahydrofolate . Sequence of GTP cyclohydrolase I from Escherichia coli; Katzenmeier G et al.; The sequence of the gene coding for GTP cyclohydrolase I of Escherichia coli and of the adjacent regions was determined . The open reading frame contains 669 nucleotides . The deduced amino-acid sequence represents a protein consisting of 223 amino-acid residues with a molecular mass of 24,873 Da . Partial amino-acid sequences of the N-terminal region and of 5 peptides obtained by trypsin and BrCN cleavage were determined by Edman degradation and were in full agreement with the sequence deduced from the nucleotide sequence . The starting methionine is removed by posttranslational modification . The protein shows extensive homology to the recently reported GTP cyclohydrolase from rats.

J Biochem (Tokyo), 1991 Nov, 110(5), 677 - 80
Conformation of 2'GMP bound to a mutant ribonuclease T1 (Y45W) determined by X-ray diffraction and NMR methods; Itoh T et al.; The crystal structure of a mutant ribonuclease T1 (Y45W) complexed with a specific inhibitor, 2'GMP, has been determined by X-ray diffraction and refined at 1.9 A resolution to a conventional R-factor of 0.164 . The mode of recognition of the guanine base by the enzyme is similar to that found for the wild-type ribonuclease T1 complexed with 2'GMP . The binding of the guanine base is clearly enhanced by maximum overlapping of the indole ring of Trp45 and the base . The glycosyl torsion angle of the inhibitor is in the syn conformation and the sugar exhibits a C3'-endo type pucker, which differs from that observed in the crystal of the complex between the wild-type ribonuclease T1 and 2'GMP . Analysis of 500-MHZ NMR spectra has also indicated that the 2'GMP molecule as bound to the mutant enzyme in solution exhibits a C3'-endo type pucker, similar to that bound to the wild-type enzyme in solution {Inagaki, Shimada, & Miyazawa (1985) Biochemistry 24, 1013-1020}.

Mol Microbiol, 1991 Nov, 5(11), 2569 - 73
Tn7: a target site-specific transposon; Craig NL; The bacterial transposon Tn7 is an unusual mobile DNA segment . Most transposable elements move at low-frequency and display little target site-selectivity . By contrast, Tn7 inserts at high-frequency into a single specific site in the chromosomes of many bacteria . In the absence of this specific site, called attTn7 in Escherichia coli where Tn7 has been most extensively studied, Tn7 transposes at low-frequency and inserts into many different sites . Much has recently been learned about Tn7 transposition from both genetic and biochemical studies . The Tn7 recombination machinery is elaborate and includes a large number of Tn7-encoded proteins, probably host-encoded proteins and also rather large cis-acting transposition sequences at the transposon termini and at the target site . Dissection of the Tn7 transposition mechanism has revealed that the DNA strand breakage and joining reactions that underlie the translocation of Tn7 have several unusual features.

Circ Shock, 1991 Nov, 35(3), 152 - 8
Lack of effect of ACTH in porcine Escherichia coli septic shock; Donaldson MD et al.; The cardiovascular and metabolic responses to treatment with ACTH(1-24) were investigated in a porcine model of septicaemia . Sixteen anaesthetised, instrumented animals were infused with live Escherichia coli over 2 hr . During the first hour of the infusion, significant reductions in cardiac index, mean arterial pressure, and pH were observed together with a significant increase in mixed venous blood lactate concentrations and packed cell volumes . ACTH(1-24) 160 micrograms kg-1, given 1 hr after starting the E . coli infusion (n = 8), had no significant effect on these haemodynamic or metabolic measurements when compared with the control group (n = 8) . These results do not support the suggestion that intravenous ACTH(1-24) reverses cardiovascular depression in septicaemic shock.

J Surg Res, 1991 Nov, 51(5), 382 - 91
Rapid reduction of intestinal cytochrome a,a3 during lethal endotoxemia; Schaefer CF et al.; In vivo near-infrared spectrophotometry was used to determine whether lethal endotoxemia impairs small intestinal oxidative phosphorylation as reflected by the redox state of mitochondrial cytochrome a,a3 (AA3) . Adult male Sprague-Dawley rats were anesthetized with 2.1% isoflurane in 30% O2:70% N2O, and the small intestine was partially exteriorized for spectrophotometric monitoring (OMNI-3) . By 5 min after an iv bolus of Escherichia coli endotoxin (40 mg/kg, LD90, n = 7) a significant shift toward reduction in intestinal AA3 had occurred in association with hypotension and a marked fall in both superior mesenteric artery blood flow (SMAF) and cardiac output . In a separate group (n = 7) SMAF was kept at the baseline level by periodic infusions of donor rat plasma begun 1 min after endotoxin injection, and the reduction in AA3 was again found despite the fluid loading intervention which successfully maintained not only organ blood flow, but also cardiac output and mean arterial pressure in their normal ranges . Further experiments (n = 34) measuring SM vascular bed oxygen consumption indicated that intestinal VO2 remained unchanged during early endotoxemia . These findings suggest a rapid impairment of oxidative phosphorylation by endotoxin which seems to occur through direct (and/or indirect) toxic cellular effects rather than through impaired tissue perfusion.

DNA Cell Biol, 1991 Nov, 10(9), 689 - 94
Cloning of a cDNA encoding a novel putative G-protein-coupled receptor expressed in specific rat brain regions; Meyerhof W et al.; A cDNA clone encoding a novel putative G-protein-coupled receptor was isolated from a rat brain cDNA library using a PCR-amplified cDNA fragment as a hybridization probe . The 3,615-bp-long nucleotide sequence predicts a single open reading frame of 1,173 bp coding for 391 amino acids, giving a calculated molecular weight of 42.75 kD . The amino acid sequence shares features common to many other receptors, including the seven membrane-spanning hydrophobic regions and putative asparagine-linked glycosylation and phosphorylation sites . Northern blot analysis reveals that a corresponding approximately 3.7-kb mRNA is expressed in specific brain regions such as hypothalamus, cortex, hippocampus, and thalamus but not in other organs analyzed . Although the ligand for this receptor has not yet been identified, it shares some similarities with the vascular type-1 angiotensin II receptor, the vasoactive intestinal peptide (VIP) receptor, and the chemotactic receptors for human C5a anaphylatoxin and the formyl peptide fMet-Leu-Phe.

Plasmid, 1991 Nov, 26(3), 222 - 4
Tn5tac1 insertion polarity in Escherichia coli; Llosa M et al.; The Tn5-derived transposon Tn5tac1 carries the strong tac promoter (Ptac) facing outward at one of its ends . Expression of Ptac is under the control of the lacIq gene, also contained within the transposon . By inserting Tn5tac1 upstream from a promoterless galK gene we determined the basal level of transcription from both ends of the transposon in the absence of IPTG to be about 4% relative to the lactose promoter (Plac) . As a result, derivatives of strain N100 containing these plasmids produce red colonies in MacConkey-galactose plates . Deletion of the BamHI fragment including Ptac causes galactokinase levels to drop to less than 1% of Plac, enough to render white colonies in MacConkey-galactose plates . Thus, Tn5tac1 can be used for genetic analysis under conditions in which it shows no polarity (+ IPTG), low polarity (- IPTG), or strong polarity (delta Ptac).

Genetics, 1991 Nov, 129(3), 647 - 58
Mutant bias in nonlethal selections results from selective recovery of mutants; Benson SA et al.; We have characterized a nonlethal selection for mutations that allow Escherichia coli to grow on large maltodextrins (Dex+) in the absence of the lamB encoded maltoporin LamB . These Dex+ mutations occur before and after imposition of the selection and the selection does not result in a general increase in mutagenesis . The recovered Dex+ mutations are almost exclusively mutations that alter the ompF gene that encodes a major E . coli porin, OmpF even though analogous mutations in the homologous ompC gene, which encodes the OmpC porin, can confer a Dex+ phenotype . We show that the bias for ompF mutations results from a biased recovery and that the genetic background of the starting strain and the selection itself influences the type of mutants that are recovered . When we use a strain carrying an amber mutation in the lamB gene we observe the same preference for ompF mutations as when we start with a lamB deletion strain . In addition, we show that there is no preferential mutagenesis of the lamB gene during the selection which induces transcription of the lamB gene . We present evidence that the biased recovery of mutants observed in this selection does not result from adaptive or directed mutagenesis and that the phenotypic fitness which allows recovery of Dex+ mutants involves more than the increased ability to take up maltodextrins.

Genetics, 1991 Nov, 129(3), 639 - 45
Large inversion in Escherichia coli K-12 1485IN between inversely oriented IS3 elements near lac and cdd; Komoda Y et al.; A companion study has shown that the inversion carried by strain 1485IN has one terminus between lac and proC and the other between his and cdd of the normal strain . Starting with this mapping data, we have done molecular work demonstrating that the inversion occurred by recombination between inversely oriented two IS3 elements, one present near lac and the other near the cdd locus; i.e., the inversion is IN(is3B-is3E) . Evidence supporting this conclusion includes: (i) Normal and inversion strains share two short regions with identical restriction maps . One of these regions is near lac and the other near cdd . (ii) IS3 homology was detected in each of the terminus regions of both the normal and inversion strains . (iii) The sequence on one side of the original IS3 element near lac has been exchanged with the sequence on one side of the IS3 near cdd . Whether the inversion has occurred by one event of homologous recombination between the two IS3 elements or has been caused by involvement of IS3 elements on an F factor is discussed . Another rearrangement, probably related to inversion and deletion, was detected between the IS3 and cdd of the inversion strain.

Genetics, 1991 Nov, 129(3), 631 - 8
Mapping by transposons of the inversion termini in Escherichia coli K-12 strain 1485IN; Enomoto M et al.; Strain 1485IN carries a chromosomal inversion which corresponds to 35% of the chromosome and includes proC, trp and his genes . The termini of the inversion lie between the lac and proC loci and between his and cdd of the normal strain . Using Tn10 and Tn5 in transduction crosses between the normal and inversion strains, the termini were mapped to sites located approximately 0.25 min and 1.6 min away from proC and his, respectively within a region of roughly 4 kb long . The crosses where the normal strains carrying Tn10 near the terminus are donors and the inversion strain is a recipient, yielded unusual Tetr His- recombinants, which arose from illegitimate recombination leading to the replacement of a chromosomal his+ region with a transducing fragment carrying proC . Another rearrangement was detected between the normal and inversion strains in a region outside the inverted segment near the cdd locus.

Proc Natl Acad Sci U S A, 1991 Nov 1, 88(21), 9473 - 7
Eukaryotic DNA polymerase amino acid sequence required for 3'----5' exonuclease activity; Morrison A et al.; We have identified an amino-proximal sequence motif, Phe-Asp-Ile-Glu-Thr, in Saccharomyces cerevisiae DNA polymerase II that is almost identical to a sequence comprising part of the 3'----5' exonuclease active site of Escherichia coli DNA polymerase I . Similar motifs were identified by amino acid sequence alignment in related, aphidicolin-sensitive DNA polymerases possessing 3'----5' proofreading exonuclease activity . Substitution of Ala for the Asp and Glu residues in the motif reduced the exonuclease activity of partially purified DNA polymerase II at least 100-fold while preserving the polymerase activity . Yeast strains expressing the exonuclease-deficient DNA polymerase II had on average about a 22-fold increase in spontaneous mutation rate, consistent with a presumed proofreading role in vivo . In multiple amino acid sequence alignments of this and two other conserved motifs described previously, five residues of the 3'----5' exonuclease active site of E . coli DNA polymerase I appeared to be invariant in aphidicolin-sensitive DNA polymerases known to possess 3'----5' proofreading exonuclease activity . None of these residues, however, appeared to be identifiable in the catalytic subunits of human, yeast, or Drosophila alpha DNA polymerases.

J Bacteriol, 1991 Nov, 173(22), 7213 - 8
Site-specific methylation of 16S rRNA caused by pct, a pactamycin resistance determinant from the producing organism, Streptomyces pactum; Ballesta JP et al.; Ribosomal resistance to pactamycin in clones of Streptomyces lividans containing DNA (pct) from Streptomyces pactum, the pactamycin producer, involves methylation of 16S RNA . The modified residue A-941 in S . lividans 16S rRNA (A-964 in the homologous Escherichia coli sequence) is converted to 1-methyladenosine, and the ribosomal ability to bind pactamycin is reduced or abolished.

J Bacteriol, 1991 Nov, 173(21), 7029 - 32
Purification and partial characterization of 7,8-dihydro-6-hydroxymethylpterin-pyrophosphokinase and 7,8-dihydropteroate synthase from Escherichia coli MC4100; Talarico TL et al.; The enzymes 7,8-dihydroxymethylpterin-pyrophosphokinase (HPPK) and 7,8-dihydropteroate synthase (DHPS), which act sequentially in the folate pathway, were purified to homogeneity from crude extracts of Escherichia coli MC4100 . The enzymes represent less than 0.01% of the total soluble protein . HPPK was purified greater than 10,000-fold; the native enzyme appears to be a monomer with a molecular mass of 25 kDa and a pI of 5.2 . DHPS was purified greater than 7,000-fold; the native enzyme has an apparent molecular mass of 52 to 54 kDa and is composed of two identical 30-kDa subunits . The amino-terminal sequences for both enzymes have been determined.

J Bacteriol, 1991 Nov, 173(21), 6910 - 8
Induction of the SOS response in Escherichia coli inhibits Tn5 and IS50 transposition; Weinreich MD et al.; In response to DNA damage or the inhibition of normal DNA replication in Escherichia coli, a set of some 20 unlinked operons is induced through the RecA-mediated cleavage of the LexA repressor . We examined the effect of this SOS response on the transposition of Tn5 and determined that the frequency of transposition is reduced 5- to 10-fold in cells that constitutively express SOS functions, e.g., lexA(Def) strains . Furthermore, this inhibition is independent of recA function, is fully reversed by a wild-type copy of lexA, and is not caused by an alteration in the levels of the Tn5 transposase or inhibitor proteins . We isolated insertion mutations in a lexA(Def) background that reverse this transposition defect; all of these mapped to a new locus near 23 min on the E . coli chromosome.

Plant Mol Biol, 1991 Nov, 17(5), 995 - 1004
Novel DNA structures resulting from dTam3 excision in tobacco; Haring MA et al.; A Tam3 two-element system has been designed by combining an immobilized Tam3 element with a non-autonomous dTam3 element inserted into the HPT gene . The phenotypic assay employed, restored hygromycin resistance, indicated that trans-activation of the non-autonomous dTam3 element occurred . Molecular analyses of the excision sites revealed that the ends of the dTam3 element remain in the empty donor sites . The predominant consequence of this type of excision appears to be that excised fragments fail to re-integrate into the tobacco genome . Only one case of dTam3 re-integration could be detected . The ends of this element had been degraded upon integration into the tobacco genome . Either the altered structure of the Tam3 derivatives or tobacco host factors are influencing the trans-activation of a dTam3 element, resulting in aberrant excision.

Plant Mol Biol, 1991 Nov, 17(5), 1089 - 93
Comparison of the primary sequences of two potato tuber ADP-glucose pyrophosphorylase subunits; Nakata PA et al.; Near-full-length cDNA clones to the small and large subunit of the heterotetrameric potato tuber ADP-glucose pyrophosphorylase have been isolated and characterized . The missing amino terminal sequence of the small subunit has also been elucidated from its corresponding genomic clone . Primary sequence comparisons revealed that each potato subunit had less identity to each other than to their homologous subunit from other plants . It also appeared that the smaller subunit is more conserved among the different plants and the larger subunit more divergent . Amino acid comparisons of both potato tuber sequences to the Escherichia coli ADP-glucose pyrophosphorylase sequence revealed conserved regions important for both catalytic and allosteric function of the bacterial enzyme.

Arch Biochem Biophys, 1991 Nov 1, 290(2), 381 - 6
One-electron reduction of carcinogen chromate by microsomes, mitochondria, and Escherichia coli: identification of Cr(V) and .OH radical; Shi XL et al.; Earlier studies have shown that a long-lived Cr(V) species is produced during the reduction of chromate (Cr(VI} by microsomes/NADPH, mitochondria, and other cellular constituents and that this Cr(V) species plays a significant role in the mechanism of Cr(VI) toxicity . The present work indicates that this species is a Cr(V) complex involving the diol moieties of NADPH as the ligand . Additionally, ESR spin trapping investigations show that the hydroxyl (.OH) radical is also generated in the reduction process . Hydrogen peroxide (H2O2) enhances the .OH generation but suppresses the Cr(V)-NADPH complex formation . Catalase decreases the .OH radical generation and enhances the Cr(V)-NADPH formation . Measurements under anaerobic atmosphere show decreased .OH radical generation, indicating that during the cellular Cr(VI) reduction process molecular oxygen is reduced to H2O2, which reacts with the Cr(V)-NADPH complex to generate the .OH radical via a Fenton-like mechanism.

Virology, 1991 Nov, 185(1), 428 - 31
Expression of Epstein-Barr virus nuclear antigen 2 in insect cells from a baculovirus vector; Schubach WH et al.; The open reading frame encoding the Epstein-Barr virus nuclear antigen 2 (EBNA-2) has been expressed in a recombinant baculovirus vector . The resulting product migrates with the same apparent molecular weight as EBNA-2 from latently infected or converted B cell lines . Rabbit antisera derived from the innoculation of this material immunoprecipitated EBNA-2 from cell extracts of EBV-containing cells . The high level of protein expression obtained in insect cells stands in sharp contrast to that seen in a number of mammalian cell lines using a variety of promoters including the endogenous EBNA-2 promoter, the Moloney MuLV LTR, the murine immunoglobulin heavy chain promoter, the human cytomegalovirus immediate early promoter, and the adenovirus major late promoter.

Virology, 1991 Nov, 185(1), 419 - 23
The HSV-1 UL45 gene product is not required for growth in Vero cells; Visalli RJ et al.; We have constructed a HSV-1 UL45 null mutant (UL45 delta) by inserting a TK-lacZ cassette into a BclI site near the 5' end of the UL45 gene . A polyclonal antiserum produced to an Escherichia coli trpE:UL45 fusion protein was used to show that an 18-kDa polypeptide corresponding to the predicted UL45 gene product was produced in HSV-1 strain KOS-infected Vero cells but was not detected in UL45 delta-infected Vero cells . The absence of the 18-kDa protein had only a slight effect on viral growth in cell culture, indicating that the UL45 gene product is not essential for growth in Vero cells . However, the burst size of UL45 delta was smaller than HSV-1 KOS in Vero and HeLa cells . UL45 delta also had a smaller plaque size and an altered plaque morphology.

Virology, 1991 Nov, 185(1), 411 - 8
Detection of hepatitis A virus proteins in infected BS-C-1 cells; Updike WS et al.; Hepatitis A virus (HAV) is distinguished from other picornaviruses by its tropism for the liver in infected hosts, a nonlytic infection in hepatocytes, and a slow and nonlytic growth cycle in cultured cells . Although the genome structure and organization of HAV appear to be similar to those of the other picornaviruses, the viral proteins synthesized in infected cells have not been previously characterized . We have utilized specific antisera raised in rabbits to recombinant HAV proteins expressed in Escherichia coli in an effort to identify both structural and nonstructural proteins in BS-C-1 cells throughout the course of a viral replication cycle . Replication was monitored by dot blot hybridization of viral genomes . Structural proteins VP0, VP1, VP2, and VP3 were found to accumulate during the infection cycle as did viral RNA . Nonstructural proteins 2C and 3D were not detected on immunoblots, although a minor amount of 2C could be detected by immunoprecipitation of lysates of radiolabeled, infected cells . The relative sensitivities of the various antisera were determined, and the failure to observe nonstructural proteins was shown not to be due to decreased sensitivity of the detection reagents . Thus, it appears that HAV nonstructural proteins do not accumulate in infected cells to levels comparable to those of capsid proteins.

Virology, 1991 Nov, 185(1), 140 - 50
Purification, properties, and mutagenesis of poliovirus 3C protease; Baum EZ et al.; Poliovirus protease 3C, type 1 Mahoney strain, was expressed in Escherichia coli under phage T7 promoter control and purified to homogeneity from resolubilized inclusion bodies . The renatured protein was as enzymatically active as the protease found in the soluble portion of the bacterial lysate . Proteolytic activity was assayed using as substrate either {35S}methionine-labeled recombinant poliovirus proteins 2C3AB or a truncated version of 3ABC, or synthetic peptide 16-mers corresponding to the cleavage sites at 2C/3A and 3A/3B . Poliovirus protein 3CD (protease-polymerase) was also expressed in bacteria . About 25% of this protein apparently autodigested in vivo, releasing immunoprecipitable protein 3D (polymerase) . No further autodigestion of 3CD could be detected in vitro, nor could addition of purified protein 3C effect digestion in trans . Both the serine protease inhibitors PMSF, TPCK, and 3,4-dichloroisocoumarin, and the cysteine protease inhibitors cystatin and zinc, were effective inhibitors of the 3C protease . Six new mutants of the protease, with altered or no enzymatic activity, were identified based on the observation that low level expression of wild type enzyme severely retards growth of bacterial colonies harboring the expression plasmid.

J Virol, 1991 Nov, 65(11), 6365 - 70
Long-term in vivo expression of genes introduced by retrovirus-mediated transfer into mammary epithelial cells; Smith GH et al.; Nonimmortalized mouse mammary epithelial cells expressing Escherichia coli beta-galactosidase from a murine amphotropic packaged retroviral vector were injected into the epithelium-divested mammary fat pads of syngeneic mice . Mammary glands formed from the injected mammary epithelial cells contained ductal and lobular cells, both of which expressed beta-galactosidase when examined in situ more than 12 months later . These results indicate that stable recombinant gene expression can be achieved in vivo in the mammary gland without altering the growth properties of normal mammary epithelium.

J Virol, 1991 Nov, 65(11), 6194 - 204
Translation of poliovirus RNA: role of an essential cis-acting oligopyrimidine element within the 5' nontranslated region and involvement of a cellular 57-kilodalton protein; Pestova TV et al.; Translation of poliovirus RNA is initiated by cap-independent internal entry of ribosomes into the 5' nontranslated region . This process is dependent on elements within the 5' nontranslated region (the internal ribosomal entry site) and may involve novel translation factors . Systematic mutation of a conserved oligopyrimidine tract has revealed a cis-acting element that is essential for translation in vitro . The function of this element is related to its position relative to other cis-acting domains . This element is part of a more complex structure that interacts with several cellular factors, but changes in protein binding after mutation of this element were not detected in a UV cross-linking assay . A 57-kDa protein from the ribosomal salt wash fraction of HeLa cells was identified that binds upstream of the oligopyrimidine tract . Translation of poliovirus mRNA in vitro was strongly and specifically inhibited by competition with the p57-binding domain (nucleotides 260 to 488) of the 5' nontranslated region of encephalomyocarditis virus, indicating a probable role for p57 in poliovirus translation . p57 is likely to be identical to the ribosome-associated factor that binds to and is necessary for the function of the internal ribosomal entry site of encephalomyocarditis virus RNA.

J Virol, 1991 Nov, 65(11), 6077 - 83
cis- and trans-cleavage activities of poliovirus 2A protease expressed in Escherichia coli; Alvey JC et al.; The poliovirus protease, 2Apro, was produced in Escherichia coli from plasmids that encode a fusion protein consisting of the N-terminal portion of the bacterial TrpE protein linked to poliovirus 2Apro . This fusion protein underwent efficient autocatalytic cleavage at the N terminus of 2Apro, generating the mature protease . Extracts of bacteria expressing 2Apro induced the specific cleavage of the p220 subunit of the eukaryotic translation initiation factor 4F, similar to the 2Apro-mediated reaction that occurs in poliovirus-infected HeLa cells . A portion of the poliovirus polyprotein containing the 2Apro cleavage site at the P1/P2 junction was produced by translation of cDNA transcripts in rabbit reticulocyte lysates and then tested as a substrate for 2Apro-mediated cleavage . The protein was partially cleaved by 2Apro in trans . Finally, a 16-amino-acid synthetic peptide, representing the P1/P2 junction sequence, was analyzed as a substrate for 2Apro . The peptide was labeled with fluorescein at a lysine residue to facilitate its detection . Recombinant 2Apro cleaved the synthetic peptide into two half-peptide molecules which were resolved by high-pressure liquid chromatography . Direct sequence analysis of the isolated peptide products demonstrated that cleavage occurred at the expected tyrosine-glycine pair . A rapid cleavage assay for 2Apro activity on the synthetic peptide was developed, using separation of the fluorescein-labeled 8-amino-acid product from the 16-residue substrate by electrophoresis on sodium dodecyl sulfate-polyacrylamide gels.

Biotechnology (N Y), 1991 Nov, 9(11), 1080 - 5
Micro-targeting: high efficiency gene transfer using a novel approach for the acceleration of micro-projectiles; Sautter C et al.; We have constructed a novel micro-projectile accelerating system for efficient gene transfer into cells in situ that avoids binding DNA to micro-projectiles and keeps the DNA in solution . Further, instead of a macro-projectile (or the equivalent), it accelerates the particles in a Bernoulli air stream . The micro-targeting approach directs highly dispersed particles to sites with diameters as little as 0.15 mm, allowing precise aiming to restricted tissues . The system is physically flexible and should therefore be adaptable to different tissues and species . Transient expression of the Escherichia coli beta-glucuronidase gene in immature wheat embryo scutella was obtained at a frequency of up to 3% of the treated cells in the surface layer . In tobacco SR1, we achieved many transgenic plants, and the efficiency of stable transformation with the neomycin phosphotransferase (NPTII) gene was approximately 10(-3) per exposed cell.

Appl Microbiol Biotechnol, 1991 Nov, 36(2), 211 - 5
In-vivo processing of the initiator methionine from recombinant methionyl human interleukin-6 synthesized in Escherichia coli overproducing aminopeptidase-P; Yasueda H et al.; Human interleukin 6 (hIL-6) overproduced in Escherichia coli HB101 was found to partially retain the initiator methionine (Met) residue (Met-hIL-6) . In order to remove the residual N-terminal Met in vivo, an attempt was made to express hIL-6 in aminopeptidase-P (Ap-P)-hyperproducing strains, since the N-terminus Met-Pro- structure of nascent recombinant hIL-6 has been shown to be a favoured substrate of the enzyme in vitro . Using a mutant with duplicated Ap-P genes (pepP) on a chromosome or some recombinant strains overproducing Ap-P, we have succeeded in removing the initiator Met from Met-hIL-6 in vivo . The content of the mature product without the initiator Met in the pepP recombinant strains could be increased to approximately 99% from 85%.

J Ind Microbiol, 1991 Nov, 8(4), 253 - 8
Expression of streptomycete cholesterol oxidase in Escherichia coli; Solaiman DK et al.; A streptomycete gene coding for extracellular cholesterol oxidase (choA) was subcloned and expressed in Escherichia coli . The pUCO series recombinants were obtained by inserting the choA gene into the unique KpnI site of pUC19 vector . Expression was observed with pUCO192A and pUCO193 constructs in which the cloned gene(s) were aligned with the upstream lacZ promoter . Isopropyl beta-D-thioglucopyranoside (IPTG) enhanced this expression up to 2.5-fold . Specific Cho activity in the cell extracts of the stable pUCO193 transformant were 0.004 U and 0.007 U per mg protein without and with IPTG induction, respectively . Cho activity was detected in the spent medium of this culture, suggesting possible secretion of the enzyme.

J Biotechnol, 1991 Nov, 21(1-2), 83 - 92
Production of homogeneous basic fibroblast growth factor by specific enzymatic hydrolysis of larger microheterogeneous molecular forms; Betbeder D et al.; The 146-amino acid form of basic fibroblast growth factor (bFGF) was expressed in Escherichia coli and purified by a two step process including ion exchange and heparin-Sepharose chromatographies . However, the resulting protein consisted of a mixture of 146- and 145-amino acid forms, indicating that, besides the initial methionine, also the following residue (proline) was removed from the N-terminus . The same phenomenon was observed when the 155-amino acid form, which is biologically equivalent to the shorter one, was expressed in E . coli . Taking into account the previously known data concerning the possible mechanism of cleavage of the extended forms of bFGF in vivo, we developed an efficient enzymatic process that allows the production of an homogeneous 146-amino acid form from recombinant NH2-end extended forms . This process takes advantage of the protecting effect that heparin exerts on bFGF . Accordingly, when bFGF, complexed to heparin, is treated with pepsin A, an aspartic protease with a broad specificity, only the Leu9-Pro10 peptide bond is cleaved generating the 146-amino acid form . Quantitative yields of this reaction are also achieved when bFGF is bound to a heparin-Sepharose column, allowing the integration of this enzymatic step directly during purification of the recombinant extended forms of bFGF.

J Biotechnol, 1991 Nov, 21(1-2), 127 - 36
Large scale expression and purification of recombinant HIV-1 proteinase from Escherichia coli; Singh OM et al.; The availability of target proteins in sufficient quantity is a limiting factor in crystallographic studies and therefore in rational drug design . Even after optimisation, expression of recombinant proteins may be low and the only way to produce enough protein is by large scale cell growth/purification . HIV-1 proteinase in Escherichia coli, which due to its toxicity is expressed as a soluble protein only at around 0.1% of total protein, is a paradigm for this . In this paper a detailed process for large scale expression and purification of HIV-1 proteinase which delivers material of suitable quantity (30 mg from 500 g of wet weight of cells) and quality for crystallographic studies is described.

Biochem Biophys Res Commun, 1991 Oct 31, 180(2), 887 - 93
Cloning and sequence analysis of the human liver rhodanese: comparison with the bovine and chicken enzymes; Pallini R et al.; The cDNA for the human rhodanese (thiosulfate: cyanide sulfurtransferase, EC 2.8.1.1), a nuclearly encoded protein of the mitochondrial matrix, was isolated from a human fetal liver cDNA library . Nucleotide sequence revealed an open reading frame coding for a polypeptide of 295 amino acids, which presented a 57% and 58% identity with the bovine and avian rhodanese, respectively . The analysis of the 5'-ends of the coding region gave no evidence for the presence of a cleavable signal sequence as found in other mitochondrial proteins . A comparison with two available amino acid sequences (cow and chicken) showed that sequence similarity is not restricted to the alpha-helices and beta-structures motifs which are remarkably superimposable in the two halves of bovine rhodanese, but extends to adjacent regions.

Biochem Biophys Res Commun, 1991 Oct 31, 180(2), 774 - 81
Effect of rec mutations on viability and processing of DNA damaged by methylmethane sulfonate in xth nth nfo cells of Escherichia coli; Wang TC et al.; The role of recombination genes in the processing of DNA damaged by methlymethane sulfonate (MMS) was examined in an xth nth nfo strain of Escherichia coli K-12 . Introduction of a recQ mutation did not increase the cell's sensitivity to MMS treatment . The presence of recF, recJ or recN mutation slightly increased the cell's sensitivity to MMS treatment . The introduction of recA or recB mutation into the cells led to inviability . Taken together, we suggest that replication of DNA containing apurinic/apyrimidinic (AP) sites in vivo will lead to the formation of secondary lesions . The repair of these secondary lesions requires the function of recA and recB genes, but does not appear to require recF, recJ, recQ or recN genes.

Biochem Biophys Res Commun, 1991 Oct 31, 180(2), 913 - 9
Induction kinetics of RNA and proteins in exponentially growing organisms; Harayama S; A mathematical model of the induction kinetics of RNAs and proteins in exponentially growing organisms is derived, and the cellular concentrations of the induced macromolecules at a given time after induction are related to three parameters: the fraction of the synthesis of these macromolecules in total synthesis, the half life of the inducible macromolecules, and the generation time of the organisms . The model predicts that the concentrations of the inducible macromolecules reach one half of the maximum induction level within one generation time after the onset of the induction . The model also predicts that induction curves of proteins are parabolic when their mRNAs are short-lived, but sigmoid when they are stable . Observed induction curves of beta-galactosidase in Escherichia coli cells fit in the theoretical induction curves.

Biochem Biophys Res Commun, 1991 Oct 31, 180(2), 538 - 44
Overexpression of dimeric guanylyl cyclase cores of an atrial natriuretic peptide receptor; Thorpe DS et al.; Using a bacterial expression system, large amounts of the catalytic core of an atrial natriuretic peptide receptor guanylyl cyclase were produced and purified . After refolding the protein from a buffer containing urea, the enzyme had positively cooperative kinetics with a Hill coefficient, nH = 1.42 +/- 0.08 . Size exclusion chromatography and denaturing polyacrylamide gel electrophoresis demonstrated that the enzyme is composed of homodimers with interacting catalytic sites.

Biochem Biophys Res Commun, 1991 Oct 31, 180(2), 994 - 1001
Expression of a full-length nonstructural protein NS1 of bluetongue virus serotype 17 in Escherichia coli; He XS et al.; The relative abundance of the nonstructural protein NS1 in bluetongue virus (BTV)-infected cells, the existence of NS1 in the BTV particles and the highly conserved NS1 gene among BTV serotypes indicate the diagnostic potential of using NS1 in detecting BTV infections . In this study a NS1 gene was expressed with the T7 RNA polymerase expression system to produce a full-length NS1 protein . Sheep anti-NS1 antibodies were raised with the E . coli-produced NS1 and used to show that the NS1 proteins of the five BTV serotypes in the Unites States were immunologically indistinguishable.

Gene, 1991 Oct 30, 107(1), 19 - 25
Introduction of single-copy sequences into the chromosome of Escherichia coli: application to gene and operon fusions; Resnik E et al.; We have developed a general method for the introduction of any cloned sequence into the chromosome of Escherichia coli . This method employs an Hfr strain which carries a fragment of bla (the pBR322 gene imparting ampicillin resistance) between lacI and lacZ . Plasmid-borne inserts which are flanked by sequences from bla and lacZ can be introduced at this locus by homologous recombination . The isolation of recombinants is enhanced by selection for transfer of an integrated copy of the plasmid during conjugation . Once introduced into the chromosome, the inserted sequences can be transferred to other strains by conventional methods such as P1 transduction or conjugation . This method is suitable for the transfer of any cloned sequence to the chromosome and is particularly well suited to the construction of chromosomal gene and operon fusions with lacZ.

Gene, 1991 Oct 30, 107(1), 133 - 8
Identification of a unique specificity determinant of the colicin E3 immunity protein; Masaki H et al.; Plasmid immunity to a nuclease-type colicin is defined by the specific binding of an immunity (or inhibitor) protein, Imm, to the C-terminal nuclease domain, T2A, of the colicin molecule . Whereas most regions of colicin operons exhibit extensive sequence identity, the small plasmid region encoding T2A and Imm is exceptionally varied . Since immunity is essential for the survival of the potentially lethal colicin plasmid (Col), we inferred that T2A and Imm must have co-evolved, retaining their mutual binding specificities . To evaluate this co-evolution model for the col and imm genes of ColE3 and ColE6, we attempted to obtain a stabilized clone from a plasmid which had been destabilized with a non-cognate immunity gene . A hybrid Col, in which the immE3 gene of the ColE3 was replaced with immE6 from ColE6, was lethal to the host cells upon SOS induction . From among this suicidal cell population, we isolated a stabilized, i.e., evolved, clone which produced colicin E3 (E3) stably and exhibited immunity to E3 . This change arose from only a single mutation in ImmE6, from Trp48 to Cys, the same residue as in the ImmE3 sequence . In addition, we constructed a series of chimeric genes through homologous recombination between immE3 and immE6 . Characterization of these chimeric immunity genes confirmed the above finding that colicins E3 and E6 are mostly distinguished by only Cys48 of the ImmE3 protein.

Gene, 1991 Oct 30, 107(1), 127 - 32
pUBEX/pUBSEX: a versatile expression vector system for production of fusion and nonfusion proteins in Escherichia coli; Banting G et al.; Despite the large number of expression vectors now available, none provide the facility of allowing fusion and nonfusion protein production from the same vector system . In some situations it is preferable to obtain an insoluble fusion protein, in others a soluble nonfusion protein may be required . We have designed, constructed and tested a modification of the pEX vectors, in which it is possible to express the product of a suitably inserted cDNA either as part of a Cro-beta-galactosidase (Cro-beta Gal) fusion or as a delta Cro fusion which contains only nine noninsert-encoded amino acids at its N terminus . The conversion from Cro-beta Gal to delta Cro fusion protein production is achieved by a simple intramolecular deletion of lacZ sequence from the pUBEX vector, to create the pUBSEX variant . Plasmid pUBEX can be induced to produce large amounts of insoluble Cro-beta Gal fusion proteins, whereas pUBSEX will produce predominantly soluble delta Cro fusion proteins.

Gene, 1991 Oct 30, 107(1), 27 - 35
Direct cloning of a long restriction fragment aided with a jumping clone; Matsuoka T et al.; Long-range physical mapping with rare-cutting restriction enzymes (rare cutters) is an important step for structural analysis of complex genomes . Combination of two types of DNA clones bearing the rare-cutter sites, linking clones and jumping clones (Fig . 1a), facilitates the physical mapping {Poustka et al., Nature 325 (1987) 353-355} . A step followed by the physical mapping is the cloning of the large (rare-cutter-generated) restriction fragment of interest . For facilitating this step, we devised a method to directly clone a long restriction fragment without constructing the whole genomic DNA library using the jumping clone as starting material . The short DNA segments of a jumping clone, which are derived from the 5' and 3' terminal regions of the large restriction fragment, are inserted into the yeast artificial chromosome plasmid (pYAC) vector, and then converted into single strands with T7 gene 6-encoded 5'----3' exonuclease . The total genomic DNA digested with the restriction enzyme is also treated with the exonuclease to convert the terminal regions of the restriction fragments into single strands . In the resulting products, only the fragment corresponding to the jumping clone can form hybrids with the just-mentioned, single-stranded DNAs, which are connected to the pYAC, and only this fragment is cloned in yeast . We describe the protocol of this method with Escherichia coli DNA as a model experiment . Judging from the cloning efficiency, this method could be applied to cloning single-copy regions of the human genome, provided a jumping clone is available . The instability of inserts in the pYAC vector is also discussed.

Biochemistry, 1991 Oct 29, 30(43), 10420 - 7
Expression and mutagenesis of human poly(ADP-ribose) polymerase as a ubiquitin fusion protein from Escherichia coli; Cherney BW et al.; The cDNA of human poly(ADP-ribose) polymerase (pADPRP), encoding the entire protein, was subcloned into the Escherichia coli expression plasmid pYUb . In this expression system, the carboxyl terminus of ubiquitin is fused to the amino terminus of a target protein, in this case pADPRP, stabilizing the accumulation of the cloned gene product . Following induction of the transformed cells, the sonicated extract contained a unique protein immunoreactive with both pADPRP and ubiquitin antibodies and corresponding to the predicted mobility of the fusion protein in SDS-PAGE . Fusion of ubiquitin to pADPRP increased the yield of pADPRP approximately 10-fold compared to that of the unfused enzyme . The resulting recombinant fusion protein had catalytic properties which were nearly identical to those of native pADPRP obtained from mammalian tissues . These properties included specific activity, Km for NAD, response to DNA strand breaks, response to Mg2+, inhibition by 3-aminobenzamide, and activity in activity gel analysis . An initial analysis by deletion mutagenesis of pADPRP's functional domains revealed that deletions in the NAD binding domain eliminated all activity; however, partial polymerase activity resulted from deletion in the DNA binding or automodification domains . The activities were not enhanced by breaks in DNA . We further report a colony filter screening procedure designed to identify functional polymerase molecules which will facilitate structure/function studies of the polymerase.

Biochemistry, 1991 Oct 29, 30(43), 10388 - 98
Structural basis for broad specificity in alpha-lytic protease mutants; Bone R et al.; Binding pocket mutants of alpha-lytic protease (Met 192----Ala and Met 213----Ala) have been constructed recently in an effort to create a protease specific for Met just prior to the scissile bond . Instead, mutation resulted in proteases with extraordinarily broad specificity profiles and high activity {Bone, R., Silen, J . L., & Agard, D . A . (1989) Nature 339, 191-195} . To understand the structural basis for the unexpected specificity profiles of these mutants, high-resolution X-ray crystal structures have been determined for complexes of each mutant with a series of systematically varying peptidylboronic acids . These inhibitory analogues of high-energy reaction intermediates provide models for how substrates with different side chains interact with the enzyme during the transition state . Fifteen structures have been analyzed qualitatively and quantitatively with respect to enzyme-inhibitor hydrogen-bond lengths, buried hydrophobic surface area, unfilled cavity volume, and the magnitude of inhibitor accommodating conformational adjustments (particularly in the region of another binding pocket residue, Val 217A) . Comparison of these four parameters with the Ki of each inhibitor and the kcat and Km of the analogous substrates indicates that while no single structural parameter consistently correlates with activity or inhibition, the observed data can be understood as a combination of effects . Furthermore, the relative contribution of each term differs for the three enzymes, reflecting the altered conformational energetics of each mutant . From the extensive structural analysis, it is clear that enzyme flexibility, especially in the region of Val 217A, is primarily responsible for the exceptionally broad specificity observed in either mutant . Taken together, the observed patterns of substrate specificity can be understood to arise directly from interactions between the substrate and the residues lining the specificity pocket and indirectly from interactions between peripheral regions of the protein and the active-site region that serve to modulate active-site flexibility.

Eur J Pharmacol, 1991 Oct 29, 204(1), 63 - 70
Endotoxin-induced impairment of vasodepressor responses in the pithed rat; Guc MO et al.; The effects of Eschericia coli endotoxin on vascular responsiveness were compared with those of sodium nitroprusside in pithed rats . Infusion of endotoxin (250 micrograms kg-1 h-1) produced a fall in mean arterial blood pressure (11 mmHg) and impaired vasodepressor responses to endothelin, 5-hydroxytryptamine, acetylcholine, bradykinin, sodium nitroprusside and salbutamol . Prevention of endotoxin-induced hypotension with vasopressin infusion (0.64 i.u . kg-1 h-1 i.v.) restored responsiveness to bradykinin, tended to restore responsiveness to endothelin and sodium nitroprusside but failed to restore responsiveness to acetylcholine, 5-HT or salbutamol . Infusion of sodium nitroprusside at a rate (400 micrograms kg-1 h-1) producing a similar fall in blood pressure to that produced by endotoxin markedly impaired vasodepressor responsiveness to 5-HT . However, this was fully restored when the hypotension was prevented by vasopressin infusion . Vasodepressor responsiveness to either acetylcholine or salbutamol was not impaired by sodium nitroprusside in vasopressin-infused rats . The impairment of vasodepressor responsiveness by endotoxin is not due to endotoxin-induced hypotension and does not fit clearly with an endotoxin-mediated impairment of endothelial function.

J Biol Chem, 1991 Oct 15, 266(29), 19265 - 8
Electron transfer associated with oxygen activation in the B2 protein of ribonucleotide reductase from Escherichia coli; Elgren TE et al.; Each of the two beta peptides which comprise the B2 protein of Escherichia coli ribonucleotide reductase (RRB2) possesses a nonheme dinuclear iron cluster and a tyrosine residue at position 122 . The oxidized form of the protein contains all high spin ferric iron and 1.0-1.4 tyrosyl radicals per RRB2 protein . In order to define the stoichiometry of in vitro dioxygen reduction catalyzed by fully reduced RRB2 we have quantified the reactants and products in the aerobic addition of Fe(II) to metal-free RRB2apo utilizing an oxygraph to quantify oxygen consumption, electron paramagnetic resonance to measure tyrosine radical generation, and Mossbauer spectroscopy to determine the extent of iron oxidation . Our data indicate that 3.1 Fe(II) and 0.8 Tyr122 are oxidized per mol of O2 reduced . Mossbauer experiments indicate that less than 8% of the iron is bound as mononuclear high spin Fe(III) . Further, the aerobic addition of substoichiometric amounts of 57Fe to RRB2apo consistently produces dinuclear clusters, rather than mononuclear Fe(III) species, providing the first direct spectroscopic evidence for the preferential formation of the dinuclear units at the active site . These stoichiometry studies were extended to include the phenylalanine mutant protein (Y122F)RRB2 and show that 3.9 mol-equivalents of Fe(II) are oxidized per mol of O2 consumed . Our stoichiometry data has led us to propose a model for dioxygen activation catalyzed by RRB2 which invokes electron transfer between iron clusters.

Nucleic Acids Res, 1991 Oct 25, 19(20), 5755 - 61
The mouse Pgk-1 gene promoter contains an upstream activator sequence; McBurney MW et al.; The Pgk-1 gene encodes the housekeeping enzyme, 3-phosphoglycerate kinase, and is ubiquitously expressed . This gene resides on the X chromosome in mammals and is always expressed except where it is silenced along with most other genes on the inactive X chromosome of female somatic cells or male germ cells . The Pgk-1 promoter is in a region rich in nucleotides G and C . This promoter can efficiently drive high levels of expression of reporter genes such as E . coli lacZ and neo . We have determined that the 120 bp upstream of the transcription start site functions as a core promoter . Upstream of this is a 320 bp region which enhances transcription from the core promoter in an orientation and position independent fashion . This 320 bp region does not enhance transcription from the core promoter of the SV40 early region . Nuclear proteins bind to this 320 bp fragment although the restricted regions to which binding can be demonstrated with gel mobility shift assays suggests that the activity of the enhancer may be mediated by factors which bind at multiple sites each with low affinity.

Nucleic Acids Res, 1991 Oct 25, 19(20), 5703 - 5
The inhibition of restriction endonuclease PvuII cleavage activity by methylation outside its recognition sequence; Chen DF et al.; The recombinant plasmid pGEM4Z-ras DNA which was methylated on dam and dcm sites outside the PvuII recognition sequence was digested with restriction endonuclease PvuII, and one of the three PvuII sites was about 16-fold less efficient to cleave than either of the other two . On the contrary, the three PvuII sites were cleaved at about the same rate on the unmethylated DNA molecule . The results show that the cleavage inhibition of the methylated DNA on the certain PvuII site was caused by methylation outside the PvuII recognition sequence . Maybe a adjacent methylated dam site *A was responsible for the less efficient cleavage . This observation suggests that methylation outside the recognition sequence may be considered a new factor in the kinetic experiment of restriction endonuclease.

Nucleic Acids Res, 1991 Oct 25, 19(20), 5597 - 601
Cloning and expresion of cDNA for rat O6-methylguanine-DNA methyltransferase; Sakumi K et al.; cDNA for O6-methylguanine-DNA methyltransferase was isolated by screening rat liver cDNA libraries, using as a probe the human cDNA sequence for methyltransferase . The rat cDNA encodes a protein with 209 amino acid residues . The predicted amino acid sequence of the rat methyltransferase exhibits considerable homology with those of the human, yeast and bacterial enzymes, especially around putative methyl acceptor sites . When the cDNA was placed under control of the lac promoter and expressed in methyltransferase-deficient Escherichia coli (ada-, ogt-) cells, a characteristic methyltransferase protein was produced . The rat DNA methyltransferase thus expressed could complement the biological defects of the E . coli cell caused by lack of its own DNA methyltransferases; e.g . increased sensitivity to alkylating agents in terms of both cell death and mutation induction.

J Immunol Methods, 1991 Oct 25, 143(2), 231 - 40
Preparation of clinical grade proteins produced by recombinant DNA technologies; Takacs BJ et al.; Methods were developed for the production of clinical grade malaria vaccine candidates expressed in E . coli by recombinant DNA technologies . The essential features of the purification protocol consist of (1) mechanical breakage of host cells and solubilization of the recombinant proteins in 6 M guanidine hydrochloride; (2) ammonium sulfate fractionation; (3) affinity chromatography on a Ni(2+)-chelate gel in the presence of 6 M guanidine hydrochloride; and (4) ion exchange chromatograph on a Phospho Ultrogel column in the presence of 6 M urea . The use of undesirable chemicals (PMSF, DFP, TFA, acetonitrile, etc.) was avoided rather than demonstrating their complete removal after the purification steps . Testing of chromatographic fractions for host-cell proteins and the elimination of fractions with E . coli protein content was found necessary to obtain a final product that contained less than 0.01% of host derived proteins . The recombinant proteins were renatured either from 8 M urea or from 6 M guanidine hydrochloride by increasing the pH to 10.5 in the presence of glycine and EDTA, reduction with DTT, dilution to a protein concentration below 1 mg.ml-1, and dialysis against 0.9% NaCl . The method presented here can be tailor-fit, with minor modification, for the purification of almost any recombinant protein and the final product satisfies current regulations concerning the production of clinically acceptable therapeutic products.

J Biol Chem, 1991 Oct 25, 266(30), 20447 - 52
Identification, expression, and deduced primary structure of transketolase and other enzymes encoded within the form II CO2 fixation operon of Rhodobacter sphaeroides; Chen JH et al.; Previous studies had indicated that the form II or B cluster of CO2 fixation structural genes is part of a large operon in Rhodobacter sphaeroides (Gibson, J . L., Chen, J.-H., Tower, P . A., and Tabita, F . R . (1990) Biochemistry 29, 8085-8093) . In this investigation, we have sequenced the DNA between the prkB and rbpL genes and provide evidence for three distinct open reading frames which encode additional structural genes of the Calvin reductive pentose phosphate pathway; these genes encode the enzymes transketolase, glyceraldehyde phosphate dehydrogenase, and aldolase . Noteworthy is transketolase, which may be expressed to high levels in Escherichia coli . This study thus represents the initial description of the primary structure of bacterial transketolase, a key enzyme of the reductive and the oxidative pentose phosphate pathways . Each of the genes are separated by short stretches of intergenic sequence, consistent with earlier evidence which suggested that these genes are cotranscribed and part of a large operon controlled by sequences upstream from fbpB.

J Biol Chem, 1991 Oct 25, 266(30), 20375 - 9
The length of the aminoacyl-acceptor stem of the selenocysteine-specific tRNA(Sec) of Escherichia coli is the determinant for binding to elongation factors SELB or Tu; Baron C et al.; Mutations in selC, which reduce the 8-base pair aminoacyl-acceptor helix to the canonical 7-base pair length (tRNA(Sec)(delAc} or which replace the extra arm of tRNA(Sec) by that of a serine acceptor tRNA species (tRNA(Sec)(ExS), block the function in selenoprotein synthesis in vivo (Baron, C., Heider, J., and Bock, A . (1990) Nucleic Acids Res . 18, 6761-6766) . tRNA(Sec), tRNA(Sec)(delAc), and tRNA(Sec)(ExS) were purified and analyzed for their interaction with purified seryl-tRNA synthetase, selenocysteine synthase and translation factors SELB and EF-Tu . It was found that seryl-tRNA synthetase displays 10-fold impaired Km and Kcat values for tRNA(Sec) in comparison to tRNA(Ser), decreasing the overall charging efficiency (Kcat/Km) of tRNA(Sec) to 1% of that characteristic for tRNA(Ser) . tRNA(Sec)(ExS) was a less efficient substrate for the enzyme (Kcat/Km 0.2% of the tRNA(Ser) value) whereas the tRNA(Ser)(delAc) variant was charged with an approximately 2-3-fold improved rate compared to wild-type tRNA(Sec) . Both mutant tRNA variants, when charged with L-serine, were able to interact with selenocysteine synthase to give rise to selenocysteyl-tRNA with tRNA(Sec)(ExS) being as efficient as wild-type tRNA(Sec) . Seryl-tRNA(Sec)(delAc), on the other hand, was selenylated very slowly . Reduction of the length of the aminoacyl-acceptor stem to 7 base pairs prevented the interaction with translation factor SELB but allowed binding to EF-Tu, irrespective of whether tRNA(Sec)(delAc) was charged with serine or selenocysteine . The aminoacyl-acceptor helix of tRNA(Sec), therefore, is a major determinant directing binding to SELB and precluding interaction with EF-Tu.

J Biol Chem, 1991 Oct 25, 266(30), 20223 - 31
The N-terminal acidic domain of heparin cofactor II mediates the inhibition of alpha-thrombin in the presence of glycosaminoglycans; Van Deerlin VM et al.; Heparin cofactor II (HCII) is a glycoprotein in human plasma that inhibits thrombin and chymotrypsin . Inhibition occurs when the protease attacks the reactive site peptide bond in HCII (Leu444-Ser445) and becomes trapped as a covalent 1:1 complex . Dermatan sulfate and heparin increase the rate of inhibition of thrombin, but not of chymotrypsin, greater than 1000-fold . The N-terminal portion of HCII contains two acidic repeats (Glu56-Asp-Asp-Asp-Tyr-Leu-Asp and Glu69-Asp-Asp-Asp-Tyr-Ile-Asp) that may bind to anion-binding exosite I of thrombin to facilitate covalent complex formation . To examine the importance of the acidic domain, we have constructed a series of 5' deletions in the HCII cDNA and expressed the recombinant HCII (rHCII) in Escherichia coli . Apparent second-order rate constants (k2) for inhibition of alpha-thrombin and chymotrypsin by each variant were determined . Deletion of amino acid residues 1-74 had no effect on the rate of inhibition of alpha-thrombin or chymotrypsin in the absence of a glycosaminoglycan . Similarly, the rate of inhibition of alpha-thrombin in the presence of a glycosaminoglycan was unaffected by deletion of residues 1-52 . However, deletion of residues 1-67 (first acidic repeat) or 1-74 (first and second acidic repeats) greatly decreased the rate of inhibition of alpha-thrombin in the presence of heparin, dermatan sulfate, or a dermatan sulfate hexasaccharide that comprises the minimum high-affinity binding site for HCII . Deletion of one or both of the acidic repeats increased the apparent affinity of rHCII for heparin-Sepharose, suggesting that the acidic domain may interact with the glycosaminoglycan-binding site of native rHCII . The stimulatory effect of glycosaminoglycans on native rHCII was decreased by a C-terminal hirudin peptide which binds to anion-binding exosite I of alpha-thrombin . Furthermore, the ability of native rHCII to inhibit gamma-thrombin, which lacks the binding site for hirudin, was stimulated weakly by glycosaminoglycans . These results support a model in which the stimulatory effect of glycosaminoglycans on the inhibition of alpha-thrombin is mediated, in part, by the N-terminal acidic domain of HCII.

J Biol Chem, 1991 Oct 25, 266(30), 20205 - 12
Enzymatic properties of mutant Escherichia coli tryptophan synthase alpha-subunits; Lim WK et al.; Thirty-nine mutant tryptophan synthase alpha subunits have been purified and analyzed (in the presence of the beta 2-subunit) for their enzymatic (kcat, Km) behavior in the reactions catalyzed by the alpha 2.beta 2 complex, the fully constituted form of this enzyme . The mutant alpha subunits, obtained by in vitro random, saturation mutagenesis of the encoding trpA gene, contain single amino acid substitutions at sites within the first 121 residues of the alpha polypeptide . Four categories of altered residues have been tentatively assigned roles in the catalytic functions of this enzyme: 1) catalytic residues (Glu49 and Asp60); 2) residues involved in substrate binding or orientation (Phe22, Thr63, Gln65, Tyr102, and Leu105); 3) residues involved in alpha.beta subunit interactions (Gly51, Pro53, Asp56, Asp60, Pro62, Ala67, Phe72, Thr77, Pro78, Tyr102, Asn104, Leu105, and Asn108); and 4) residues with no apparent catalytic roles . Catalytic residue alterations result in no detectable activity in the alpha-subunit specific reactions . Substrate binding/orientation roles are detected enzymatically primarily as rate defects; alterations only at Tyr102 result in apparent Km effects . alpha.beta interaction roles are detected as rate defects in all tryptophan synthase reactions plus Km increases for the alpha-subunit substrate, indole-3-glycerol phosphate, only when L-serine is present at the beta 2-subunit active site . A substitution at only one site, Asn104, appears to be unique in its potential effect on intersubunit channeling of indole, the product of the alpha-subunit specific reaction, to the beta 2-subunit active site.

J Biol Chem, 1991 Oct 25, 266(30), 19976 - 80
NADPH-cytochrome P-450 oxidoreductase . The role of cysteine 566 in catalysis and cofactor binding; Shen AL et al.; Site-directed mutagenesis was employed to investigate the role of Cys566 in the catalytic mechanism of rat liver NADPH-cytochrome P-450 oxidoreductase . Rat NADPH-cytochrome P-450 oxidoreductase and mutants containing either alanine or serine at position 566 were expressed in Escherichia coli and purified to homogeneity . Substitution of alanine at position 566 had no effect on enzymatic activity with the acceptors cytochrome c and ferricyanide but did increase trans-hydrogenase activity with 3-acetylpyridine adenine dinucleotide phosphate by 79% . The Km for NADPH was increased 2.5-fold, and the NADP+ KI was increased 4.8-fold compared with that found for the wild-type enzyme . The conservative substitution, Ser566, produced a 50% decrease in cytochrome c reductase activity whereas activity with ferricyanide was decreased 57%, and 3-acetylpyridine adenine dinucleotide phosphate activity was unaffected . The NADPH Km was increased 4.6-fold, and the NADP+ KI increased 7.6-fold . The dependence of cytochrome c reductase activity on the KCl concentration was markedly altered by the Cys566 substitutions . Maximum activity for the wild-type enzyme was observed at approximately 0.18 M KCl whereas maximum activity for the mutant enzymes was observed between 0.04 and 0.09 M KCl . The pH dependence of cytochrome c reductase activity, cytochrome c Km, and flavin content were unaffected by these substitutions . These results demonstrate that Cys566 is not essential for activity of rat liver NADPH-cytochrome P-450 oxidoreductase although the cysteine side chain does affect the interaction of NADPH with the enzyme.

J Biol Chem, 1991 Oct 25, 266(30), 19925 - 9
Characterization of a mutation affecting the function of Escherichia coli folylpolyglutamate synthetase-dihydrofolate synthetase and further mutations produced in vitro at the same locus; Keshavjee K et al.; The folC gene from mutant strain SF4 was cloned into a pUC19 plasmid . Expression of the mutant gene from the lac promoter of the plasmid complemented the auxotrophy for methionine of the SF4 strain . The only difference in sequence between the mutant and wild-type genes was a G925A base change resulting in an A309T amino acid change . The mutant enzyme had a 30-fold higher Km for 10-formyltetrahydrofolate as well as a 60-fold higher Km for glutamate and a 200-fold higher Km for dihydropteroate of the dihydrofolate synthetase activity . Site-specific mutagenesis was used to substitute other amino acids at codon 309 . Mutants with glycine, isoleucine, and valine substitutions at this position, when expressed from multicopy plasmids, complemented the SF4 strain . The glycine mutant had properties similar to the wild-type enzyme, whereas the isoleucine and valine mutants had properties similar to the threonine mutant, SF4 . Mutant genes with arginine, glutamate, and leucine substitutions, which did not complement the SF4 strain, could complement a folC deletion strain, but produced smaller colonies on complex plates and did not grow on minimal medium . In the deletion strain, an increasing requirement for folate product supplements was observed as the folylpolyglutamate synthetase-dihydrofolate synthetase activities of the complementing mutants decreased.

Biochim Biophys Acta, 1991 Oct 25, 1080(2), 96 - 102
Recombinant mouse leukotriene A4 hydrolase: a zinc metalloenzyme with dual enzymatic activities; Wetterholm A et al.; Recombinant mouse leukotriene A4 hydrolase was expressed in Escherichia coli as a fusion protein with ten additional amino acids at the amino terminus and was purified to apparent homogeneity by means of precipitation, anion exchange, hydrophobic interaction and chromatofocusing chromatographies . By atomic absorption spectrometry, the enzyme was shown to contain one mol of zinc/mol of enzyme . Apparent kinetic constants (Km and Vmax) for the conversion of leukotriene A4 to leukotriene B4 (at 0 degree C, pH 8) were 5 microM and 900 nmol/mg per min, respectively . The purified enzyme also exhibited significant peptidase activity towards the synthetic amide alanine-4-nitroanilide . Km and Vmax for this reaction (at 37 degrees C, pH 8) were 680 microM and 365 nmol/mg per min, respectively . Apo-leukotriene A4 hydrolase, prepared by treating the enzyme with 1,10-phenanthroline, was virtually inactive with respect to both enzymatic activities, but could be reactivated by addition of stoichiometric amounts of zinc or cobalt . Exposure of the enzyme to leukotriene A4 resulted in a dose-dependent inactivation of both enzyme activities.

Biochim Biophys Acta, 1991 Oct 25, 1080(2), 173 - 80
Immunoreactive alpha A crystallin in rat non-lenticular tissues detected with a sensitive immunoassay method; Kato K et al.; For the quantitative analysis of the A subunit of alpha crystallin (alpha A) in the lens and for the survey of possible existence of alpha A in the non-lenticular tissues, we have established a highly sensitive and specific immunoassay method for alpha A . Antisera to alpha A were raised in rabbits with alpha A purified from bovine lens, or the C-terminal decapeptide (EEKPSSAPSS) of alpha A (alpha Apep) . The antibodies to alpha A and alpha Apep were purified by the use of an alpha A-coupled Sepharose 4B column . The F(ab')2 fragments of purified anti-alpha A IgG were immobilized on polystyrene balls and the Fab' fragments of purified anti-alpha Apep IgG were labeled with beta-D-galactosidase from Escherichia coli . The minimum detection limit of the sandwich-type immunoassay using the two antibody preparations was less than 10 pg alpha A without any cross-reactivity with alpha B . By employing the present methods, it was found that a significant amount of immunoreactive alpha A was present in rat spleen and thymus . Very low levels of immunoreactive alpha A were detected in the rectum, caecum, liver, kidney, adrenal, cerebellum and brainstem . The immunoreactive alpha A in the spleen extract was purified partially (about 50% purity) by the use of anti-alpha Apep-coupled Sepharose . The concentration of alpha A in the spleen was less than 1 ng/mg protein before 3 weeks of age . After 5 weeks of age, however, it increased lineally reaching about 20 ng/mg protein by 18 weeks of age . Immunohistochemically, the alpha A was localized in the reticular cells in the spleen and thymus.

Nucleic Acids Res, 1991 Oct 25, 19(20), 5569 - 74
Processing in vitro of an abasic site reacted with methoxyamine: a new assay for the detection of abasic sites formed in vivo; Rosa S et al.; In this study we demonstrate that the different substrate recognition properties of bacterial and human AP endonucleases might be used to quantify and localize apurinic (AP) sites formed in DNA in vivo . By using a model oligonucleotide containing a single AP site modified with methoxyamine (MX), we show that endonuclease III and IV of E . coli are able to cleave the alkoxyamine-adducted site whereas a partially purified HeLa AP endonuclease and crude cell-free extracts from HeLa cells are inhibited by this modification . In addition MX-modified AP sites in a DNA template retain their ability to block DNA synthesis in vitro . Since MX can efficiently react with AP sites formed in mammalian cells in vivo we propose that the MX modified abasic sites thus formed can be quantitated and localized at the level of the individual gene by subsequent site specific cleavage by either E . coli endonuclease III or IV in vitro.

Nucleic Acids Res, 1991 Oct 25, 19(20), 5491 - 6
Divergent genes for translation initiation factor eIF-4A are coordinately expressed in tobacco; Owttrim GW et al.; Three cDNA clones coding for eukaryotic translation initiation factor 4A, eIF-4A, were isolated from a Nicotiana plumbaginifolia root cDNA library by heterologous screening . The clones comprise two distinct gene classes as two clones are highly similar while the third is divergent . The genes belong to a highly conserved gene family, the DEAD box supergene family, although the divergent clone contains a DESD box rather than the characteristic DEAD box . The two clones are representatives of separate small multigene families in both N . plumbaginifolia and N . tabacum . Representatives of each family are coordinately expressed in all plant organs examined . The 47 kD polypeptide product of one clone, overexpressed in E . coli, crossreacts immunologically with a rabbit reticulocyte eIF-4A polyclonal antibody . Taken together the data suggest that the two Nicotiana eIF-4A genes encode translation initiation factors . The sequence divergence and the coordinate expression of the two Nicotiana eIF-4A families provide an excellent system to determine if functionally distinct eIF-4A polypeptides are required for translation initiation in plants.

Nucleic Acids Res, 1991 Oct 25, 19(20), 5743 - 8
Effects of phosphorothioate capping on antisense oligonucleotide stability, hybridization and antiviral efficacy versus herpes simplex virus infection; Hoke GD et al.; Efforts have been made to improve the biological stability of phosphodiester (PO) oligonucleotides by the addition of various modifications to either the 3', 5' or both the 3' and 5' ends of an oligonucleotide . ISIS 1080, a phosphorothioate (PS) 21-mer oligonucleotide complementary to the internal AUG codon of UL13 mRNA in HSV-1, reduces the infectious yield of HSV-1 in HeLa cells to 9.0% +/- 11% . PO analogs of ISIS 1080 containing three PS linkages placed on the 3' (ISIS 1365), 5' (ISIS 1370), both the 3' and 5' (ISIS 1364) ends or with four linkages in the middle (ISIS 1400) demonstrated reduced antiviral efficacy compared to fully PS ISIS 1080 . Thermal denaturation profiles demonstrated that these oligonucleotides hybridized to complementary DNA or RNA with equivalent binding affinities . All were able to support E . coli RNAse H cleavage of the HSV mRNA to which they were targeted . The stability of the congeners in cell culture medium containing 10% fetal calf serum (FCS), HeLa cytosolic extract, HeLa nuclear extract and in intact HeLa cells revealed that ISIS 1080 was most resistant to nucleolytic digestion through 48 hours . Partial PS oligonucleotides exhibited increased degradation compared to the fully thioated oligonucleotide by exonuclease activity in FCS and endonuclease activity in cell extracts or intact cells . Thus, the reduced efficacy of partial compared to fully PS oligonucleotides against HSV-1 in HeLa cells may result from increased degradation of the mixed PO/PS oligonucleotides.

J Biol Chem, 1991 Oct 25, 266(30), 20232 - 7
Role of alpha beta receptor heterodimer formation in beta platelet-derived growth factor (PDGF) receptor activation by PDGF-AB; Heidaran MA et al.; We investigated the ability of highly purified recombinant platelet-derived growth factor (PDGF) AB to interact with the products of alpha and beta receptor genes expressed in cells independently or concurrently . Although PDGF-AB lacked any detectable ability to bind or activate beta receptors in cells expressing only this receptor, efficient beta receptor activation by this ligand was readily observed in cells coexpressing alpha platelet-derived growth factor receptors (alpha PDGFRs) . beta receptor activation induced by PDGF-AB was shown to be dependent upon in vivo physical association of this receptor with alpha PDGFRs . Moreover, cross-linking analysis established the existence of PDGF-AB-induced beta PDGFR dimers in vivo . All of these findings argue that initial PDGF-AB interaction with the alpha PDGFR induces conformational changes in the ligand or receptor that facilitates efficient recruitment of beta PDGFR by this PDGF isoform.

Nature, 1991 Oct 24, 353(6346), 762 - 5
Structure of domain 1 of rat T lymphocyte CD2 antigen; Driscoll PC et al.; The CD2 antigen is largely restricted to cells of the T-lymphocyte lineage and has been established as an important adhesion molecule in interactions between human T lymphocytes and accessory cells . In the adhesion reaction, CD2 on T cells binds to LFA-3 on other cells, with binding through domain 1 of CD2 . CD2 can also be a target for the delivery of mitogenic signals to T lymphocytes cultured with combinations of anti-CD2 antibodies . Two predictions that are contradictory have been made for the structure of CD2 domain 1 . One suggests an immunoglobulin (Ig) fold, on the basis of sequence patterns conserved in the Ig-superfamily (IgSF), whilst the other proposes a pattern of alternating alpha-helices and beta-strands, on the basis of secondary structure predictions . Thus CD2 domain 1 is an important test case for the validity of IgSF assignments based on sequence patterns . We report here the expression of domain 1 of rat CD2 in an Escherichia coli expression system and have determined a low-resolution solution structure by NMR spectroscopy.

Biochemistry, 1991 Oct 22, 30(42), 10337 - 43
Synthesis and expression in Escherichia coli of a gene for kappa-bungarotoxin; Fiordalisi JJ et al.; A gene which codes for the 66-residue polypeptide of kappa-bungarotoxin has been chemically synthesized by linking together 3 synthetic double-stranded oligonucleotides in a bacterial plasmid . The synthesis incorporated six unique silent restriction sites spaced throughout the gene for use in cassette mutagenesis . Direct expression of the kappa-bungarotoxin polypeptide by itself in Escherichia coli failed to result in a stable product . The toxin polypeptide was stabilized and expressed in E . coli as part of a fusion protein with rat intestinal fatty acid binding protein under control of the nalidixic acid inducible recA promoter . Two fusion protein constructs were prepared that differed only in the cleavage site between the fatty acid binding protein and the toxin polypeptide . One contained a factor Xa cleavage site, and the other, since the toxin itself is devoid of methionine, contained a methionyl residue that served as a cyanogen bromide cleavage site . The fusion proteins were isolated by ion-exchange chromatography and reverse-phase HPLC . The construct containing the factor Xa cleavage site could not be cleaved under nondenaturing conditions . On the other hand, kappa-bungarotoxin was efficiently cleaved from the methionyl fusion protein with CNBr . The toxin polypeptide was isolated by reverse-phase HPLC and ion-exchange chromatography and produced a complete and specific blockade of neuronal nicotinic acetylcholine receptors in chick ciliary ganglia which was indistinguishable from that produced by a comparable amount of venom-purified kappa-bungarotoxin.

Biochemistry, 1991 Oct 22, 30(42), 10200 - 6
Fluorescence study of a mutant cytochrome b5 with a single tryptophan in the membrane-binding domain; Ladokhin AS et al.; Fluorescence studies of cytochrome b5 are complicated by the presence of three tryptophans, at positions 108, 109, and 112, in the membrane-binding domain . The cDNA for rabbit liver cytochrome b5, isolated from a lambda gt11 library, was used to generate a mutated mRNA where the codons for tryptophans-108 and -112 were replaced by codons for leucine . The sequence was expressed in Escherichia coli and the mutant protein was isolated . This mutant protein had the expected absorption spectrum, and its amino acid composition was confirmed by amino acid analysis and by DNA sequencing of the construct . The fluorescence emission spectrum of the mutant is blue-shifted and is narrower than that of the native protein . The quantum yield of the mutant protein, per molecule, is only 60% of that of the native protein, and the enhancement when bound to lipid vesicles or detergent micelles is higher for the mutant . Fluorescence anisotropy measurements and quenching studies using brominated lipids suggest that the fluorescence of the native protein is due to tryptophans-109 and -108 while tryptophan-112 does not emit but undergoes nonradiative energy transfer to tryptophan-108 . With this mutant, it was shown that incomplete energy transfer from tyrosines-126 and -129 to tryptophan-109 occurs when the membrane binding domain is inserted into lipid vesicles, which suggests that the membrane-binding domain does not exist in a tight hairpin loop.

Biochemistry, 1991 Oct 22, 30(42), 10155 - 63
Hydrophobic content and lipid interactions of wild-type and mutant OmpA signal peptides correlate with their in vivo function; Hoyt DW et al.; Peptides corresponding to the wild-type signal sequence of the Escherichia coli outer membrane protein OmpA and several mutants have been synthesized and characterized biophysically . The mutations were designed collaboratively with Inouye and co-workers to test the understanding of the critical characteristics of signal sequences required for their functions . The in vivo results for these mutants have been reported {Lehnhardt, S., Pollitt, S., & Inouye, M . (1987) J . Biol . Chem . 262, 1716-1719; Goldstein, J., Lehnhardt, S., & Inouye, M . (1990) J . Bacteriol . 172, 1225-1231; Goldstein, J., Lehnhardt, S., & Inouye, M . (1991) J . Biol . Chem . 266, 14413-14417}, and the present paper compares the conformational and membrane-interactive properties of six of the OmpA signal peptides . Peptides corresponding to functional OmpA signal sequences in vivo are predominantly alpha-helical in membrane-mimetic environments and insert readily into phospholipid bilayers . Nonfunctional OmpA signal peptides may have high helical content but do not penetrate deeply into the acyl chain region of bilayers . The ability of the signal peptides to insert into membranes and their in vivo function correlate with the residue-average hydrophobicity of their hydrophobic cores . The results obtained on OmpA signal peptides parallel closely our previous observations on peptides corresponding to the LamB signal sequence and mutants, arguing that the critical biophysical properties of signal sequences are general despite their lack of primary sequence identity.

Biochemistry, 1991 Oct 22, 30(42), 10150 - 5
Effect of heme binding on the structure and stability of Escherichia coli apocytochrome b562; Feng YQ et al.; The structure and stability of apocytochrome b562 were explored using absorption and circular dichroism spectroscopic methods . The polypeptide chain retains a well-defined structure when the prosthetic heme group is removed from cytochrome b562 . Circular dichroism measurements estimate 60% helicity for apocytochrome b562, compared with 80% helicity found in holocytochrome b562 . At low pH, apocytochrome b562 displays a midpoint pH of 2.9, while ferricytochrome b562 displays a midpoint pH of 2.3 . The unfolding of the apoprotein by urea and heat can be well approximated by the two-state transition model . The stability of apocytochrome b562 is significantly reduced from that of the holoprotein . The free energy of stabilization (delta G degrees) and the midpoint transition temperature (Tm) for apocytochrome b562 are found to be 3.2 +/- 0.5 kcal/mol and 52.3 +/- 0.9 degrees C, respectively, compared with 6.6 +/- 0.5 kcal/mol and 67.2 +/- 0.5 degrees C for ferricytochrome b562 . The smaller heat capacity change upon unfolding of apocytochrome b562 than that of ferricytochrome b562, estimated from the thermodynamic parameters, indicates that apocytochrome b562 possesses a smaller hydrophobic core than holocytochrome b562 . Size-exclusion chromatography studies indicate that the apoprotein is slightly more extended in molecular dimension than ferricytochrome b562 . The data suggest that apocytochrome b562 resembles a "molten globule" or a "collapsed form" of the holoprotein, in which secondary structure formation is largely complete while the global folding is either only partially complete or dynamically expanded.

Biochemistry, 1991 Oct 22, 30(42), 10280 - 7
Fluorescence studies on the interaction of inhibitor 2 and okadaic acid with the catalytic subunit of type 1 phosphoprotein phosphatases; Picking WD et al.; Phosphatase inhibitor 2 was mutagenized and expressed in Escherichia coli to produce a protein with a single cysteinyl residue at position 129 . The newly introduced sulfhydryl group was labeled with a maleimide derivative of coumarin (CPM) . The resulting fluorescent inhibitor 2 molecule (CPM-I2) retains biological activity and binds to the catalytic subunit of type 1 phosphatase (PP1-C) with a Kd similar to the Ki of native I2 (2-3 nM) . Fluorescence anisotropy data indicate that kinase FA (glycogen synthase kinase 3) does not dissociate the CPM-I2.PP1-C complex but rather causes a conformational change in the I2 molecule that is retained even after the CPM-I2 is displaced by an excess of native I2 . The fluorescence data presented here also indicate that okadaic acid and I2 are competitive for binding to PP1-C, even after kinase FA treatment of the CPM-I2.PP1-C complex.

FEBS Lett, 1991 Oct 21, 291(2), 259 - 63
A coupled in vitro transcription-translation system for the exclusive synthesis of polypeptides expressed from the T7 promoter; Nevin DE et al.; A coupled transcription-translation in vitro system has been developed in Escherichia coli specifically for the expression of genes under the exclusive control of the T7 promoter . This system consists of an E . coli crude extract (prepared from cells containing endogenous T7 RNA polymerase), rifampicin (an E . coli RNA polymerase inhibitor) and a labelled amino acid . When primed with a plasmid template encoding the target gene under exclusive control of the T7 promoter, this system has the capability to synthesize relatively large amounts of a unique, labelled polypeptide . This paper describes the characteristics and use of such a T7 RNA polymerase/T7-promoter specific in vitro system.

FEBS Lett, 1991 Oct 21, 291(2), 225 - 8
Acetylcholine interactions with tryptophan-184 of the alpha-subunit of the nicotinic acetylcholine receptor revealed by transferred nuclear Overhauser effect; Fraenkel Y et al.; Acetylcholine interactions with three genetically engineered fusion proteins containing peptides from the nicotinic acetylcholine receptor were studied by 1D and 2D nuclear magnetic resonance methods . The three proteins were Torpedo alpha 184-200, Torpedo alpha 186-198, and human alpha 183-204 of the acetylcholine receptor fused to the first 323 residues of the E . coli protein trpE . Nuclear Overhauser effect studies revealed interactions of bound acetylcholine with tryptophan-184 present in the Torpedo alpha 184-200, and the human alpha 183-204 sequences . These interactions are between the N(CH3)3+ and CH3 groups of acetylcholine with the aromatic protons of tryptophan . The appearance of these cross-peaks indicates a distance of less than 5 A between tryptophan and the bound ligand; however, direct contact has yet to be proven.

FEBS Lett, 1991 Oct 21, 291(2), 222 - 4
Protein aggregation and inclusion body formation in Escherichia coli rpoH mutant defective in heat shock protein induction; Gragerov AI et al.; Mutations in the rpoH gene, encoding sigma 32, an alternative factor required for transcription of the heat shock genes, result in the extensive aggregation of virtually all cellular proteins and formation of inclusion bodies both under stress and non-stress conditions . Inhibitors of protein synthesis suppress this aggregation, suggesting that newly synthesized proteins preferentially aggregate in rpoH mutants . These data suggest that the heat shock proteins are involved in acquisition of the soluble state (i.e . correct conformation) of the bulk of intracellular proteins after their translation.

FEBS Lett, 1991 Oct 21, 291(2), 319 - 21
Glutamic acid-112 of the A subunit of heat-labile enterotoxin from enterotoxigenic Escherichia coli is important for ADP-ribosyltransferase activity; Tsuji T et al.; A mutant strain of enterotoxigenic Escherichia coli (E . coli pTUH 6A) produced an abnormal heat-labile enterotoxin (LT), the A subunit of which has a single amino acid substitution at position 112 (Glu-112 to Lys-112) . As already reported, this mutant LT had no ileal loop and vascular permeability activities {(1990) J . Biol . Chem . 265, 22520-22525} . In this paper we report that the mutant LT showed no CHO cell elongation activity and did not activate adenylate cyclase of target cells . Moreover, no ADP-ribosyltransferase activity was detected in the mutant LT . It is concluded that the amino acid substitution at position 112 abolished the ADP-ribosyltransferase activity of the A subunit and this leads to the loss of toxic activities of LT.

J Mol Biol, 1991 Oct 20, 221(4), 1367 - 78
An efficient algorithm for identifying matches with errors in multiple long molecular sequences; Leung MY et al.; An efficient algorithm is described for finding matches, repeats and other word relations, allowing for errors, in large data sets of long molecular sequences . The algorithm entails hashing on fixed-size words in conjunction with the use of a linked list connecting all occurrences of the same word . The average memory and run time requirement both increase almost linearly with the total sequence length . Some results of the program's performance on a database of Escherichia coli DNA sequences are presented.

J Mol Biol, 1991 Oct 20, 221(4), 1311 - 24
Sequence-specific 1H n.m.r . assignments and determination of the three-dimensional structure of reduced Escherichia coli glutaredoxin; Sodano P et al.; The determination of the nuclear magnetic resonance structure of reduced E . coli glutaredoxin in aqueous solution is described . Based on nearly complete, sequence-specific resonance assignments, 813 nuclear Overhauser effect distance constraints and 191 dihedral angle constraints were employed as the input for the structure calculations, for which the distance geometry program DIANA was used followed by simulated annealing with the program X-PLOR . The molecular architecture of reduced glutaredoxin is made up of three helices and four-stranded beta-sheet . The first strand of the beta-sheet (residues 2 to 7) runs parallel to the second strand (32 to 37) and antiparallel to the third strand (61 to 64), and the sheet is extended in an antiparallel fashion with a fourth strand (67 to 69) . The first helix with residues 13 to 28 and the last helix (71 to 83) run parallel to each other on one side of the beta-sheet, with their direction opposite to that of the two parallel beta-strands, and the helix formed by residues 44 to 53 fills space available due to the twist of the beta-sheet and the reduced length of the last two beta-strands . The active site Cys11-Pro-Tyr-Cys14 is located after the first beta-strand and occupies the latter part of the loop connecting this strand with the first helix.

J Mol Biol, 1991 Oct 20, 221(4), 1433 - 42
Mutations in the MotA protein of Escherichia coli reveal domains critical for proton conduction; Blair DF et al.; The MotA protein of Escherichia coli is an essential component of the torque-generating units that drive the flagellar rotary motor . A variety of evidence indicates that MotA is involved in transmembrane proton conduction . We have now mapped a number of MotA mutants, focusing primarily on those previously shown to be dominant . Fifty-six mutations (all dominant), each causing severe or complete impairment of function, were sequenced and found to correspond to 31 different alleles . All except two of these encoded amino acid substitutions clustered in four hydrophobic, presumably membrane-spanning segments, that together make up only one-third of the length of the polypeptide chain . In contrast, eight mutations (5 dominant), each causing only slight impairment of function (slow alleles), were sequenced and found to specify amino acid substitutions in three hydrophilic domains . The clustering of the mutations provides independent support for the suggestion that MotA is a transmembrane proton channel and places significant constraints on models for the molecular mechanism of ion conduction.

J Mol Biol, 1991 Oct 20, 221(4), 1139 - 51
Structure and assembly of the Escherichia coli transcription termination factor rho and its interactions with RNA . II . Physical chemical studies; Seifried SE et al.; Transcription termination factor rho from Escherichia coli is comprised of a hexamer of identical protein monomers . Hydrodynamic and light-scattering studies have shown the fully assembled rho to be a doughnut-shaped structure . Semi-denaturing gels, protein crosslinking, and spectroscopic studies, as well as other functional and binding determinations have established that the rho hexamer displays D3 symmetry (i.e . it exists as a trimer of dimers) . In the accompanying paper we visualize rho directly in the absence of cofactor and show that binding of RNA it into the hexameric form . In this paper we examine the pathway and association constants involved in rho oligomer assembly . Sedimentation and fluorescence-detected size exclusion chromatography are used to demonstrate three steps in the assembly process . These steps can be differentiated by subunit association affinity and kinetic properties . The kinetics of the monomer-dimer equilibrium are fast and an apparent association constant of 1.3 x 10(6) M-1 is measured for this process . In contrast, the dimer-tetramer and tetramer-hexamer association processes appear to be slower (of the order of seconds) and to involve association constants that are smaller than that of the monomer-dimer reaction . This behaviour is consistent with a hexamer of D3 symmetry . Such a particle displays two kinds of subunit interactions; one associated with an intra-dimer A:A interface and the other with an inter-dimer B:B interface . The closure of the circular hexamer does not appear to contribute additional free energy to the assembly process . Fluorescence and sedimentation studies show the association steps to be sensitive to salt concentration . Consistent with earlier work, we find that assembly to the hexameric state is driven by RNA binding.

J Mol Biol, 1991 Oct 20, 221(4), 1127 - 38
Structure and assembly of the Escherichia coli transcription termination factor rho and its interaction with RNA . I . Cryoelectron microscopic studies; Gogol EP et al.; Cryoelectron microscopy has been used to visualize the Escherichia coli transcription termination protein rho in a vitreously frozen state, without the use of strains, fixatives or other chemical perturbants . In the absence of RNA cofactor, a variety of structures are observed, reflecting the heterogeneity of complexes formed by rho at protein concentrations near the physiological range (3 to 10 microM) . One of the most common structural motifs we see is a six-membered ring of rho subunits (present as either a closed or "notched" circle), which corresponds to the predominant hexameric association state of the protein . Also visible are smaller oligomeric structures, present as curved lines of rho subunits, which probably represent the lower association states of the protein that coexist with the hexamer at these protein concentrations . Addition of oligomers of ribocytosine (rC) of defined lengths (23-mers and 100-mers) results in the generation of more homogeneous populations of rho oligomers . In the presence of (rC)23, all identifiable particles appear either as closed or as notched hexameric circles . A small fraction of these particles are of visibly higher density, and are identified with the dodecamers expected as a subpopulation of rho under these conditions . Binding of (rC)100, an oligomer of length greater than that needed to span the entire hexamer binding site, results in a uniform population of closed circular hexamers . In some images additional features are visible at either the centers or the peripheries of the particles . These features may correspond to the excess length of the rC strands bound to the hexamers . The distributions of particles observed under the various experimental conditions used correlate well to those deduced from physical biochemical studies Seifried et al., accompanying paper).

J Mol Biol, 1991 Oct 20, 221(4), 1453 - 60
Mapping electrostatic interactions in macromolecular associations; Rodgers KK et al.; In the association of electron transfer proteins, electrostatics has been proposed to play a role in maintaining the stability and specificity of the biomolecular complexes formed . An excellent model system is the interaction between mammalian cytochrome b5 and cytochrome c, in which the X-ray structures of the individual components reveal a complementary asymmetry of charges surrounding their respective redox centers . Determining the exact extent of the electrostatic interactions and identifying the specific residues involved in the formation of the electron transfer complex has proved more elusive . We report herein the utilization of high-pressure techniques, together with site-directed mutagenesis, to provide a map of the interaction domains in biomolecular complex formation . The application of high pressure disrupts macromolecular associations since dissociation of the complex results in a decreased volume of the system due to the solvation of charges that had been previously sequestered in the interface region and force solvation of hydrophobic surfaces . Site-directed mutagenesis of a totally synthetic gene for rat liver cytochrome b5, which expresses this mammalian protein in Escherichia coli as a hemecontaining soluble component, was used to selectively alter negatively charged residues of cytochrome b5 to neutral amide side-chains . We have demonstrated that the interaction domain of cytochrome b5 with cytochrome c can be mapped from a comparison of dissociation volumes of these modified cytochrome b5-cytochrome c complexes with the native complex . Using these techniques we can specifically investigate the role of particular residues in the equilibrium association of these two electron transfer proteins . Single-point mutations in the interaction domain give nearly identical effects on the measured dissociation volumes, yet removal of acidic residues outside the recognition surface yield volumes similar to wild-type protein . Multiple mutations in the proposed protein-protein interaction site are found to allow greater solvent-accessibility of the interface as reflected in a diminution in the volume changes on subsequent charge removal . This is indicative that the interprotein salt-bridges in this complex provide a mechanism for a greater exclusion of solvent from the interfacial domain of the complex, resulting in a more stable association.

Cell, 1991 Oct 18, 67(2), 365 - 76
Adenovirus E1A activation domain binds the basic repeat in the TATA box transcription factor; Lee WS et al.; The adenovirus large E1A protein is a potent activator of transcription . We use several different experimental approaches to demonstrate that the large E1A protein binds specifically and stably to the TATA box-binding factor (TFIID), the general polymerase II transcription factor that initiates assembly of transcription complexes . Sedimentation velocity centrifugation revealed that TFIID and E1A form a heterodimer in vitro . We demonstrate that the activation domain of E1A (conserved region 3) binds to TFIID . E1A interacts with a 51 residue region from the conserved C-terminal domain of TFIID that includes a repeat of basic residues between the homologous direct repeats of TFIID . Analysis of TFIID binding by various E1A mutants indicates that TFIID binding is necessary, although not sufficient, for E1A transactivation.

Cell, 1991 Oct 18, 67(2), 377 - 88
Transcriptional repression by YY1, a human GLI-Krüppel-related protein, and relief of repression by adenovirus E1A protein; Shi Y et al.; A sequence within the transcription control region of the adeno-associated virus P5 promoter has been shown to mediate transcriptional activation by the adenovirus E1A protein . We report here that this same element mediates transcriptional repression in the absence of E1A . Two cellular proteins have been found to bind to overlapping regions within this sequence element . One of these proteins, YY1, is responsible for the repression . E1A relieves repression exerted by YY1 and further activates transcription through its binding site . A YY1-specific cDNA has been isolated . Its sequence reveals YY1 to be a zinc finger protein that belongs to the GLI-Kruppel gene family . The product of the cDNA binds to YY1 sites . When fused to the GAL4 DNA-binding domain, it is capable of repressing transcription directed by a promoter that contains GAL4-binding sites, and E1A proteins can relieve the repression and activate transcription through the fusion protein.

Cell, 1991 Oct 18, 67(2), 293 - 302
The retinoblastoma gene product regulates progression through the G1 phase of the cell cycle; Goodrich DW et al.; The RB gene product is a nuclear phosphoprotein that undergoes cell cycle-dependent changes in its phosphorylation status . To test whether RB regulates cell cycle progression, purified RB proteins, either full-length or a truncated form containing the T antigen-binding region, were injected into cells . Injection of either protein early in G1 inhibits progression into S phase . Co-injection of anti-RB antibodies antagonizes this effect . Injection of RB into cells arrested at G1/S or late in G1 has no effect on BrdU incorporation, suggesting that RB does not inhibit DNA synthesis in S phase . These results indicate that RB regulates cell proliferation by restricting cell cycle progression at a specific point in G1 and establish a biological assay for RB activity . Neither co-injection of RB with a T antigen peptide nor injection into cells expressing T antigen prevents cells from progressing into S phase, which supports the hypothesis that T antigen binding has functional consequences for RB.

Mol Cell Biochem, 1991 Oct 16, 107(2), 87 - 94
Cloning and expression of a new human polypeptide which regulates protein phosphorylation in Escherichia coli; Daniele A et al.; A 1,820 bp full-length clone encoding for a new human protein was isolated from a lambda gt11 placental cDNA library using anti-human hexokinase antibodies . The cDNA complete sequence includes a 12 bp 5' non-coding region, a single open reading frame encoding a protein of 55 KDa (HP-10) and a 177 bp non-coding with two putative polyadenylation signals upstream of 3' poly(A)tail . The deduced amino acid sequence reveals a sequence of 492 amino acids that contains a stretch of 7 glutamic acid from position 169 and one potential glycosylation site at position 274 . Although antibodies against hexokinase recognize the fusion protein and antibodies against the fusion protein recognize hexokinase, HP-10 is not human hexokinase, by a number of criteria including the alignment of determined amino acid sequences . In searching for a possible functional role of HP-10 its cDNA was inserted into a procaryotic vector which allows the expression of the non-fused protein . Bacteria expressing the HP-10 encoded protein were isolated and found to have a dramatic increase in endogenous phosphorylated proteins . Since HP-10 does not have a protein kinase activity per se it should be considered a new regulatory phosphorylation protein which is active in E . coli.

Eur J Biochem, 1991 Oct 15, 201(2), 373 - 83
Functional characterization of human recombinant apolipoprotein AIV produced in Escherichia coli; Duverger N et al.; Apolipoprotein AIV (apoAIV), a protein which is known to activate the enzyme lecithin: cholesterol acyltransferase, to bind to apoAI/AII receptor sites and also to promote cholesterol efflux from adipose cells, may play an important role in reverse cholesterol transport . In this report, the high-level production of soluble recombinant mature human apoAIV (isoform 1) in Escherichia coli is described . The recombinant protein was purified by avoiding lipid extraction or denaturation . The apoAIV preparation was analysed by its reactivity with antibodies raised against human apoAIV, SDS-gel electrophoresis, isoelectric focusing and N-terminal sequencing . The purified recombinant protein retains an extra methionine at the N-terminus . Purified recombinant and natural apoAIV proteins were indistinguishable with regard to their denaturation properties, thermo-stability or their fluorescence emission properties in the presence of various quantities of a quenching agent . Complexes of ApoAIV with L-alpha-dimyristoyl-glycerophosphocholine (Myr2GroPCho), glycerophosphocholine (GroPCho), or L-alpha-1-palmitoyl-2-oleoylglycerophosphocholine (PamOleGroPCho) prepared from plasmatic and from recombinant apoAIV proteins have similar densities as revealed by analytical centrifugation . They also share the same cofactor properties for the lecithin:cholesterol acyltransferase reaction . Recombinant apoAIV complex with Myr2GroPCho was also able to bind to the same apoAI/AII receptor sites and to promote cholesterol efflux to an equal extent from adipose cells . It is concluded that the recombinant protein is functionally identical to the plasmatic apoAIV and may therefore be very useful in helping to elucidate the physiological role of apoAIV.

Proc Natl Acad Sci U S A, 1991 Oct 15, 88(20), 9272 - 6
Four sites in the acceptor helix and one site in the variable pocket of tRNA(Ala) determine the molecule's acceptor identity; McClain WH et al.; The structural features that determine tRNA(Ala) acceptor identity have been studied with amber-suppressor tRNAs in Escherichia coli cells . Previous work established that a wobble pair composed of guanosine at position 3 and uridine at position 70 (G3-U70) in the acceptor helix of tRNA(Ala) is a determinant of the molecule's acceptor identity . We show that additional determinants are located at three other sites in the acceptor helix and at one site in the variable pocket of tRNA(Ala) . These latter determinants are less important than G3.U70 since their individual alterations in mutants of tRNA(Ala) have smaller degrading effects on the functions of the molecules, and subsets of the determinants, when combined with G3.U70, are sufficient to switch the identities of several other tRNAs to that of tRNA(Ala) . Other workers are using fragments of the tRNA(Ala) acceptor helix to study the molecule's acceptor identity . Our demonstration that the variable pocket contributes to tRNA(Ala) acceptor identity means that such fragments do not faithfully replicate the structure-function relationship of the cellular process.

Proc Natl Acad Sci U S A, 1991 Oct 15, 88(20), 9188 - 92
Alanine scanning site-directed mutagenesis of the zinc fingers of transcription factor ADR1: residues that contact DNA and that transactivate; Thukral SK et al.; To identify functionally important amino acids in the two zinc fingers of transcription factor ADR1 {alcohol dehydrogenase (ADH) II synthesis regulator}, oligonucleotide-directed mutagenesis was used to substitute alanine for the original amino acid at each position in both fingers . The effects of these mutations on DNA binding and thermal stability of ADR1 in vitro and on activation of ADH2 expression in vivo were measured . The DNA binding activity was remarkably heatstable . Amino acids that are candidates for DNA contact sites were identified in the finger-tip and alpha-helical region of each finger, three in the first finger and two in the second . Unexpectedly, an acidic residue in the first finger was essential for transactivation, but its replacement by alanine had no effect on DNA binding . Substitution at several highly conserved positions did not affect ADR1 functions . The ADR1 zinc fingers appear to make relatively few energetically significant contacts to DNA, perhaps as few as three in the first finger and one in the second.

Proc Natl Acad Sci U S A, 1991 Oct 15, 88(20), 9180 - 3
Heterotropic interactions in aspartate transcarbamoylase: turning allosteric ATP activation into inhibition as a consequence of a single tyrosine to phenylalanine mutation; Van Vliet F et al.; Aspartate transcarbamoylase (EC 2.1.3.2) is extensively studied as a model for cooperativity and allostery . This enzyme shows cooperativity between the catalytic sites, and its activity is feedback inhibited by CTP and activated by ATP . These regulatory processes involve several interfaces between catalytic and regulatory chains as well as between domains within these two types of chains . As far as the regulatory chain is concerned, its two domains are in contact through a hydrophobic interface, in which a tyrosine residue is inserted in a pocket involving two leucine residues of the allosteric domain and a valine and a leucine residue of the zinc domain . To probe the possible implication of this hydrophobic core in the CTP and ATP regulatory effect, the tyrosine was replaced by a phenylalanine through oligonucleotide-directed mutagenesis . Interestingly, the resulting mutant shows a complete inversion of the ATP effect; it is now inhibited by ATP instead of being activated by this nucleotide triphosphate . This mutant remains normally sensitive to the feedback inhibitor CTP . This result shows that the hydrophobic interface between the two domains of the regulatory chain plays an important role in the discrimination between the regulatory signals promoted by the two allosteric effectors.

Proc Natl Acad Sci U S A, 1991 Oct 15, 88(20), 9011 - 5
Antiparallel plasmid-plasmid pairing may control P1 plasmid replication; Abeles AL et al.; The copy number of the P1 plasmid replicon is stringently controlled, giving only one or two copies per newborn cell . Control is achieved by the action of the copy-control locus incA, which contains nine repeats of the 19-basepair binding site for the plasmid-encoded initiator protein RepA . A set of five similar repeats are present in the replication origin where RepA acts to trigger initiation . Using an in vitro replication system consisting of an Escherichia coli extract, the P1 origin as a template, and purified RepA protein, we show that supercoiled DNA circles containing the incA locus block origin function in trans . Shutdown becomes complete at a 1:1 ratio of origin to incA sequences . This is not due to titration of the RepA protein, as an excess of RepA can be added without restoring activity . Rather, the incA sequences appear to block the origin by direct contact in a plasmid-plasmid pairing event . When both the origin and the incA locus are present on one plasmid, trans contacts with daughter molecules appear to predominate over cis looping . The results are consistent with a model for replication control where daughter plasmids block their own replication by a pairing in which each origin is in contact with the incA locus of its partner.

Proc Natl Acad Sci U S A, 1991 Oct 15, 88(20), 9001 - 5
A region of the Ada DNA-repair protein required for the activation of ada transcription is not necessary for activation of alkA; Shevell DE et al.; The adaptive response of Escherichia coli protects cells against the mutagenic and toxic effects of alkylating agents . This response is controlled by the Ada protein, which not only functions as the transcriptional activator of the ada and alkA genes but also possesses two DNA methyltransferae activities . Ada is converted into an efficient transcriptional activator by transferring a methyl group from a DNA methylphosphotriester to its own Cys-69 residue and then binds to a DNA sequence (the Ada box) present in both the ada and alkA promoters . Although the Ada protein initially appeared to regulate the ada and alkA genes in a similar fashion, our studies show that the wild-type Ada protein and its truncated derivatives can differentially regulate ada and alkA transcription . In vivo, lower levels of wild-type methylated Ada are needed to activate ada transcription than alkA transcription . In cells exposed to alkylating agents, the N-terminal half of Ada, which contains the DNA-binding domain, is sufficient for efficient activation of alkA, but not ada, transcription . Moreover, truncated derivatives containing 80-90% of Ada are extremely strong constitutive activators of ada but are only inducible activators of alkA transcription . These results suggest that the mechanism by which Ada activates ada transcription differs from that by which it activates alkA transcription.

J Biol Chem, 1991 Oct 15, 266(29), 19833 - 41
Constitution of the twin polymerase of DNA polymerase III holoenzyme; Studwell-Vaughan PS et al.; It is speculated that DNA polymerases which duplicate chromosomes are dimeric to provide concurrent replication of both leading and lagging strands . DNA polymerase III holoenzyme (holoenzyme), is the 10-subunit replicase of the Escherichia coli chromosome . A complex of the alpha (DNA polymerase) and epsilon (3'-5' exonuclease) subunits of the holoenzyme contains only one of each protein . Presumably, one of the eight other subunit(s) functions to dimerize the alpha epsilon polymerase within the holoenzyme . Based on dimeric subassemblies of the holoenzyme, two subunits have been elected as possible agents of polymerase dimerization, one of which is the tau subunit (McHenry, C . S . (1982) J . Biol . Chem . 257, 2657-2663) . Here, we have used pure alpha, epsilon, and tau subunits in binding studies to determine whether tau can dimerize the polymerase . We find tau binds directly to alpha . Whereas alpha is monomeric, tau is a dimer in its native state and thereby serves as an efficient scaffold to dimerize the polymerase . The epsilon subunit does not associate directly with tau but becomes dimerized in the alpha epsilon tau complex by virtue of its interaction with alpha . We have analyzed the dimeric alpha epsilon tau complex by different physical methods to increase the confidence that this complex truly contains a dimeric polymerase . The tau subunit is comprised of the NH2-terminal two-thirds of tau but does not bind to alpha epsilon, identifying the COOH-terminal region of tau as essential to its polymerase dimerization function . The significance of these results with respect to the organization of subunits within the holoenzyme is discussed.

J Biol Chem, 1991 Oct 15, 266(29), 19725 - 30
Tandem transcription termination sites in the dnaN gene of Escherichia coli; Armengod ME et al.; The dnaN gene of Escherichia coli encodes the beta-subunit of DNA polymerase III and maps between the dnaA and recF genes . We demonstrated previously that dnaN and recF constitute a transcriptional unit under control of the dnaN promoters . However, the recF gene has its own promoter region located in the middle of the dnaN structural gene . In this report, we use S1 mapping of mRNAs, transcriptional and translational fusions to the galK and lacZ genes, and in vitro mutagenesis to identify and characterize three tandem transcription termination sites responsible for transcriptional polarity in the dnaN-recF operon . These sites are located in the dnaN gene, downstream from the recF promoter region . Cumulatively, they terminate about 80% of the untranslated transcripts started at the recF promoters . As expected, they do not reduce transcription coming from the dnaN promoters unless dnaN translation was prematurely disrupted by the presence of a nonsense codon . The particular arrangement of regulatory elements (promoters and terminators) in the dnaN-recF region provides an exceptional in vivo system to confirm the latent termination site model of transcriptional polarity . In addition, our results contribute to the understanding of the complex regulation of the dnaA, dnaN, and recF genes . We propose that these three genes constitute an operon and that the terminators described in this work could be used to reduce expression of the distal genes of the operon under circumstances in which the dnaN translation happens to be slowed down.

J Biol Chem, 1991 Oct 15, 266(29), 19636 - 44
Oligomeric binding of T3 receptor is required for maximal T3 response; Williams GR et al.; Receptors in the thyroid-steroid hormone superfamily bind preferentially as dimers to palindromic response elements containing two hexameric half-sites . The 23-base pair rat growth hormone (rGH) T3 response element (T3RE), however, contains three hexameric binding domains, all of which are required for maximal T3 response . We examined the binding of purified T3 receptor alpha (T3R alpha), overexpressed in Escherichia coli, to wild-type and up and down mutations of the rGH T3RE to evaluate whether transcriptional potency correlates with changes in T3R binding . T3R binds to the rGH T3RE as a monomer, dimer, or higher order oligomer . Cooperative T3R dimer binding was demonstrated to two hexameric domains of the rGH T3RE arranged as either direct or inverted repeats . Decreased binding was seen with point mutations in each domain as well as with mutations which altered hexamer orientation and spacing within the site . These results demonstrate that all three hexamers of the rGH T3RE are involved in binding T3R . Occupancy of all three hexamers by T3R in the gel shift assay was observed with functional up mutations of the T3RE, increasing receptor concentration or addition of nuclear extract . The transcriptional response potencies of T3RE up or down mutants in a transient transfection assay correlated closely with T3R binding . These results confirm our earlier hypothesis that all three hexamers of the rGH T3RE bind T3R in a novel binding arrangement and provide a model for the interaction of T3R and other nuclear proteins with the DNA sequences of thyroid hormone-regulated genes.

J Biol Chem, 1991 Oct 15, 266(29), 19551 - 7
Biochemical and ultrastructural evidence for the association of basic fibroblast growth factor with cardiac gap junctions; Kardami E et al.; Basic fibroblast growth factor (bFGF) is a ubiquitous and multifunctional polypeptide that is believed to have a role in tissue repair and to act as a morphogen in embryonic development . Here, we have used immunohistochemical and biochemical methods with antibodies directed against the amino-terminal domain of bFGF, designated IS2, which recognize native and denatured bFGF, to demonstrate that in addition to its known intracellular and extracellular localization in heart, bFGF is also associated with cardiomyocyte gap junctions . In tissue sections, IS2 labeled regions of intercalated discs, producing an immunofluorescence pattern virtually indistinguishable from that obtained with antibodies against the heart gap junction protein connexin-43 . By electron microscopy, gap junctions but not other regions of plasma membrane were heavily immunolabeled with this antibody . By solid phase immunoassay, bFGF was found to be more concentrated in a fraction enriched in cardiac gap junctions than in whole sarcolemmal preparations . Finally, an 18-kDa protein was recognized by several different antibodies specific for bFGF on Western blots of heart subcellular fractions enriched in gap junctions . We suggest that bFGF-like peptides are either an integral part of, or exist in close association with, cardiac gap junctions and thus may play a role in modulating gap junctional intercellular communication.

J Biol Chem, 1991 Oct 15, 266(29), 19475 - 9
Are the histidine residues of glutathione S-transferase important in catalysis? An assessment by 13C NMR spectroscopy and site-specific mutagenesis; Zhang PH et al.; To test the proposition that a histidine residue is essential in the catalytic mechanism of glutathione S-transferase, rat liver isoenzyme 3-3 specifically labeled with {ring-2-13C}histidine was prepared . The 13C NMR spectrum of the labeled enzyme revealed four resonances corresponding to the 4 histidine residues in the mu gene class type 3 subunit . Titration of the four resonances in the range of pH 4-9 both in the presence and absence of glutathione gave pK alpha values of much less than 4, 5.2, 7.1, and 7.8 for the four side chains that were identified by site-specific mutagenesis as His14, His83, His84, and His167, respectively . The magnetic resonance properties and titration behavior of His14 suggest that this residue is buried in a hydrophobic environment . Conservative replacement of each histidine with asparagine results in mutant enzymes that have catalytic properties very close to the native protein as assessed with three different substrates, 1-chloro-2,4-dinitrobenzene, 4-phenyl-3-buten-2-one, and phenanthrene 9,10-oxide . The results indicate clearly that none of the histidine residues of isoenzyme 3-3 is essential for stabilization of the bound glutathione thiolate or for any other aspect of catalysis.

J Biol Chem, 1991 Oct 15, 266(29), 19450 - 8
The malZ gene of Escherichia coli, a member of the maltose regulon, encodes a maltodextrin glucosidase; Tapio S et al.; We have characterized a maltodextrin glucosidase, previously described as a maltose-inducible, cytoplasmic enzyme that cleaves p-nitrophenyl-alpha-maltoside in Escherichia coli . The gene encoding the enzyme activity, referred to as malZ, is located at 9.3 min on the chromosomal map . We cloned the gene in a high copy number vector and purified the enzyme . It is a monomer, with an apparent molecular weight of 65,000 . The enzyme degrades maltodextrins, ranging from maltotriose to maltoheptaose, to shorter oligosaccharides, the final hydrolysis products being maltose and glucose . We measured the kinetic parameters, Km and Vmax, for the hydrolysis to glucose of the five different substrates . The binding of the substrate is enhanced by increasing the number of glucosyl residues in the maltodextrin . In contrast, the maximum rate of hydrolysis (Vmax) is fastest for maltotriose . To study the mode of action of the enzyme, we quantitatively measured the amount of free glucose liberated from the different maltodextrin substrates after a long incubation . More glucose is liberated from the long dextrins, as compared to the shorter ones, showing that the primary hydrolysis product was glucose, not maltose . Furthermore, {14C}maltotriose, specifically labeled at the reducing end, was hydrolyzed to {14C}glucose and unlabeled maltose . These data demonstrate that the malZ gene product is a maltodextrin glucosidase, liberating glucose from the reducing end of malto-oligosaccharides . The nucleotide sequence of malZ and the deduced amino acid sequence showed that malZ encodes a protein with a molecular weight of 68,960 . Homology to glucosidases, alpha-amylases, and pullulanases were observed . Conserved regions thought to represent active sites in dextrin hydrolases were found in the MalZ protein.

J Biol Chem, 1991 Oct 15, 266(29), 19419 - 25
Gene expression by a hypovirulence-associated virus of the chestnut blight fungus involves two papain-like protease activities . Essential residues and cleavage site requirements for p48 autoproteolysis; Shapira R et al.; Proteolytic processing plays a fundamental role in gene expression of a recently characterized viral-like double-stranded RNA associated with biological control of the chestnut blight fungus . Polypeptide p29, a papain-like protease, was shown to autocatalytically release itself from the NH2 terminus of the polyprotein specified by the first of two encoded open reading frames, ORF A (Choi, G . H., Shapira, R., and Nuss, D . L . (1991) Proc . Natl . Acad . Sci . U.S.A . 88, 1167-1171; Choi, G . H., Pawlyk, D . M., and Nuss, D . L . (1991) Virology 183, 747-752) . The characterization of a second autocatalytic protease, p48, which is encoded by ORF B, is the subject of this report . Deletion analysis revealed that the catalytic domain resides within the carboxyl-terminal region, while site-specific mutational analysis identified Cys-341 and His-388 as residues essential for autoproteolysis . Autoproteolytic processing by p48 was also demonstrated when expressed in Escherichia coli and microsequence analysis of the recovered COOH-terminal cleavage product indicated that cleavage occurred between Gly-418 and Ala-419 . The requirements for a functional cleavage site, including confirmation of the cleavage dipeptide, were defined by amino acid substitution analysis . Similarities between p29 and p48 suggest that the respective coding domains could have arisen as a result of a gene duplication event.

J Biol Chem, 1991 Oct 15, 266(29), 19320 - 7
The yeast general transcription factor TFIIA is composed of two polypeptide subunits; Ranish JA et al.; The general transcription factor TFIIA was purified from yeast . A key step in the purification was affinity chromatography using a column containing the adenovirus major late promoter with bound recombinant TFIID to which TFIIA binds with high affinity . TFIIA activity copurifies with two polypeptides of molecular mass 32 and 13.5 kDa . Elution and renaturation of these two polypeptides from sodium dodecyl sulfate-polyacrylamide gels showed that both polypeptides were required for TFIIA activity . TFIIA activity was measured by both a native gel shift assay and by in vitro complementation of transcription using yeast nuclear extracts depleted of TFIIA . The purified renatured yeast TFIIA also complements basal level transcription using a mammalian transcription system depleted of TFIIA . Native TFIIA has an apparent molecular mass of approximately 90 kDa measured by gel filtration chromatography . TFIIA binds to a TFIID.TATA element.DNA complex with an apparent equilibrium dissociation constant (KD) of 20 pM . This affinity is about 100-fold greater than the affinity of TFIID for TATA elements and much greater than the affinity of TFIIA for TFIID not bound to DNA.

J Biol Chem, 1991 Oct 15, 266(29), 19186 - 91
The expression of a catalytically active cholesterol 7 alpha-hydroxylase cytochrome P450 in Escherichia coli; Li YC et al.; We have recently cloned a full-length cDNA encoding the rat hepatic cholesterol 7 alpha-hydroxylase cytochrome P450 (P450c7) (Li, Y . C., Wang, D . P., and Chiang, J . Y . L . (1990) J . Biol . Chem . 265, 12012-12019), which catalyzes the rate-limiting reaction of bile acid synthesis in the liver . By using the polymerase chain reaction, we have designed two P450c7 cDNAs . One has the second Met codon deleted and the third Thr codon replaced with an Ala . The other lacks codons for the NH2-terminal hydrophobic sequence of amino acids 2-24 (P450c7 delta 2-24) . The cDNAs were separately cloned into the expression vector pKK233-2 and transformed into Escherichia coli . After induction with isopropyl-beta-D-thiogalactopyranoside, bacteria harboring recombinant plasmids expressed a polypeptide which reacted with the antibody against cholesterol 7 alpha-hydroxylase in immunoblots . The slightly modified full-length enzyme was expressed to 0.2% of the total bacterial lysate and was located in the membrane fraction, whereas P450c7 delta 2-24 was expressed at a 10-fold higher level (2%), of which 85% was in the cytosol and the remaining associated with the membranes . We have purified P450c7 delta 2-24 which showed a typical reduced-CO difference spectrum of cytochrome P450 and reconstituted cholesterol 7 alpha-hydroxylase activity in the presence of NADPH-cytochrome P450 reductase . P450c7 delta 2-24 has a similar Km for cholesterol (24.6 microM) but a lower Vmax (0.10 nmol/min) and a lower turnover number (1.93 min-1) as compared with the enzyme isolated from rat liver microsomes . The purified P450c7 delta 2-24 has an unique hydrophilic NH2 terminus and contains monomers and dimers in equal amounts . This is the first report demonstrating that a genetically engineered cytochrome P450 enzyme lacking a typical NH2-terminal hydrophobic sequence is mainly cytosolic and catalytically active.

J Biol Chem, 1991 Oct 15, 266(29), 19180 - 5
Isolation of a functional transferase component from the rat fatty acid synthase by limited trypsinization of the subunit monomer . Formation of a stable functional complex between transferase and acyl carrier protein domains; Rangan VS et al.; Limited trypsinization of rat fatty acid synthase monomers results in cleavage at sites protected in the native dimer . A 47,000-Da polypeptide containing the transferase component was isolated from the digest and its location in the multifunctional polypeptide established . Both acetyl and malonyl moieties are transferred stoichiometrically from CoA ester to this polypeptide and each can replace the other, confirming that a single common site is utilized in the loading of these substrates onto the fatty acid synthase . Transferase activity of the 47,000-Da polypeptide decreases with increasing acyl donor chain length (malonyl = acetyl greater than butyryl greater than hexanoyl greater than octanoyl) . Activity is inhibited by certain thiol-directed reagents, and protection is afforded by substrate suggesting the presence of a sensitive cysteine residue near the substrate binding site . The transferase was also able to utilize as acyl acceptor the Escherichia coli acyl carrier protein and the acyl carrier protein domain of the multifunctional fatty acid synthase . When the fatty acid synthase monomer was trypsinized under milder conditions, the 47,000-Da transferase domain could be isolated in association with the 8,000-Da acyl carrier protein domain . The transferase was capable of translocating substrate moieties from CoA ester donors to the associated acyl carrier protein . The results provide the first direct evidence that, in the head-to-tail oriented fatty acid synthase homodimer, functional communication between the transferase domain located near the end of one polypeptide and the acyl carrier protein domain located at the opposite end of the other polypeptide is facilitated by a stable physical interaction between these domains.

Biochemistry, 1991 Oct 15, 30(41), 10043 - 57
1H, 15N, and 13C NMR signal assignments of IIIGlc, a signal-transducing protein of Escherichia coli, using three-dimensional triple-resonance techniques; Pelton JG et al.; IIIGlc is an 18.1-kDa signal-transducing phosphocarrier protein of the phosphoenolpyruvate:glycose phosphotransferase system (PTS) of Escherichia coli . Virtually complete (98%) backbone 1H, 15N, and 13C nuclear magnetic resonance (NMR) signal assignments were determined by using a battery of triple-resonance three-dimensional (3D) NMR pulse sequences . In addition, nearly complete (1H, 95%; 13C, 85%) side-chain 1H and 13C signal assignments were obtained from an analysis of 3D 13C HCCH-COSY and HCCH-TOCSY spectra . These experiments rely almost exclusively upon one- and two-bond J couplings to transfer magnetization and to correlate proton and heteronuclear NMR signals . Hence, essentially complete signal assignments of this 168-residue protein were made without any assumptions regarding secondary structure and without the aid of a crystal structure, which is not yet available . Moreover, only three samples, one uniformly 15N-enriched, one uniformly 15N/13C-enriched, and one containing a few types of amino acids labeled with 15N and/or 13C, were needed to make the assignments . The backbone assignments together with the 3D 15N NOESY-HMQC and 13C NOESY-HMQC data have provided extensive information about the secondary structure of this protein {Pelton, J.G., Torchia, D.A., Meadow, N.D., Wong, C.-Y., & Roseman, S (1991) Proc . Natl . Acad . Sci . U.S.A . 88, 3479-3488} . The nearly complete set of backbone and side-chain atom assignments reported herein provide a basis for studies of the three-dimensional structure and dynamics of IIIGlc as well as its interactions with a variety of membrane and cytoplasmic proteins.

Biochemistry, 1991 Oct 15, 30(41), 10019 - 26
Mutations in the small subunit of ribulosebisphosphate carboxylase affect subunit binding and catalysis; Paul K et al.; Fully functional Synechococcus PCC 6301 ribulose 1,5-bisphosphate carboxylase-oxygenase (kcat = 11.8 s-1) was assembled in vitro following separate expression of the large- and small-subunit genes in different Escherichia coli cultures . The small subunits were expressed predominantly as monomers, in contrast to the large subunits which have been shown to be largely octameric when expressed separately {Andrews, T . J . (1988) J . Biol . Chem . 263, 12213-12219} . This separate expression system was applied to the study of mutations in the amino-terminal arm of the small subunit, which is one of the major sites of contact with the large subunit in the assembled hexadecamer . It enabled the effects of a mutation on the tightness of binding of the small subunit to the large-subunit octamer to be distinguished from the effects of the same mutation on catalysis carried out by the assembled complex when fully saturated with mutant small subunits . This important distinction cannot be made when both subunits are expressed together in the same cell . Substitutions of conserved amino acid residues at positions 14 (Ala, Val, Gly, or Asp instead of Thr) and 17 (Cys instead of Tyr), which make important contacts with conserved large-subunit residues, were introduced by site-directed mutagenesis . All mutant small subunits were able to bind to large subunits and form active enzymes . A potential intersubunit hydrogen bond involving the Thr-14 hydroxyl group is shown to be unimportant . However, the binding of Gly-14, Asp-14, and Cys-17 mutant small subunits was weaker, and the resultant mutant enzymes had reduced catalytic rates compared to the wild type.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1991 Oct 15, 30(41), 10012 - 8
Differences in thermal stability between reduced and oxidized cytochrome b562 from Escherichia coli; Fisher MT; The thermal stabilities of ferri- and ferrocytochrome b562 were examined . Thermally induced spectral changes, monitored by absorption and second-derivative spectroscopies, followed the dissociation of the heme moiety and the increased solvation of tyrosine residue(s) located in close proximity to the heme binding site . All observed thermal transitions were independent of the rate of temperature increase (0.5-2 degrees C/min), and the denatured protein exhibited partial to near-complete reversibility upon return to ambient temperature . The extent of renaturation of cytochrome b562 is dependent on the amount of time the unfolded conformer is exposed to temperatures above the transition temperature, Tm . All thermally induced spectra changes fit a simple two-state model, and the thermal transition was assumed to be reversible . The thermal transition for ferrocytochrome b562 yielded Tm and van't Hoff enthalpy (delta HvH) values of 81.0 degrees C and 137 kcal/mol, respectively . In contrast, Tm and delta HvH values obtained for the ferricytochrome were 66.7 degrees C and 110 kcal/mol, respectively . The estimated increase in the stabilization free energy at the Tm of ferricytochrome b562 following the one-electron reduction to the ferrous form, where delta delta G = delta Tm delta Sm {delta Sm = 324 cal/(K.mol), delta Tm = 14.3 degrees C} {Becktel, W . J., & Schellman, J . A . (1987) Biopolymers 26, 1859-1877}, is 4.6 kcal/mol.

Proc Natl Acad Sci U S A, 1991 Oct 15, 88(20), 8958 - 62
Functional map of the alpha subunit of Escherichia coli RNA polymerase: two modes of transcription activation by positive factors; Igarashi K et al.; The role of the alpha subunit of Escherichia coli RNA polymerase in transcription activation by positive factors was investigated using two reconstituted mutant RNA polymerases (containing C-terminally truncated alpha subunits) and three positive factors {the cAMP receptor protein (CRP), OmpR, and PhoB} . The mutant RNA polymerases did not respond to transcription activation by activator proteins that bind upstream of the respective promoters . Transcription by these mutant enzymes was, however, activated in the cases where activators bind to target sites that overlap the promoter -35 region . Two different mechanisms are proposed for the positive control of transcription by activator proteins, one requiring the C-terminal domain of the alpha subunit, and the other not requiring it.

FEMS Microbiol Lett, 1991 Oct 15, 67(3), 317 - 21
Construction of two stable bifunctional plasmids for Streptomyces spp . and Escherichia coli; Durajlija S et al.; Two bifunctional plasmid vectors pZG5 (7.45 kb) and pZG6 (6.95 kb), for gene transfer between Streptomyces spp . and Escherichia coli have been constructed by fusion of the multicopy broad-host-range Streptomyces plasmid pIJ350 with E . coli plasmids Bluescribe M13- (pZG5) or pUC18 (pZG6) . Both plasmids possess several unique restriction sites suitable for DNA cloning . Stable transformants of Streptomyces rimosus R6 and S . lividans 66 were obtained, harboring intact plasmids regardless of colony age or multiple subculturing . Moreover, pZG5 and pZG6 were successfully used to introduce several homologous transfer RNA genes into S . rimosus.

FEMS Microbiol Lett, 1991 Oct 15, 67(3), 277 - 82
Development of a new host vector system in mycobacteria; Goto Y et al.; The hybrid plasmid pYT72/pYT92 constructed from an Escherichia coli plasmid pACYC177 and mycobacterial plasmid pMSC262 isolated from Mycobacterium scroflaceum strain W262 transformed both E . coli and BCG . Phage-sensitive mutants S-10 and S-20 isolated from BCG Tokyo strain showed higher frequency of transformation than the wild-type strain . Frequency of transformation was dependent on age of the culture and the electroporation condition used . Several deletion mutants were generated from pYT72/92 to determine the minimum region for the replication in the mycobacteria . A 2.3-kb fragment of pMSC262 was found to contain an essential region . Using this fragment and pACYC177, a small shuttle vector pYT937 containing two drug-resistance markers, kanamycin- and ampicillin-resistance, was constructed . pYT937 contains AatII, BamHI, BbvII, GsuI, HincII, PstI, ScaI and XbaI cloning sites.

FEMS Microbiol Lett, 1991 Oct 15, 67(3), 255 - 9
Dimethyl sulfoxide reductase is not required for trimethylamine N-oxide reduction in Escherichia coli; Daruwala R et al.; Deletion mutants of Escherichia coli lacking dimethyl sulfoxide (DMSO) reductase activity and consequently unable to utilize DMSO as an electron acceptor for anaerobic growth have been isolated . These mutants retained the ability to use trimethylamine N-oxide (TMAO) as an electron acceptor and the TMAO reductase activity was found to be unaltered . Heating the cell-free extract of the wild-type strain at 70 degrees C for 15 min selectively inactivated the DMSO reductase activity while the TMAO reductase activity remained unchanged for at least 1 h.

Proc Natl Acad Sci U S A, 1991 Oct 15, 88(20), 9262 - 6
Immunocytochemical localization of the cystic fibrosis gene product CFTR; Crawford I et al.; Antisera against two peptides, corresponding to different domains of the cystic fibrosis gene product CFTR, have been raised and extensively characterized . Both antisera recognize CFTR as a 165-kDa polypeptide in Western analysis of cells transfected with CFTR cDNA as well as in epithelial cell lines . The cell and tissue distribution of CFTR has been studied by immunocytochemistry . CFTR is abundant in epithelial cells, including those lining sweat ducts, small pancreatic ducts, and intestinal crypts . Unexpectedly, the level of CFTR in lung epithelia is relatively low, while it is abundant in the epithelia of kidney tubules . The protein appears to be restricted to the apical, rather than basolateral, regions of epithelial cells and at least a proportion is associated with the plasma membrane . The cell and tissue distributions of CFTR are consistent with a function for this protein as a chloride channel or as a regulator of channel activity.

Proc Natl Acad Sci U S A, 1991 Oct 15, 88(20), 9112 - 6
Bleomycin-resistance gene derived from the transposon Tn5 confers selective advantage to Escherichia coli K-12; Blot M et al.; The plasmid pRAB2 contains a silent operon derived from the transposon Tn5 and carrying the gene neo for neomycin-kanamycin resistance and a truncated ble gene (ble333) for bleomycin resistance . Spontaneous mutants that express the two resistances provide Escherichia coli cells an improved fitness during the phase of decline in the absence of the antibiotics . It is shown that the ble333 gene product is responsible for this better fitness . These results can explain a previously described selective advantage attributed to the presence of Tn5 . The improved fitness of bleomycin-resistant bacteria is proposed to relate to DNA repair by the ble gene product . The consequences of the presence of an accessory gene improving fitness are discussed in terms of evolutionary stable strategy of a transposon in populations of E . coli.

J Immunol, 1991 Oct 15, 147(8), 2547 - 52
Complete sequence of the allergen Amb alpha II . Recombinant expression and reactivity with T cells from ragweed allergic patients; Rogers BL et al.; This study defines the complete primary structure of Amb alpha II, an important allergen produced by short ragweed (Ambrosia artemisiifolia) . The deduced amino acid sequence derived from the cDNA indicates that Amb alpha II shares approximately 65% sequence identity with the Amb alpha I multigene family of allergens . Full-length cDNA encoding Amb alpha I.1 and Amb alpha II have been expressed in E . coli and purified . An in-frame linker encoding polyhistidine has been added to the 5' end of the cDNA to facilitate purification using Ni2+ ion affinity chromatography, yielding greater than 90% pure recombinant protein in a single step . T cells from patients allergic to ragweed proliferate in response to pollen extract as well as purified recombinant Amb alpha I.1 and Amb alpha II . T cell lines established using either Amb alpha I.1 or II as the stimulating Ag exhibit a high level of cross-reactivity to both proteins . This result is entirely consistent with the extensive primary sequence identity shared by these two proteins . These data suggest that allergic humans recognize shared T cell epitopes on these two related molecules.

FEMS Microbiol Lett, 1991 Oct 15, 67(3), 341 - 6
The nucleotide sequence of a regulatory gene present on a plasmid in an enterotoxigenic Escherichia coli strain of serotype O167:H5; de Haan LA et al.; A DNA fragment that can functionally substitute for cfaD, the positive regulatory gene involved in expression of CFA/I fimbriae, has recently been cloned from an Escherichia coli strain of serotype O167:H5 that produces CS5 fimbriae . Nucleotide sequence determination showed that the fragment contained a gene, csvR (Coli Surface Virulence factor Regulator) homologous to the cfaD gene, which encoded for a protein of 301 amino acid residues . The csvR gene was found to be located between two different insertion sequences . Comparison of the amino acid sequence of the CsvR and CfaD proteins showed that CsvR is 34 amino acid residues longer at the C-terminus and, in the sequence, it also contains an insertion of two amino acid residues . The similarity between CfaD and Rns, the positive regulator of CS1 and CS2 expression, is much higher (97%) than between CsvR and CfaD (87%) . This is reflected by the fact that the level of expression of CFA/I fimbriae induced by CsvR is not as high as when expression is induced by CfaD or Rns.

Proc Natl Acad Sci U S A, 1991 Oct 15, 88(20), 9340 - 4
Saccharide orientation at the cell surface affects glycolipid receptor function; Stromberg N et al.; Three allelic variants of P-pilus-associated G-adhesins (lectins) with different cell-binding properties were recently described . Here we have analyzed Escherichia coli HB101 strains expressing the recombinant G-adhesin variants for their ability to agglutinate erythrocytes from various species as this relates to the glycosphingolipid (GSL) composition in the erythrocyte membranes . All three variants exhibit similar specificities for the globo-series GSLs affixed to artificial surfaces . However, only the PapGJ96 adhesin induces agglutination of erythrocytes having globotriaosylceramide (GbO3) {Gal(alpha 1-4)LacCer} as the major GSL . Furthermore, only PapGAD110 induces strong agglutination of erythrocytes having globotetraosylceramide (GbO4) {GalNAc(beta 1-3)Gal(alpha 1-4)LacCer} as the major GSL, while PrsGJ96 alone agglutinates those containing globopentaosylceramide (GbO5) {GalNAc(alpha 1-3)GalNAc(beta 1-3)Gal(alpha 1-4)LacCer} . Molecular modeling of these globo-GSLs demonstrates different saccharide orientations to the membrane surface for these isoreceptors . We suggest that the differential binding of the three G-adhesin variants results from differences in epitope presentation at the membrane among these globo-GSLs.

Biochem Biophys Res Commun, 1991 Oct 15, 180(1), 124 - 30
A new catalytic function of halohydrin hydrogen-halide-lyase, synthesis of beta-hydroxynitriles from epoxides and cyanide; Nakamura T et al.; Halohydrin hydrogen-halide-lyase, which catalyzes the interconversion of halohydrins to epoxides, purified from a recombinant E . coli was found to catalyze the transformation of 1,2-epoxybutane into beta-hydroxyvaleronitrile in the presence of cyanide . Chloride inhibited competitively the formation of beta-hydroxyvaleronitrile . The enzyme also catalyzed the transformation of some other epoxides into the corresponding beta-hydroxynitriles in the presence of cyanide.

Proc Natl Acad Sci U S A, 1991 Oct 15, 88(20), 9315 - 9
Human vitamin D receptor is selectively phosphorylated by protein kinase C on serine 51, a residue crucial to its trans-activation function; Hsieh JC et al.; The vitamin D receptor (VDR) is known to be a phosphoprotein and inspection of the deduced amino acid sequence of human VDR (hVDR) reveals the conservation of three potential sites of phosphorylation by protein kinase C (PKC)--namely, Ser-51, Ser-119, and Ser-125 . Immunoprecipitated extracts derived from a rat osteoblast-like osteosarcoma cell line that contains the VDR in high copy number were incubated with the alpha, beta, and gamma isozymes of PKC, and VDR proved to be an effective substrate for PKC-beta, in vitro . When hVDR cDNAs containing single, double, and triple mutations of Ser-51, Ser-119, and Ser-125 were expressed in CV-1 monkey kidney cells, immunoprecipitated and phosphorylated by PKC-beta, in vitro, the mutation of Ser-51 selectively abolished phosphorylation . Furthermore, when transfected CV-1 cells were treated with phorbol 12-myristate 13-acetate, a PKC activator, phosphorylation of wild-type hVDR was enhanced, whereas that of the Ser-51 mutant hVDR was unaffected . Therefore, Ser-51 is the site of hVDR phosphorylation by PKC, both in vitro and in vivo . To evaluate the functional role of Ser-51 and its potential phosphorylation, hVDR-mediated transcription was tested using cotransfection with expression plasmids and a reporter gene that contained a vitamin D response element . Mutation of Ser-51 markedly inhibited transcriptional activation by the vitamin D hormone, suggesting that phosphorylation of Ser-51 by PKC could play a significant role in vitamin D-dependent transcriptional activation . Therefore, the present results link the PKC signal transduction pathway of growth regulation and tumor promotion to the phosphorylation and function of VDR.

Proc Natl Acad Sci U S A, 1991 Oct 15, 88(20), 9248 - 52
Evidence for increased in vitro recombination with insertion of human hepatitis B virus DNA; Hino O et al.; Chromosomal translocation, deletion, and inversion/duplication directly linked to hepatitis B virus (HBV) DNA integration occur frequently in host DNA of human hepatocellular carcinomas . To test the possible recombinogenic effect of HBV DNA, we have utilized an in vitro recombination assay . Fragments containing the region spanning DR1, which is believed to be the origin of viral replication and a preferred site in the viral genome for integration, increased the recombination events reproducibly in the presence of extracts from actively dividing cells (e.g., hepatocellular carcinoma) but not resting cells (e.g., normal liver) . Moreover, in these extracts we have found a protein(s) that specifically binds to these HBV DNA fragments . These results support the notion that in some instances integrated HBV DNAs cause further genomic instability, possibly involving specific cellular protein(s) . The fact that extracts from nondividing, normal liver did not increase recombination events suggests that genomic instability depends upon active cellular growth, a feature more commonly found subsequent to HBV-induced hepatocellular injury than in healthy liver . Our results offer an explanation for the high incidence of liver cancer that accompanies chronic hepatitis and add HBV to the list of agents that can cause genetic recombination.

Proc Natl Acad Sci U S A, 1991 Oct 15, 88(20), 9141 - 5
Purification and properties of a shortened form of cytochrome P-450 2E1: deletion of the NH2-terminal membrane-insertion signal peptide does not alter the catalytic activities; Larson JR et al.; As reported previously, alcohol-inducible cytochrome P-450 2E1 lacking the hydrophobic NH2-terminal segment is located primarily in the inner cell membrane when expressed in Escherichia coli and is active with a typical substrate . To study the catalytic properties in detail, we have purified the truncated P-450 lacking residues 3-29 to electrophoretic homogeneity from the solubilized bacterial membrane fraction in the presence of 4-methylpyrazole as a stabilizing agent . The resulting heme protein with a specific content of 15.8 nmol of P-450 per mg of protein has a reduced CO difference spectrum identical to that of the full-length enzyme, with a Soret maximum at 452 nm . The rates of catalysis of four reactions in the reconstituted enzyme system, including the oxygenation of ethanol to give acetaldehyde, the oxidative dealkylation of N-nitrosodiethylamine to give ethylene and acetaldehyde, and the ring hydroxylation of aniline and p-nitrophenol, are the same with the shortened and full-length enzymes . The apparent Km of p-nitrophenol is also the same, as is that for NADPH-cytochrome P-450 reductase and for cytochrome b5, which stimulates p-nitrocatechol formation about 3-fold . Moreover, the requirement for phosphatidylcholine for full catalytic activity is unchanged despite the absence of the NH2-terminal segment . Although this highly hydrophobic segment is believed to play a role in the intact cell as a membrane-insertion signal sequence, we conclude that it has no function in the catalytic activity of the cytochrome as an oxygenase, including interactions with the other components of the enzyme system.

Proc Natl Acad Sci U S A, 1991 Oct 15, 88(20), 9031 - 5
DNase protection analysis of the stable synaptic complexes involved in Mu transposition; Mizuuchi M et al.; Several critical steps in phage Mu transposition involve specialized protein-DNA complexes . Cleavage of Mu donor DNA by MuA protein leads to the formation of the stable cleaved donor complex (CDC), in which the two Mu DNA ends are held together by MuA . In the subsequent strand-transfer reaction the CDC attacks a target DNA to generate the strand-transfer complex, in which the donor and the target DNAs are covalently joined . We have carried out DNase I protection experiments on these protein-DNA complexes and found that only three MuA binding sites (L1, R1, and R2 of the six total) at the two Mu ends are stably bound by MuA to maintain the paired Mu end structure . The protection extends beyond the ends of the Mu sequence for different lengths (7-20 nucleotides) depending on the strand and the type of complex . After formation of the CDC, the other MuA binding sites (L2, L3, and R3) and internal activation sequence become dispensable for the subsequent strand-transfer reaction.

J Biol Chem, 1991 Oct 15, 266(29), 19334 - 41
Small substrates and cytochrome c are oxidized at different sites of cytochrome c peroxidase; DePillis GD et al.; Modeling studies suggest that electrons are transferred from cytochrome c to cytochrome c peroxidase (CcP) with cytochrome c predominantly bound at a site facing the gamma-meso edge of the CcP prosthetic heme group (Poulos, T.L., and Kraut, J . (1980) J . Biol . Chem . 255, 10322-10330) . As shown here, guaiacol and ferrocyanide are oxidized at a different site of CcP . Thus, the oxidations of cytochrome c and guaiacol are differentially inactivated by phenylhydrazine and sodium azide . The loss of guaiacol oxidation activity correlates with covalent binding of 1 equivalent of {14C}phenylhydrazine to the protein, whereas the slower loss of cytochrome c activity correlates with the appearance of a 428-nm absorbance maximum attributed to the formation of a sigma-phenyl-iron heme complex . The delta-meso-phenyl and 8-hydroxymethyl derivatives of heme are formed as minor products . Catalytic oxidation of azide to the azidyl radical results in inactivation of CcP and formation of delta-meso-azidoheme . Reconstitution of apo-CcP with delta-meso-azido-, -ethyl-, and -(2-phenylethyl)heme yields holoproteins that give compound I species with H2O2 and exhibit 80, 59, and 31%, respectively, of the control kcat value for cytochrome c oxidation but little or no guaiacol or ferrocyanide oxidizing activity . Conversely, CcP reconstituted with gamma-meso-ethylheme is fully active in the oxidation of guaiacol and ferrocyanide but only retains 27% of the cytochrome c oxidizing activity . These results indicate that guaiacol and ferrocyanide are primarily oxidized near the delta-meso-heme edge rather than, like cytochrome c, at a surface site facing the gamma-meso edge.

J Biol Chem, 1991 Oct 15, 266(29), 19328 - 33
Superoxide sensitivity of the Escherichia coli aconitase; Gardner PR et al.; Mutants of Escherichia coli lacking superoxide dismutase (SOD) activity were used to explore the sensitivity of aconitase toward O2 and O2- . The aconitase activity in SOD-free extracts was rapidly lost under aerobic conditions and exogenous SOD afforded a concentration-dependent protection . The rate of the inactivating reaction between O2- and aconitase was estimated to be of the order of 10(9) M-1 s-1 . The competitive inhibitors fluorocitrate and tricarballylate provided some protection, and at saturating concentrations, they decreased the rate of the inactivating reaction by 100- and 10-fold, respectively . Aconitase was markedly less sensitive to O2 than it was to O2- . Aerobic growth on succinate involves a greater dependence upon aconitase than does growth on glucose and, as expected, the deleterious consequences of SOD deficiency were more pronounced on succinate than on glucose . Moreover, aconitase activity was lower in extracts of aerobically grown SOD mutants, than it was in the parental strain . We suppose that inactivation of aconitase by O2- involves oxidative attack on the prosthetic iron-sulfur cluster . The extreme sensitivity of aconitase to inactivation by O2- suggests that its inactivation will be an early event in the oxidative stress imposed by hyperoxia, ultraviolet irradiation or redox-cycling agents, such as viologens or quinones.

Biochim Biophys Acta, 1991 Oct 14, 1069(1), 1 - 4
A comment on the preparation of liposomes from and on the beta in equilibrium alpha acyl chain melting behaviour of rough mutant lipopolysaccharide; Brandenburg K et al.; We would like to comment on the investigations of Vaara, M., Plachy, W.Z . and Nikaido, H . (Vaara, M . et al . (1990) Biochim . Biophys . Acta 1024, 152-158) on the partitioning of hydrophobic probes in lipopolysaccharide bilayers . These authors reported that they did not succeed in preparing closed vesicles (liposomes) from rough mutant lipopolysaccharide . We describe the conditions under which lipopolysaccharide liposomes are formed most readily . We, furthermore, summarize data which strongly support the existence of thermotropic phase transitions of lipopolysaccharides (with transition temperatures lying in the range of 30-36 degrees C) contradictory to Vaara et al . who argue that such transitions are artefacts . Exemplary measurements of the beta in equilibrium alpha acyl chain melting for lipopolysaccharide from Escherichia coli deep rough mutant (strain F515) as compared to synthetic and natural phospholipids are presented using fluorescence spectroscopy, Fourier-transform infrared spectroscopy and differential scanning calorimetry . These results unequivocally prove the necessity to perform experiments at 37 degrees C for a determination of the outer membrane permeability under physiological conditions.

Carbohydr Res, 1991 Oct 14, 219, 193 - 201
Escherichia coli O9:K38 capsular antigen: another ribofuranose-containing glycan; Hackland PL et al.; The primary structure of the O-deacetylated acidic capsular antigen of Escherichia coli O9:K38 was shown by glycose analysis, methylation analysis, and one- and two-dimensional 1H- and 13C-n.m.r . spectroscopy to be composed of repeating linear pentasaccharide units having the structure.

Lymphokine Cytokine Res, 1991 Oct, 10(5), 397 - 403
Differential regulation of interleukin-1 alpha and interleukin-1 beta mRNA expression in human monocytes: evidence for protein kinase C-dependent and -independent pathways; Smith MF Jr et al.; The expression of mRNA coding for IL-1 alpha and IL-1 beta was examined in human peripheral blood monocytes (PBM) to determine if the two genes are under the same mechanisms of transcriptional control and whether or not they can be regulated independently . In response to E . coli lipopolysaccharide (LPS), PBM express approximately 10-fold more IL-1 beta-specific mRNA than IL-1 alpha . However, treatment of these cells with phorbol myristate acetate (PMA) resulted in the expression of IL-1 beta mRNA . Likewise, treatment of PBM with phorbol dibutyrate (PdBu), phorbol diacetate (PDA), or mezerein, which, similar to PMA, were able to induce the translocation of protein kinase C (PKc) to the monocyte plasma membrane, resulted in predominantly IL-1 beta mRNA expression . The inactive tumor promoter 4 alpha-phorbol didecanoate (4 alpha-PDD) did not cause the translocation of PKc or induce the expression of either form of IL-1 mRNA . Following 18 h pretreatment with PMA to downregulate PKc activity, LPS was capable of inducing the expression of both forms of IL-1 mRNA, demonstrating that at least part of the response of PBM to LPS is PKc independent . These results suggest that the activation of PKc alone is sufficient to induce a high level expression of IL-1 beta but not IL-1 alpha mRNA . Furthermore, the possibility exists that another, as yet unknown, signal transduction mechanism is involved in inducing the expression of both IL-1 alpha and IL-1 beta mRNA in response to LPS.

J Bacteriol, 1991 Oct, 173(20), 6647 - 9
rRNA transcription rate in Escherichia coli; Gotta SL et al.; The rate of in vivo transcription elongation for Escherichia coli rRNA operons was determined by electron microscopy following addition of rifampin to log-phase cultures . Direct observation of RNA polymerase positions along rRNA operons 30, 40, and 70 s after inhibition of transcription initiation yielded a transcription elongation rate of 42 nucleotides per s.

Biochim Biophys Acta, 1991 Oct 11, 1080(1), 19 - 28
Topological analysis of the pyridine nucleotide transhydrogenase of Escherichia coli using proteolytic enzymes; Tong RC et al.; The pyridine nucleotide transhydrogenase of Escherichia coli has an alpha 2 beta 2 structure (alpha: Mr, 54,000; beta: Mr, 48,700) . Hydropathy analysis of the amino acid sequences suggested that the 10 kDa C-terminal portion of the alpha subunit and the N-terminal 20-25 kDa region of the beta subunit are composed of transmembranous alpha-helices . The topology of these subunits in the membrane was investigated using proteolytic enzymes . Trypsin digestion of everted cytoplasmic membrane vesicles released a 43 kDa polypeptide from the alpha subunit . The beta subunit was not susceptible to trypsin digestion . However, it was digested by proteinase K in everted vesicles . Both alpha and beta subunits were not attacked by trypsin and proteinase K in right-side out membrane vesicles . The beta subunit in the solubilized enzyme was only susceptible to digestion by trypsin if the substrates NADP(H) were present . NAD(H) did not affect digestion of the beta subunit . Digestion of the beta subunit of the membrane-bound enzyme by trypsin was not induced by NADP(H) unless the membranes had been previously stripped of extrinsic proteins by detergent . It is concluded that binding of NADP(H) induces a conformational change in the transhydrogenase . The location of the trypsin cleavage sites in the sequences of the alpha and beta subunits were determined by N- and C-terminal sequencing . A model is proposed in which the N-terminal 43 kDa region of the alpha subunit and the C-terminal 30 kDa region of the beta subunit are exposed on the cytoplasmic side of the inner membrane of E . coli . Binding sites for pyridine nucleotide coenzymes in these regions were suggested by affinity chromatography on NAD-agarose columns.

Nucleic Acids Res, 1991 Oct 11, 19(19), 5403 - 8
The core element of the EcoRII methylase as defined by protease digestion and deletion analysis; Friedman S et al.; Binding of the EcoRII DNA methyltransferase to azacytosine-containing DNA protects the enzyme from digestion by proteases . The limit digest yields a product having a Mr on SDS-PAGE 20% less than the intact protein . The N terminus of the tryptic digestion product was sequenced and found to be missing the N terminal 82 amino acids . Under the conditions used unbound enzyme was digested to small peptides . Protection of the enzyme from protease digestion implies that the enzyme undergoes major conformational changes when bound to DNA . The trypsin sensitive region of the EcoRII methyltransferase occurs prior to the first constant region shared with other procaryotic DNA(cytosine-5)methyltransferases . To determine if this region played a role in substrate binding or specificity, N-terminal deletion mutants were studied . Deletion of 97 amino acids resulted in a decrease of enzyme activity . Further deletions caused a complete loss of activity . Enzyme deleted through amino acid 85 was purified and found to have the same specificity as wild type however there was an increase in Km for both S-adenosylmethionine (AdoMet) and DNA of 27 and 18 fold respectively . The N-terminus of the EcoRII methylase, although a variable region present in many procaryotic DNA(cytosine-5)methylases, plays no role in determining enzyme specificity, although it does contribute to the interaction with both AdoMet and DNA.

Nucleic Acids Res, 1991 Oct 11, 19(19), 5363 - 70
The yeast RNA polymerase I promoter: ribosomal DNA sequences involved in transcription initiation and complex formation in vitro; Kulkens T et al.; Using an in vitro transcription system for Saccharomyces cerevisiae RNA polymerase I, we have analyzed Pol I promoter deletion mutants and mapped the boundaries of the promoter between positions -155 and +27 . The 5'-boundary of the minimal core promoter capable of transcription initiation, however, was found to lie between -38 and -26 . The 3'-deletion extending to -2 and -5 still allowed some transcription, suggesting that the positioning of Pol I is directed by upstream sequences . The results of in vitro analysis of linker scanning mutants (LSMs) combined with the deletion analysis showed that the promoter consists of three domains: two essential core domains (I: -28 to +8 and II: -76 to -51) and a transcription modulating upstream domain (III: -146 to -91) . These results are in general agreement with those obtained in vivo (1) . Using a template competition assay we also analyzed these mutant promoters for their ability to form a stable preinitiation complex . We found that the ability of 5'-deletion mutants to sequester an essential factor(s) correlates with their transcriptional activity . In contrast, several 3'-deletions and some LSMs in domain I and II decrease transcription activity greatly without significantly decreasing competition ability . The results indicate that the stimulatory function of domain III is achieved through its interaction with an essential transcription factor(s), although the other domains also participate in this interaction, perhaps directly or through another protein factor.

Nucleic Acids Res, 1991 Oct 11, 19(19), 5293 - 300
A chemical interference study on the interaction of ribosomal protein L11 from Escherichia coli with RNA molecules containing its binding site from 23S rRNA; Karaoglu D et al.; The interaction between ribosomal protein L11 from Escherichia coli and in vitro synthesized RNA containing its binding site from 23S rRNA was characterized by identifying nucleotides that interfered with complex formation when chemically modified by diethylpyrocarbonate or hydrazine . Chemically modified RNA was incubated with L11 under conditions appropriate for specific binding of L11 and the resulting protein-RNA complex was separated from unbound RNA on Mg(2+)-containing polyacrylamide gels . The ability to isolate L11 complexes on such gels was affected by the extent of modification by either reagent . Protein-bound and free RNAs were recovered and treated with aniline to identify their content of modified bases . Exclusion of RNA containing chemically altered bases from L11-associated material occurred for 29 modified nucleotides, located throughout the region corresponding to residues 1055-1105 in 23S rRNA . Ten bases within this region did not reproducibly inhibit binding when modified . Multiple bands of RNA were consistently observed on the nondenaturing gels, suggesting that significant intermolecular RNA-RNA interactions had occurred.

Nucleic Acids Res, 1991 Oct 11, 19(19), 5233 - 6
Orientation of the Lac repressor DNA binding domain in complex with the left lac operator half site characterized by affinity cleaving; Shin JA et al.; Lac repressor (LacR) is a helix-turn-helix motif sequence-specific DNA binding protein . Based on proton NMR spectroscopic investigations, Kaptein and co-workers have proposed that the helix-turn-helix motif of LacR binds to DNA in an orientation opposite to that of the helix-turn-helix motifs of lambda repressor, lambda cro, 434 repressor, 434 cro, and CAP {Boelens, R., Scheek, R., van Boom, J . and Kaptein, R., J . Mol . Biol . 193, 1987, 213-216} . In the present work, we have determined the orientation of the helix-turn-helix motif of LacR in the LacR-DNA complex by the affinity cleaving method . The DNA cleaving moiety EDTA.Fe was attached to the N-terminus of a 56-residue synthetic protein corresponding to the DNA binding domain of LacR . We have formed the complex between the modified protein and the left DNA half site for LacR . The locations of the resulting DNA cleavage positions relative to the left DNA half site provide strong support for the proposal of Kaptein and co-workers.

Nucleic Acids Res, 1991 Oct 11, 19(19), 5191 - 8
A DNA binding domain is contained in the C-terminus of wild type p53 protein; Foord OS et al.; In the present study we evaluated the DNA binding activity of wild type and mutant p53 proteins that were isolated from bacterial expression vectors . A comparison of the binding activities of the various purified p53 proteins, assessed by their ability to bind DNA cellulose columns, indicated that wild type p53 has a higher affinity to DNA than have mutant p53 forms . Furthermore, only wild type p53 was able to bind genomic DNA upon electrophoretic protein blotting . As specific deletion of the C-terminal region of wild type p53 totally abolished binding to genomic DNA, it was concluded that the 47 C-terminal amino acids contain the DNA binding region . The fact that the N-terminus contains a transcription activation region whereas the C-terminus contains a DNA binding domain places p53 in the family of typical transcription factors . Our experiments show that the topographical positioning of these domains plays an important role in the activity of wild type p53.

Nucleic Acids Res, 1991 Oct 11, 19(19), 5117 - 23
Identification and expression of the cDNA of KIN17, a zinc-finger gene located on mouse chromosome 2, encoding a new DNA-binding protein; Angulo JF et al.; We report the cloning of KIN17 cDNA, 1414 bp long with an ORF of 391 residues showing a zinc finger and nuclear localization signals . By recloning the cDNA into an appropriate vector, we produced kin17 protein in E . coli, purified it partially and shown that kin17 protein binds to double-stranded DNA . The KIN17 gene was localized by cytogenetic mapping in mouse chromosome 2, band A . Genomic sequences homologous to KIN17 cDNA were detected also in rat and human DNAs . KIN17 mRNA is highly expressed in rodent transformed AtT-20 neuroendocrine cells whereas it can be detected only in the total RNA of mouse embryos and various normal adult tissues by reverse transcription and PCR amplification . The mouse nuclear kin17 protein was identified by a local small structural similarity with E.coli recA protein . Kin17 and recA have only 39 amino acid residues in a region that might be involved in DNA-binding.

Nucleic Acids Res, 1991 Oct 11, 19(19), 5359 - 62
Cloning and functional characterization of a eucaryotic DNA photolyase gene from Neurospora crassa; Yajima H et al.; We cloned a genomic fragment of a photolyase gene from Neurospora crassa by polymerase chain reaction using synthesized oligonucleotide primers designed from the most conserved amino acid sequences among photolyases of various organisms . Using the cloned fragment as a hybridization probe we isolated a genomic fragment and cDNA clones encoding the complete photolyase gene of this organism . The amino acid sequence of the photolyase deduced from the determined nucleotide sequence indicates a protein consisting of 615 amino acid residues (Mr 69,971), which is most similar to that of Saccharomyces cerevisiae . Like yeast photolyase it contains a protruding amino terminus which is missing in photolyases of bacterial origin . Comparison of amino acids sequences among six photolyases suggests that the Neurospora crassa photolyase is more similar to photolyases of pterin type than those of deazaflavin type.

Nucleic Acids Res, 1991 Oct 11, 19(19), 5247 - 51
Codon choice and potential complementarity between mRNA downstream of the initiation codon and bases 1471-1480 in 16S ribosomal RNA affects expression of glnS; Faxen M et al.; A cis-acting expression mutation, GAG to GAA, in the third codon of the glnS gene is analyzed . Both codons code for glutamic acid but the mutation is known to increase gene expression by four fold . We show that the mutation has an effect only if it is located in the beginning of a gene but not if located internally . Data are presented that suggest that the reason for the increased expression by the mutation is the potential formation of one more base pair between the mRNA and 16S ribosomal RNA . Gene expression varies about 16 fold as the number of potential base pairs within the sequence 1471-1480 in 16S RNA increase from two to ten . We also give evidence that supports the idea that the presence of rare codons near the beginning of the mRNA can affect expression.

Nucleic Acids Res, 1991 Oct 11, 19(19), 5285 - 91
An erythroid specific enhancer upstream to the gene encoding the cell-type specific transcription factor GATA-1; Nicolis S et al.; The transcription factor GATA-1 is expressed in a subset of hemopoietic cells, where it mediates the cell-type specific expression of several genes . We have cloned the mouse and human GATA-1 genes . A region upstream to the first exon, and highly conserved between mouse and man, acts as an erythroid specific enhancer in transient assays, if linked to the GATA-1 or to the SV40 promoter . The activity of the enhancer is almost completely dependent on the integrity of a dimeric GATA-1 binding site.

Biochim Biophys Acta, 1991 Oct 10, 1075(2), 154 - 61
Characterisation of the binding sites for Escherichia coli heat-labile toxin type I in intestinal brush borders; Griffiths SL et al.; Intestinal brush borders from Wistar rats contained a total of 20-30-times more binding sites for Escherichia coli heat-labile enterotoxin (LT-1) than for cholera toxin (CT) . The results suggest that LT-1 binds to sites in addition to ganglioside GM1, the binding site for CT . Brush border proteins were separated by SDS-PAGE, blotted to nitrocellulose and the filters incubated with 125I-labeled toxins . {125I}LT-1 was shown to bind to a series of brush border galactoproteins ranging in size from 130-140 kDa . Binding was inhibited by unlabeled LT-1 (but not CT), and by ricin and free galactose . A number of brush border enzymes are large glycoproteins which can be solubilised by papain . The papain-solubilised sucrase-isomaltase complex was purified by affinity chromatography and shown to bind LT-1, as did the proteins in fractions enriched in maltase activity . However, such brush border galactoproteins do not account for all of the additional LT-1 binding sites . Thus, brush borders prepared from 1-15-day-old rabbits contained many more binding sites for LT-1 than CT despite the absence of any sucrase-isomaltase activity, and no {125I}LT-1 binding proteins could be detected by blotting . There was a marked variation in the number of LT-1 binding sites in different strains of rat, and between different species.

Nature, 1991 Oct 10, 353(6344), 569 - 71
Binding of general transcription factor TFIIB to an acidic activating region; Lin YS et al.; A central issue in eukaryotic transcriptional regulation is the mechanism by which promoter-specific transcription factors (activators) stimulate transcription . Two lines of evidence indicate that the general transcription factor TFIIB is a pivotal component in the mechanism by which an acidic activator functions . First, during assembly of the preinitiation complex TFIIB binding is a rate-limiting step enhanced by an acidic activator . Second, the TFIIB activity in a HeLa cell nuclear extract is specifically retained on a column containing an acidic activating region . But because our previous study monitored only TFIIB activity, it remains possible that the interaction between TFIIB and the acidic activating region is mediated through additional proteins, for example, those designated as adaptors, coactivators or mediators . A complementary clone encoding TFIIB has recently been isolated and shown to encode a polypeptide of relative molecular mass 35,000 . Here we report that TFIIB expressed in and purified from Escherichia coli (recombinant TFIIB) binds directly to the potent acidic activating region of the herpes simplex virus-1 VP16 protein.

Biochem Pharmacol, 1991 Oct 9, 42(9), 1739 - 44
Interaction of metronidazole with Escherichia coli deoxyribonucleic acid; Malliaros DP et al.; To define the characteristics of the reported binding of metronidazole to DNA, we isolated the DNA from hypoxic incubation mixtures that contained both {14C}metronidazole and metronidazole-susceptible strains of Escherichia coli . Thus, either {2-14C}metronidazole or {1',2'-14C}metronidazole was incubated with either wild-type E . coli (strain AB1157) or a DNA repair mutant (strain SR58) that is highly susceptible to metronidazole . Approximately 0.02% of the radiolabel in the metronidazole was found to be associated with DNA isolated from both strains of bacteria, a percentage similar to that found to be associated with DNA from mammalian sources in a variety of in vitro and in vivo experiments performed by other investigators . The bound radioactivity was not diminished, however, when a great excess of non-radiolabeled metronidazole was included in the incubation mixture, indicating that the binding we observed was probably due to impurities in the radiolabeled metronidazole . We also examined the binding to DNA of a possible surrogate for the partially reduced form of metronidazole, 1-methyl-4-phenyl-5-nitrosoimidazole (5NO), that has been described previously . The binding of the tritiated form of 5NO to DNA was also found to be undiminished by the addition of carrier 5NO (a finding which does not refute the hypothesis that 5NO may serve as a surrogate for the study of the active form of metronidazole) . These studies do not exclude the binding to DNA of either metronidazole or a possible surrogate of its active functionality, but they indicate that if such binding occurs, it must be limited to very few sites on DNA and hence will be difficult to characterize.

Biochemistry, 1991 Oct 8, 30(40), 9657 - 64
Construction, purification, and characterization of a hybrid protein comprising the DNA binding domain of the LexA repressor and the Jun leucine zipper: a circular dichroism and mutagenesis study; Schmidt-Dorr T et al.; An increasing number of eukaryotic transcription factors interacting specifically with DNA comprise a dimerization motif called the "leucine zipper" . These leucine zipper proteins form homodimers and/or heterodimers with another protein containing a leucine zipper motif . The leucine zipper of the oncoprotein Jun is particular in that Jun may form homodimers as well as heterodimers with the oncoprotein Fos, which are however more stable than the Jun-Jun homodimers . Leucine zipper dimerization is thought to occur through a coiled-coil arrangement of parallel alpha-helices, but the rules governing the specificity of homo- and/or heterodimerization are still largely unknown . To address this question in the case of the Jun leucine zipper, we constructed a fusion protein containing the amino-terminal DNA binding domain of the LexA repressor from Escherichia coli fused to the Jun leucine zipper . This hybrid protein (LexA-JunZip) is stable in E . coli and confers much tighter repression in vivo than the DNA binding domain of LexA alone . DNA binding competition experiments with synthetic Jun and Fos leucine zipper peptides in vitro showed that the leucine zipper mediated dimerization of LexA-JunZip is essential for DNA binding of the fusion protein . The purified LexA-JunZip protein dimerizes in vitro with a dimerization constant of 2 x 10(7) M-1 at 5 degrees C . Dimerization is very sensitive to temperature, since the dimerization constant drops at 20 degrees C to 2 x 10(6) M-1 and at 30 degrees C to only 3 x 10(5) M-1.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1991 Oct 8, 30(40), 9648 - 56
Motion of aromatic side chains, picosecond fluorescence, and internal energy transfer in Escherichia coli thioredoxin studied by site-directed mutagenesis, time-resolved fluorescence spectroscopy, and molecular dynamics simulations; Elofsson A et al.; We have determined the picosecond fluorescence of the four aromatic amino acid residues (W28, W31, Y49, and Y70) in wild-type Escherichia coli thioredoxin (wt Trx) and a mutant Trx with W31 replaced by phenylalanine, Trx-W28-W31F . The internal motions of the four aromatic side chains were also analyzed . We examined the possibility of using internal energy transfer from tyrosine to tryptophan as a measure of long-range distances . The major features of the lifetime distribution of tryptophan fluorescence were unchanged in the W31F mutation, indicating that the environment of W28 is similar in both wt Trx and Trx-W28-W31F . However, the mutation of W31F changed the mobility of W28, situated close to the active-site disulfide/dithiol, but not the mobility of two tyrosines, Y49 and Y70, situated on the other side of the molecule . The mobility of the two tyrosine residues increased upon reduction of the active-site disulfide, indicating a looser structure with reduction . This increased motion could also be seen from molecular dynamics simulations . The change in energy transfer rates, as judged by tyrosine fluorescence lifetimes, was in agreement with energy transfer rates calculated from the molecular dynamics simulations . The anisotropy of tryptophan and tyrosine fluorescence could be separated in three parts: (I) overall rotation of the protein (10(-9)s), (II) internal mobility of side chains (10(-10)s), and (III) a very fast relaxation (10(-12)s) . We can only experimentally detect this very fast relaxation when the internal motion is not present.

Biochemistry, 1991 Oct 8, 30(40), 9595 - 600
Construction of a functional lactose permease devoid of cysteine residues; van Iwaarden PR et al.; By use of oligonucleotide-directed, site-specific mutagenesis, a lactose (lac) permease molecule was constructed in which all eight cysteinyl residues were simultaneously mutagenized (C-less permease) . Cys154 was replaced with valine, and Cys117, -148, -176, -234, -333, -353, and -355 were replaced with serine . Remarkably, C-less permease catalyzes lactose accumulation in the presence of a transmembrane proton electrochemical gradient (interior negative and alkaline) . Thus, in intact cells and right-side-out membrane vesicles containing comparable amounts of wild-type and Cys-less permease, the mutant protein catalyzes lactose transport at a maximum velocity and to a steady-state level of accumulation of about 35% and 55%, respectively, of wild-type with a similar apparent Km (ca . 0.3 mM) . As anticipated, moreover, active lactose transport via C-less permease is completely resistant to inactivation by N-ethylmaleimide . Finally, C-less permease also catalyzes efflux and equilibrium exchange at about 35% of wild-type activity . The results provide definitive evidence that sulfhydryl groups do not play an essential role in the mechanism of lactose/H+ symport . Potential applications of the C-less mutant to studies of static and dynamic aspects of permease structure/function are discussed.

Biochemistry, 1991 Oct 8, 30(40), 9569 - 75
Activation of methionine by Escherichia coli methionyl-tRNA synthetase; Ghosh G et al.; In the present work, we have examined the function of three amino acid residues in the active site of Escherichia coli methionyl-tRNA synthetase (MetRS) in substrate binding and catalysis using site-directed mutagenesis . Conversion of Asp52 to Ala resulted in a 10,000-fold decrease in the rate of ATP-PPi exchange catalyzed by MetRS with little or no effect on the Km's for methionine or ATP or on the Km for the cognate tRNA in the aminoacylation reaction . Substitution of the side chain of Arg233 with that of Gln resulted in a 25-fold increase in the Km for methionine and a 2000-fold decrease in kcat for ATP-PPi exchange, with no change in the Km for ATP or tRNA . These results indicate that Asp52 and Arg233 play important roles in stabilization of the transition state for methionyl adenylate formation, possibly directly interacting with complementary charged groups (ammonium and carboxyl) on the bound amino acid . Primary sequence comparisons of class I aminoacyl-tRNA synthetases show that all but one member of this group of enzymes has an aspartic acid residue at the site corresponding to Asp52 in MetRS . The synthetases most closely related to MetRS (including those specific for Ile, Leu, and Val) also have a conserved arginine residue at the position corresponding to Arg233, suggesting that these conserved amino acids may play analogous roles in the activation reaction catalyzed by each of these enzymes . Trp305 is located in a pocket deep within the active site of MetRS that has been postulated to form the binding cleft for the methionine side chain.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1991 Oct 8, 30(40), 9716 - 23
Complex interactions between the chaperonin 60 molecular chaperone and dihydrofolate reductase; Viitanen PV et al.; The spontaneous refolding of chemically denatured dihydrofolate reductase (DHFR) is completely arrested by chaperonin 60 (GroEL) . This inhibition presumably results from the formation of a stable complex between chaperonin 60 and one or more intermediates in the folding pathway . While sequestered on chaperonin 60, DHFR is considerably more sensitive to proteolysis, suggesting a nonnative structure . Bound DHFR can be released from chaperonin 60 with ATP, and although chaperonin 10 (GroES) is not obligatory, it does potentiate the maximum effect of ATP . Hydrolysis of ATP is also not required for DHFR release since certain nonhydrolyzable analogues are capable of partial discharge . "Native" DHFR can also form a stable complex with chaperonin 60 . However, in this case, complex formation is not instantaneous and can be prevented by the presence of DHFR substrates . This suggests that native DHFR exists in equilibrium with at least one conformer which is recognizable by chaperonin 60 . Binding studies with 35S-labeled DHFR support these conclusions and further demonstrate that DHFR competes for a common saturable site with another protein (ribulose-1,5-bisphosphate carboxylase) known to interact with chaperonin 60.

Biochemistry, 1991 Oct 8, 30(40), 9601 - 7
Involvement of the carboxy-terminal residue in the active site of the histidine-containing protein, HPr, of the phosphoenolpyruvate:sugar phosphotransferase system of Escherichia coli; Anderson JW et al.; Histidine-containing protein, HPr, of the Escherichia coli phosphoenolpyruvate:sugar phosphotransferase system has an active site that involves His-15, which is phosphorylated to form a N delta 1-P-histidine, Arg-17, and the carboxy-terminal residue Glu-85 . Mutant HPrs with alterations to the three C-terminal residues, Glu-85, Leu-84, and Glu-83, were produced by site-directed mutagenesis . The properties of these mutants were assessed by kinetic analysis of enzyme I, enzyme IImannose, enzyme IIN-acetylglucosamine, and enzyme IImannitol, and the phosphohydrolysis properties of the HPr mutants . The results show that it is the C-terminal alpha-carboxyl of Glu-85 that is involved in the active site, and this involvement may be restricted to the phosphoryl donor action of HPr . The contribution of this alpha-carboxyl group is modest as the deletion of Glu-85 resulted in the reduction of the enzyme II activity (kcat/Km) to about 33% . Removal of both Glu-85 and Leu-84 yields an HPr that is an impaired substrate of both the enzyme I and enzyme II reactions . Glu-83 appears to have no role in the active site.

FEBS Lett, 1991 Oct 7, 291(1), 75 - 8
Molecular cloning and primary structure of Thermoactinomyces vulgaris carboxypeptidase T . A metalloenzyme endowed with dual substrate specificity; Smulevitch SV et al.; A gene coding for an extracellular Zn-carboxypeptidase of Thermoactinomyces vulgaris has been cloned and sequenced (EMBL X56901) . This enzyme named carboxypeptidase T reveals simultaneously both types of substrate specificity characteristic of mammalian carboxypeptidases A and B . The carboxypeptidase T gene is primarily expressed in E . coli as a non-active preproenzyme with an additional 98 amino acid residues at the N-terminus . Primary structure alignment of mature carboxypeptidase T and mammalian metallocarboxypeptidases demonstrated 25-30% overall identity but a full preservation of presumed catalytically important residues . These observations imply a basic uniformity of the general catalytic mechanism for enzymes of that class produced by evolutionarily remote organisms.

J Mol Biol, 1991 Oct 5, 221(3), 889 - 907
Computer modeling 16 S ribosomal RNA; Hubbard JM et al.; A three-dimensional structure for 16 S RNA has been produced with a computer protocol that is not dependent on human intervention . This protocol improves upon traditional modeling techniques by using distance geometry to fold the molecule in an objective and reproducible fashion . The method is based on the secondary structure of RNA and treats the molecule as a set of double-stranded helices that are linked by flexible single-strands of variable length . Data derived from chemical cross-linking studies of 16 S RNA and tertiary phylogenetic relationships provide the constraints used to fold the molecule into a compact three-dimensional form . Possibly subjective evaluation of the input data are transformed into verifiable quantitative parameters . Relationships based on general locations within the 30 S subunit or on protein-RNA interactions have been specifically excluded . The resolution of the model exceeds that of electron micrographs and approaches that obtained in preliminary X-ray crystal structures . The model size of 245 x 190 x 140 A is compatible with that of the 30 S subunit as determined by electron microscopy . The volume of the model is 1.87 x 10(6) A which is similar to that of the small subunit in a preliminary X-ray crystal structure . The radius of gyration of the model structure of 76 A is intermediate to that seen for partially denatured and fully folded 16 S RNA . Computer graphics are used to display the results in a manner that maximizes the opportunities for human visual interpretation of the models . A format for displaying the structures has been developed that will make it possible for researchers who have not devoted themselves to ribosomal modeling to comprehend and make use of the information that the models embody . On this basis the computer-generated models are compared with models developed by other researchers and with structural data not included in the folding parameter data set.

J Mol Biol, 1991 Oct 5, 221(3), 805 - 21
An in vitro approach to identifying specificity determinants of mutagenesis mediated by DNA misalignments; Papanicolaou C et al.; In vitro, misalignments of the newly synthesized (primer) strand during DNA polymerization lead to deletion and/or complex frameshift mutations . In vivo, similar misalignments of repeated and quasipalindromic DNA sequences are predicted to be intermediates of mutagenesis . The mutagenic misalignments are mediated by complementary pairing between the sequence at the 3'-OH end of the newly synthesized DNA strand and sequences in the template or in the newly synthesized DNA . Mutant sequences are produced when the misaligned primers act as substrates for DNA polymerization . The misalignments responsible for detected mutant sequences were compared to similar misalignments that were not implicated in mutagenesis, and all misalignment possibilities were compared to the position of pausing during polymerization by Escherichia coli polymerase I or its Klenow fragment . These comparisons revealed three characteristics of in vitro misalignment specificity . First, the termini produced by pausing are likely to be precursors to mutagenic misalignments . Second, the absence of some potential misalignments from the detected spectrum is explained well by the predicted undetectability of the mutant sequences they produce . Third, factors distinct from pausing and mutant detectability are responsible for differences in the specificity of misalignment mutagenesis mediated by E . coli DNA polymerase I and Klenow polymerase during in vitro synthesis.

J Mol Biol, 1991 Oct 5, 221(3), 751 - 4
Evidence for an ancestral core structure in nucleotide-binding proteins with the type A motif; Milner-White EJ et al.; Many proteins that bind purine nucleotide triphosphates have a type A sequence motif . Only two classes of structures for such proteins are so far available from X-ray crystallography . We examined the tertiary structures of representatives of the two classes, porcine cytoplasmic adenylate kinase and Escherichia coli translational elongation factor Tu . Comparison of the two proteins suggests that the A motif may be just one part of a larger common core structure consisting of four parallel strands of beta-sheet sandwiched between four alpha-helices . This compact core structure comprises over one half of each protein . We speculate that A motif proteins have diverged from a common ancestor having this core structure.

J Biol Chem, 1991 Oct 5, 266(28), 19056 - 62
Autoflavinylation of apo6-hydroxy-D-nicotine oxidase; Brandsch R et al.; 6-Hydroxy-D-nicotine oxidase (6-HDNO) was expressed in Escherichia coli JM109 cells from the recombinant plasmid pAX-6-HDNO as a beta-galactosidase-6-HDNO fusion protein . Affinity chromatography of the fusion protein on p-aminobenzyl-1-thio-beta-galactopyranoside-agarose and subsequent digestion with protease Xa yielded highly purified apo6-HDNO . Incubation of the purified protein with {14C}FAD demonstrated that flavinylation of apo6-HDNO proceeds autocatalytically . Phosphorylated three-carbon compounds such as glycerol-3-P, which are known to stimulate the formation of the histidyl (N3)-(8 alpha) FAD between apo6-HDNO and FAD (Brandsch, R., and Bichler, V . (1989) Eur . J . Biochem . 182, 125-128), could be replaced in their action by high concentrations of glycerol (45%) or sucrose (20%) . These substances apparently induced and stabilized a conformational state of the apoenzyme compatible with covalent attachment of FAD . In the absence of glycerol the apoenzyme readily lost the ability to form holoenzyme at temperatures above 30 degrees C . Holoenzyme formation protected the 6-HDNO polypeptide from this thermal denaturation . Autoflavinylation of 6-HDNO was inhibited by the sulfhydryl reagents dithionitrobenzoate or N-ethylmaleimide . Inhibition was prevented by mercaptoethanol or FAD, but not 6-hydroxy-D-nicotine, the substrate of the holoenzyme . A cysteine-thiol group may therefore be involved in reactions leading to the covalent attachment of FAD to apo6-HDNO . When flavinylation of apo6-HDNO proceeded under anaerobic conditions, the amount of incorporation of {14C}FAD into the polypeptide was indistinguishable from reactions performed in the presence of O2 . None of the FAD-derivatives (8-demethyl-FAD, 8-chloro-FAD, and 5-deaza-FAD) could replace FAD in holoenzyme formation . The failure of covalent attachment of 5-deaza-FAD to apo6-HDNO is in agreement with the assumption that the quinone methide form of the isolloxazine ring is an intermediate in the flavinylation reaction.

J Biol Chem, 1991 Oct 5, 266(28), 19028 - 33
Action of RecBCD enzyme on Holliday structures made by RecA; Muller B et al.; In vitro, Escherichia coli RecA protein acts upon gapped and partially homologous linear duplex DNA to generate recombination products linked by Holliday junctions . When strand exchange reactions are supplemented with purified RecBCD enzyme, we observe the formation of products that resemble "patch" recombinants . The formation of "splice" recombinant products was not observed . The individual subunits, RecB, RecC, or RecD, had no effect on RecA protein-mediated strand exchange nor on the Holliday junctions formed in the reaction . Analysis of the way in which patch products arise indicates exonucleolytic digestion of the linear arms of the recombination intermediates (alpha-structures) by RecBCD enzyme . We find no evidence for specific resolution events at the site of the Holliday junction by RecBCD enzyme using these DNA substrates.

J Biol Chem, 1991 Oct 5, 266(28), 19013 - 7
Purification and molecular cloning of succinyltransferase of the rat alpha-ketoglutarate dehydrogenase complex . Absence of a sequence motif of the putative E3 and/or E1 binding site; Nakano K et al.; Full-length cDNA clones for succinyltransferase of the rat alpha-ketoglutarate dehydrogenase complex were isolated from rat heart cDNA libraries in lambda gt11 . The cDNA clones were identified as those for rat succinyltransferase by the identity of their predicted amino acid sequence with the NH2-terminal amino acid sequence of rat succinyltransferase determined by protein chemical analysis and the known amino acid sequence of bovine succinyltransferase . The clone with the longest cDNA consisted of 2747 base pairs and coded for a leader peptide of 56 amino acid residues and a mature protein of 386 amino acid residues . The primary structure of rat succinyltransferase showed close similarity to Escherichia coli and Azotobacter vinelandii succinyltransferases, in the COOH-terminal part forming the lipoyl-binding domain and the NH2-terminal part forming the inner core-catalytic domain . However, the rat succinyltransferase did not contain a sequence motif that has been found as an E3- and/or E1-binding site in the dihydrolipoamide acyltransferases of three alpha-ketoacid dehydrogenase complexes (Hummel, K . B., Litwer, S., Bradford, A . P., Aitken, A., Danner, D . J., and Yeaman, S . J . (1988) J . Biol . Chem . 263, 6165-6168, Reed, L . J., and Hackert, M . L . (1990) J . Biol . Chem . 265, 8971-8974) . The absence of this sequence was confirmed by direct sequencing of the polymerase chain reaction product of rat heart mRNA and by computer analysis . These results show that the rat succinyltransferase does not have the sequence motif of the putative E3- and/or E1-binding site.

J Biol Chem, 1991 Oct 5, 266(28), 18895 - 906
Mechanism of DNA A protein-dependent pBR322 DNA replication . DNA A protein-mediated trans-strand loading of the DNA B protein at the origin of pBR322 DNA; Parada CA et al.; pBR322 DNA can be replicated via a DNA A-dependent pathway mediated by its binding to the two DNA A-binding sites (dnaA boxes) present near the plasmid origin . DNA synthesis requires the transcription of RNA II (the leading-strand primer precursor) to generate a specific unwound structure in the region containing the dnaA boxes . In this structure, the DNA containing the dnaA boxes can take the form of either a RNA II-parental H strand pBR322 DNA hybrid opposed by the displaced parental L strand (in the absence of RNase H and DNA polymerase I), or a nascent leading strand-parental H strand DNA duplex opposed by the displaced parental L strand (in the presence of RNase H and DNA polymerase I) . These findings defined three types of potential sites for productive DNA A binding: (i) the displaced parental L single strand, (ii) a hairpin formed by the inverted repeat of the two dnaA boxes, and (iii) either the RNA-DNA duplex or the nascent leading strand-parental DNA duplex . By using a combination of: (i) inhibition of the replication of a plasmid carrying oriC by oligonucleotides of various dnaA box sequences and conformation, (ii) a gel mobility shift assay to measure DNA A binding to the same oligonucleotide substrates, (iii) replication of pBR322 DNA templates with either one or no dnaA box, and (iv) photocross-linking to demonstrate DNA A binding to an RNA-DNA hybrid, evidence is presented here that DNA A-mediated pBR322 DNA replication proceeds by a mechanism in which DNA A binds to the duplex side of the unwound origin structures and loads the DNA B protein in trans to the displaced parental L strand DNA.

J Biol Chem, 1991 Oct 5, 266(28), 18642 - 8
Replacement of lysine 269 by arginine in Escherichia coli tryptophan indole-lyase affects the formation and breakdown of quinonoid complexes; Phillips RS et al.; Lysine 269 in Escherichia coli tryptophan indole-lyase (tryptophanase) has been changed to arginine by site-directed mutagenesis . The resultant K269R mutant enzyme exhibits kcat values about 10% those of the wild-type enzyme with S-(o-nitrophenyl)-L-cysteine, L-tryptophan, and S-benzyl-L-cysteine, while kcat/Km values are reduced to 2% or less . The pH profile of kcat/Km for S-benzyl-L-cysteine for the mutant enzyme exhibits two pK alpha values which are too close to separate, with an average value of 7.6, while the wild-type enzyme exhibits pK alpha values of 6.0 and 7.8 . The pK alpha for the interconversion of the 335 and 412 nm forms of the K269R enzyme is 8.3, while the wild-type enzyme exhibits a pK alpha of 7.4 . Steady-state kinetic isotope effects on the reaction of {alpha-2H}S-benzyl-L-cysteine with the K269R mutant enzyme (Dkcat = 2.0; D(kcat/Km) = 3.9) are larger than those of the wild-type enzyme (Dkcat = 1.4; D(kcat/Km) = 2.9) . Rapid scanning stopped-flow kinetic studies demonstrate that the K269R mutant enzyme does not accumulate quinonoid intermediates with L-alanine, L-tryptophan, or S-methyl-L-cysteine, but does form quinonoid absorption peaks in complexes with S-benzyl-L-cysteine and oxidolyl-L-alanine, whereas wild-type enzyme forms prominent quinonoid bands with all these amino acids . Single wavelength stopped-flow kinetic studies demonstrate that the alpha-deprotonation of S-benzyl-L-cysteine is 6-fold slower in the K269R mutant enzyme, while the intrinsic deuterium kinetic isotope effect is less for the K269R enzyme (Dk = 4.2) than for the wild-type (Dk = 7.9) . The decay of the K269R quinonoid intermediate in the presence of benzimidazole is 7.1-fold slower than that of the wild-type enzyme . These results demonstrate that Lys-269 plays a significant role in the conformational changes or electrostatic effects obligatory to the formation and decomposition of the quinonoid intermediate, although it is not an essential basic residue.

J Biol Chem, 1991 Oct 5, 266(28), 18626 - 34
Control of L-ornithine specificity in Escherichia coli ornithine transcarbamoylase . Site-directed mutagenic and pH studies; Goldsmith JO et al.; Escherichia coli ornithine transcarbamoylase displays a strict specificity toward its second substrate L-ornithine . After forming a binary complex with carbamoyl phosphate and undergoing an induced-fit isomerization (Miller, A . W., and Kuo, L . C . (1990) J . Biol . Chem . 265, 15023-15027), the enzyme selects only the minor, zwitterionic ornithine with an uncharged delta-amino group for transcarbamoylation . Formation of the productive ternary complex is linked to two enzymic ionizations (pK alpha 6.2 approximately 6.3 and 9.1 approximately 9.3) and two ornithine ionizations (pK alpha 8.5 and 10.6) (Kuo, L . C., Herzberg, W., and Lipscomb, W . N . (1985) Biochemistry 24, 4754-4761) . To elucidate the mechanism through which substrate specificity is achieved, the binding of L-ornithine to two site-specific point mutants (Arg-57----Gly and Cys-273----Ala) of the enzyme has been examined . For the Gly-57 mutant enzyme, which does not undergo the induced-fit isomerization, affinity for ornithine drops by a factor of 500 . The pH profile of the apparent equilibrium constant governing the association of L-ornithine to the binary complex of this mutant reveals that only two enzymic ionizations affect ornithine binding . The ionizations linked to L-ornithine are not detected . Hence, the preisomerized binary complex binds not only poorly but also indiscriminately all ionic species of L-ornithine . For the Ala-273 mutant enzyme, which exhibits the induced-fit isomerization, affinity of the amino acid is decreased by an order of magnitude . Ionizations of L-ornithine to yield a zwitterion for binding are detected in pH analyses for this mutant, but the pK alpha of 6.2 associated with the enzymic deprotonation in the wild type is absent . Therefore, Cys-273 is a binding site of L-ornithine . The D-isomer of ornithine is a very weak, deadend ligand to all three forms of the enzyme with affinities in the millimolar range . Employing the estimated affinities of D- and L-ornithine, the binding stereospecificity of the wild-type and mutant binary complexes toward the amino acid substrate may be evaluated . L-Ornithine binds preferentially over D-ornithine by two and four orders of magnitude in the absence and presence of protein isomerization, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)

J Biol Chem, 1991 Oct 5, 266(28), 18606 - 12
Site-specific mutations in human ferredoxin that affect binding to ferredoxin reductase and cytochrome P450scc; Coghlan VM et al.; Ferredoxins found in animal mitochondria function in electron transfer from NADPH-dependent ferredoxin reductase (Fd-reductase) to cytochrome P450 enzymes . To identify residues involved in binding of human ferredoxin to its electron transfer partners, neutral amino acids were introduced in a highly conserved acidic region (positions 68-86) by site-directed mutagenesis of the cDNA . Mutant ferredoxins were produced in Escherichia coli, and separate assays were used to determine the effect of substitutions on the capacity of each mutant to bind to Fd-reductase and cytochrome P450scc and to participate in the cholesterol side chain cleavage reaction . Replacements at several positions (mutants D68A, E74Q, and D86A) did not significantly affect activity, suggesting that acidic residues at these positions are not required for binding or electron transfer interactions . In contrast, substitutions at positions 76 and 79 (D76N and D79A) caused dramatic decreases in activity and in the affinity of ferredoxin for both Fd-reductase and P450scc; this suggests that the binding sites on ferredoxin for its redox partners overlap . Other substitutions (mutants D72A, D72N, E73A, E73Q, and D79N), however, caused differential effects on binding to Fd-reductase and P450scc, suggesting that the interaction sites are not identical . We propose a model in which Fd-reductase and P450scc share a requirement for ferredoxin residues Asp-76 and Asp-79 but have other determinants that differ and play an important role in binding . This model is consistent with the hypothesis that ferredoxin functions as a mobile shuttle in steroidogenic electron transfer, and it is considered unlikely that a functional ternary complex is formed.

J Biol Chem, 1991 Oct 5, 266(28), 18446 - 53
Potato tuber type H phosphorylase isozyme . Molecular cloning, nucleotide sequence, and expression of a full-length cDNA in Escherichia coli; Mori H et al.; Higher plant tissues contain two alpha-glucan phosphorylase isozymes (EC 2.4.1.1), types L and H, localized in the plastid and the cytoplasm, respectively . We already isolated and sequenced a cDNA clone encoding the type L isozyme . Presently, a cDNA clone encoding the type H counterpart was isolated from a cDNA library of immature potato tuber by plaque hybridization, using two oligonucleotide probes synthesized based on the partial amino acid sequences of the type H isozyme . The message encodes a polypeptide of 838 amino acid residues . Sequence comparison of the two potato tuber phosphorylase isozymes revealed two major distinctions; the type L isozyme contains a 78-residue insertion in the middle of the polypeptide chain as well as a 50-residue amino-terminal extension . Except for these extra portions, the two isozyme sequences show an identity of 63% . The entire structural gene for the type H isozyme was inserted 3'-downstream of the strong T7 RNA polymerase promoter in the expression plasmid pET-3b . Escherichia coli BL21 (DE3) cells carrying this plasmid produced active phosphorylase upon induction with isopropyl-beta-D-thiogalactoside at 22 degrees C . The expression is entirely dependent on the temperature; the bacteria did not produce a detectable amount of the active enzyme at 37 degrees C . Addition of pyridoxine to the culture medium was effective for the enzyme production.

J Biol Chem, 1991 Oct 5, 266(28), 18419 - 22
Carbohydrate-binding protein 35 (Mac-2), a laminin-binding lectin, forms functional dimers using cysteine 186; Woo HJ et al.; Carbohydrate-binding protein 35 (CBP35), also known as the macrophage surface antigen Mac-2, is a lactosamine-specific lectin whose extracellular properties include the ability to agglutinate cells and to bind avidly to the basement membrane glycoprotein laminin . Although these and other properties would be facilitated by dimerization of this lectin, previous studies have argued against multimeric forms of this protein . We report here that macrophage CBP35, purified by laminin affinity chromatography, exists as several distinct species (Mr 35,000, 67,000, and 80,000) when analyzed under non-reducing conditions . This unexpected finding prompted us to study the biochemistry of multimerization using recombinant CBP35 (rCBP35) . rCBP35 expressed in Escherichia coli forms disulfide-linked homodimers (Mr 67,000) . The dimeric form of CBP35 binds to laminin with higher affinity than does monomer and by a lactosamine-dependent mechanism . Site-directed mutagenesis indicated that cysteine 186, the single cysteine residue in CBP35, is required for dimerization . These results raise the possibility that homo- and heterodimeric forms of CBP35 contribute to its postulated functions in cell-matrix interactions and growth regulation.

J Mol Biol, 1991 Oct 5, 221(3), 737 - 43
Escherichia coli ribosome is inactivated by Mirabilis antiviral protein which cleaves the N-glycosidic bond at A2660 of 23 S ribosomal RNA; Habuka N et al.; Ribosome-inactivating proteins (RIPs) are known to inactivate eukaryotic ribosomes, which results in the inhibition of protein synthesis, but there has been no evidence that they inactivate the ribosomes of Escherichia coli . Recently, Mirabilis antiviral protein (MAP), a RIP, has been shown to inhibit the protein synthesis of E . coli as well as eukaryotes . To elucidate its mechanism, E . coli ribosomes treated with MAP were analyzed by polyacrylamide/agarose composite gel electrophoresis and RNA sequencing using reverse transcriptase with DNA primer . The 23 S rRNAs, with an A260 value for ribosomes of 15, were completely cleaved in vitro by a 30 minute treatment with MAP at a concentration of 100 nM at 37 degrees C and a subsequent treatment with aniline . However, they were not affected by ricin A-chain under the same conditions . The primer extension of DNA polymerization stopped before A2660 of 23 S rRNA in RNA sequencing . Furthermore, both 16 S and 23 S rRNAs were cleaved by the MAP and aniline treatments when naked E . coli rRNAs were used as substrates, and the primer extension stopped before bases A2660 and A1014, respectively, in RNA sequencing . As the A2660 region has been shown to interact with the elongation factors EF-Tu and EF-G these results indicate that MAP cleaves the N-glycosidic bond at A2660 in E . coli 23 S RNA resulting in the inactivation of the ribosome.

J Biol Chem, 1991 Oct 5, 266(28), 18942 - 8
Glycosylated and unglycosylated recombinant-derived human stem cell factors are dimeric and have extensive regular secondary structure; Arakawa T et al.; We have recently described the identification, isolation, and characterization of a factor, termed stem cell factor (SCF), which acts on primitive hematopoietic progenitors of the marrow . A soluble form of the factor was isolated from the conditioned medium of a rat cell line (Zsebo, K . M., Wypych, J., McNiece, I . K., Lu, H . S., Smith, K . A., Karkare, S . B., Sachdev, R . K., Yuschenkoff, V . N., Birkett, N . C., Williams, L . R., Satyagal, V . N., Tung, W., Bosselman, R . A., Mendiaz, E . A., and Langley, K . E . (1990) Cell 63, 195-201) and rat and human cDNAs have been cloned (Martin, F . H., Suggs, S . V., Langley, K . E., Lu, H . S., Ting, J., Okino, K . H., Morris, C . F., McNiece, I . K., Jacobsen, F . W., Mendiaz, E . A., Birkett, N . C., Smith, K . A., Johnson, M . J., Parker, V . P., Flores, J . C., Patel, A . C., Fisher, E . F., Erjavec, H . O., Herrera, C . J., Wypych, J., Sachdev, R . K., Pope, J . A., Leslie, I., Wen, D., Lin, C.-H., Cupples, R . L., and Zsebo, K . M . (1990) Cell 63, 203-211) . The cDNAs encode amino acids C-terminal to those found in the isolated natural form, including a putative transmembrane domain . This paper describes the structural characterization of soluble forms of recombinant human SCF purified from Escherichia coli (unglycosylated) and from Chinese hamster ovary (CHO) cells (glycosylated) . Fluorescence emission spectra indicate that the single Trp residue is present in a hydrophobic environment . Circular dichroism and infrared spectroscopy indicate considerable secondary structure, including both alpha-helix and beta-sheet . Molecular weight determinations by sedimentation equilibrium show that the molecules are dimeric (noncovalently associated), and gel filtration analyses are consistent with this conclusion . The CHO cell-derived SCF is about 30% carbohydrate by weight, with both N-linked and O-linked sugar . The presence or absence of the carbohydrate does not influence the results of the various structural analyses.

J Biol Chem, 1991 Oct 5, 266(28), 18525 - 9
Mapping ribosomal protein S20-16 S rRNA interactions by mutagenesis; Cormack RS et al.; Specific ribonucleotides within the 5' domain of Escherichia coli 16 S rRNA were altered by deletion and/or substitution by site-directed mutagenesis of cloned DNA to assess the importance of these particular residues in defining the binding site for ribosomal protein S20 . Gel filtration and sucrose gradient centrifugation were employed to measure the binding of ribosomal protein S20 to synthetic transcripts spanning the 5' domain of 16 S rRNA containing various alterations . In several cases, the apparent association constants for these interactions were also determined . Three essential results were obtained . First, RNA transcripts containing residues 1-402 are sufficient for high affinity S20 binding . Second, bulges at residues 250-251 and 278-280 in a stem-loop structure extending from residues 240 to 286 are critical for S20 binding, arguing that the "260 stem" directly interacts with S20 at these sites . Third, mutations in a hairpin structure spanning residues 316-337 demonstrate a strong requirement for both the specific A321*G332 bulge and for unpaired residues in the loop itself for proper S20 binding . Our results document the importance of unpaired residues in maintaining the interaction between S20 and 16 S rRNA.

J Mol Biol, 1991 Oct 5, 221(3), 1007 - 14
Mapping transition states of protein unfolding by protein engineering of ligand-binding sites; Sancho J et al.; We describe a method for probing the integrity and relative orientation of structural elements that are indirectly linked by ligands in protein complexes during protein folding . The effect of 3'-GMP on the rate constants of unfolding of wild-type barnase and several mutants has been studied . By comparing the rates of unfolding of wild-type and mutant proteins, we show that the interaction between His102 and 3'-GMP is fully retained in the transition state compared with the folded state, while the interaction between Glu60 and the ligand is partly retained and that of Lys27 is broken . Our data suggest that the transition state has a partly formed ligand binding site in which the guanine binding loop containing Glu60 and the loop containing His102 are formed at the sides of the beta-sheet but the docking of the N terminus of the second alpha-helix containing Lys27 on the beta-sheet is disrupted . The active site of barnase in complexes is thus partly retained in the transition state of unfolding . Although the ligand could in principle perturb the unfolding pathway, there is independent evidence that indicates that similar structural changes occur upon unfolding of unligated barnase.

J Biol Chem, 1991 Oct 5, 266(28), 19095 - 102
Cyclic AMP induction of phosphoenolpyruvate carboxykinase (GTP) gene transcription is mediated by multiple promoter elements; Liu JS et al.; The cis elements involved in the cAMP regulation of transcription of the gene for phosphoenolpyruvate carboxykinase (GTP) (EC 4.1.1.32) (PEPCK) were studied by introducing a series of block mutations (10-15 base pairs of random sequence) into eight of the protein binding domains in a region of the promoter between -490 and +73 . Each mutant promoter was ligated to the structural gene for chloramphenicol acetyltransferase (CAT) and transfected into HepG2 cells . Transcription of PEPCK-CAT was stimulated 4-fold by the addition of 8-bromo-cAMP (8-Br-cAMP), whereas overexpression of the catalytic subunit of protein kinase A in these cells increased transcription from the PEPCK promoter 30-fold . Several elements within the PEPCK promoter acted synergistically to mediate this effect . These include CRE-1 (-92 to -82) and a complex unit from -220 to -280 composed of multiple binding sites termed P3(I) (-250 to -234), P3(II) (-260 to -250), and P4 (-286 to -270) . Mutation of both CRE-1 and P3(I) resulted in the complete elimination of transcriptional induction by either 8-Br-cAMP or the catalytic subunit of protein kinase A . To examine the proteins involved in this response, we replaced CRE-1, which binds both C/EBP and cAMP-responsive element binding protein (CREB), with an optimal C/EBP binding sequence which significantly decreased the binding of CREB, but maintained the affinity for C/EBP . Transcription from this modified promoter was induced by 8-Br-cAMP and the catalytic subunit of protein kinase A (PKA) to a similar extent as noted with the native PEPCK promoter . However, the results of experiments involving cotransfection of PEPCK-CAT with expression vectors for PKA and either C/EBP or CREB suggest that CREB is capable of mediating a greater responsiveness to PKA than C/EBP . Our results indicate that multiple cis elements are involved in the cAMP induction of PEPCK gene transcription and that C/EBP and CREB are potentially involved in this response.

J Biol Chem, 1991 Oct 5, 266(28), 18454 - 9
Cloning and expression of Chromobacterium violaceum phenylalanine hydroxylase in Escherichia coli and comparison of amino acid sequence with mammalian aromatic amino acid hydroxylases; Onishi A et al.; The complete amino acid sequence (296 amino acids) of Chromobacterium violaceum phenylalanine hydroxylase (PAH) was determined by nucleotide analysis of a DNA clone isolated using both a synthetic oligonucleotide probe based on the NH2-terminal amino acid sequence and an antibody against this enzyme . The ApaL I fragment (approximately 1.9 kilobase pairs) containing the entire PAH gene was subcloned in pBluescript II and induced by isopropyl-beta-D-thiogalactopyranoside . In order to eliminate fusion proteins the XbaI/ClaI fragment which contained the PAH gene from the Bluescript construct was subcloned into pMAC 5-8 containing the TAC promoter . The recombinant protein reacts with antibody raised to authentic C . violaceum PAH and its NH2-terminal 20-amino acid sequence and COOH-terminal amino acid residue were identical with the wild-type protein . Key physical and chemical characteristics of the recombinant protein, i.e . its copper content and Michaelis-Menten parameters, were the same as wild-type . Comparison of amino acid sequences revealed a highly conserved region between C . violaceum PAH and three different mammalian aromatic amino acid hydroxylases . This conserved area may well be a catalytically important domain of these pterin- and metal-requiring aromatic amino acid hydroxylases . The over-expression of C . violaceum PAH in Escherichia coli will facilitate the analysis of the enzyme mechanism by various spectroscopic methods.

J Chromatogr, 1991 Oct 4, 570(2), 309 - 20
Direct determination of codeine, norcodeine, morphine and normorphine with their corresponding O-glucuronide conjugates by high-performance liquid chromatography with electrochemical detection; Verwey-van Wissen CP et al.; A high-performance liquid chromatographic method has been developed for the detection, separation and measurement of codeine and its metabolites norcodeine, morphine and normorphine, with their glucuronide conjugates . The glucuronidase Escherichia coli type VIIA hydrolyses codeine-6-glucuronide completely and is used for the construction of the calibration curves of codeine-6-glucuronide . Enzymic hydrolysis of codeine-6-glucuronide depends on the specific activity of the glucuronidase applied . Examples are shown of a volunteer who is able to form morphine from codeine and one who is unable to do so.

Diabetologia, 1991 Oct, 34(10), 687 - 94
Cardiovascular sequelae of endotoxin shock in diabetic dogs; Law WR et al.; Diabetic patients exhibit a higher incidence of post-surgical sepsis, as well as a higher rate of mortality from sepsis, than their non-diabetic counterparts . This may be a result of cardiovascular deterioration associated with diabetes mellitus . This study was designed to characterize the cardiovascular sequelae associated with endotoxin shock in a canine model of diabetes . Diabetes was induced with alloxan (50 mg/kg) and streptozotocin (30 mg/kg) in dogs weighing 19-25 kg . Thirty days later, anaesthetized dogs were instrumented to obtain blood pressures, blood samples, left ventricular chamber diameter, circumflex arterial blood flow, and aortic blood flow . Metabolic parameters were calculated according to the Fick principle, and myocardial inotropic state assessed with the end-systolic pressure-diameter relationship . After stable baseline measurements, Escherichia coli endotoxin (1 mg/kg) was infused over 1 h, and measurements were obtained every 30 min . After endotoxin administration diabetic dogs became more hypotensive than the non-diabetic dogs . Cardiac performance parameters were also depressed to a greater degree . These changes could be attributed to depressions in vascular resistance and myocardial inotropic state in diabetic dogs . Cardiac dysfunction occurred in association with a relative decrease in the supply to demand ratio for oxygen in the diabetic dogs, suggesting functional ischemia . Data indicating a decrease in pre-load and vascular resistance in the diabetic group suggest a greater degree of vascular collapse, vascular pooling, or extravasation of fluid than occurred in the non-diabetic group . These data support the hypothesis that the cardiovascular system of diabetic subjects cannot tolerate a septic insult as well as their non-diabetic counterparts.

Poult Sci, 1991 Oct, 70(10), 2094 - 101
Comparison of macrophage function in several commercial broiler genetic lines; Qureshi MA et al.; The present study was conducted to establish baseline profiles of various macrophage functions in four commercial broiler genetic lines designated as Lines 1, 2, 3, and 4 . All experiments were carried out between 2 to 3 wk of age . Total numbers of peritoneal exudate cells per bird collected 42 h after a single i.p . injection of Sephadex-G50 were comparable among the lines . Line 1 produced fewer macrophages along with reduced phagocytosis of opsonized SRBC, whereas Line 4 macrophages were depressed in unopsonized SRBC phagocytosis . Macrophages from Lines 2 and 4 killed internalized Escherichia coli earlier than macrophages from Lines 1 and 3 . Supernatants from lipopolysaccharide-treated macrophages from Lines 1 and 2 exhibited significantly higher cytolytic activity against LSCC-RP9 tumor cells when compared with supernatants from Line 3 and 4 macrophages . The current study demonstrates genetic variation among the four broiler lines for mononuclear phagocytic system functions.

Mol Gen Genet, 1991 Oct, 229(3), 413 - 20
Immunological identification of yeast SCO1 protein as a component of the inner mitochondrial membrane; Buchwald P et al.; The SCO1 gene of Saccharomyces cerevisiae encodes a 30 kDa protein which is specifically required for a post-translational step in the accumulation of subunits 1 and 2 of cytochrome c oxidase (COXI and COX-II) . Antibodies directed against a beta-Gal::SCO1 fusion protein detect SCO1 in the mitochondrial fraction of yeast cells . The SCO1 protein is an integral membrane protein as shown by its resistance to alkaline extraction and by its solubilization properties upon treatment with detergents . Based on the results obtained by isopycnic sucrose gradient centrifugation and by digitonin treatment of mitochondria, SCO1 is a component of the inner mitochondrial membrane . Membrane localization is mediated by a stretch of 17 hydrophobic amino acids in the amino-terminal region of the protein . A truncated SCO1 derivative lacking this segment, is no longer bound to the membrane and simultaneously loses its biological function . The observation that membrane localization of SCO1 is affected in mitochondria of a rho0 strain, hints at the possible involvement of mitochondrially coded components in ensuring proper membrane insertion.

J Med Microbiol, 1991 Oct, 35(4), 214 - 8
Nucleotide sequence of dihydrofolate reductase type VI; Wylie BA et al.; The complete sequence of the type VI dihydrofolate reductase (DHFR) gene from plasmid pUK672 was determined . The structural gene coded for a polypeptide of 157 amino acids which had a deduced mol . wt of 17,424 . Comparison with amino-acid sequences of the type I, type V and Escherichia coli K12 chromosomal DHFRs showed that there was 63%, 61% and 31% homology respectively . Putative RNA polymerase and ribosomal binding sites were identified proximal to the initiation codon and a feature consistent with transcription termination was present distal to the coding region . Sodium dodecyl sulphate-polyacrylamide gel electrophoresis showed that the enzyme had a subunit mol . wt of 17,500.

J Clin Microbiol, 1991 Oct, 29(10), 2250 - 2
Immunodot detection of Escherichia coli heat-stable enterotoxin b by using enhanced chemiluminescence reaction; Lortie LA et al.; An indirect immunodot assay with rabbit antibodies raised against purified heat-stable enterotoxin type b (STb) and with a Western blotting (immunoblotting) detection system (ECL; Amersham International plc, Amersham, United Kingdom) was developed for the detection of STb toxin . Culture supernatants of 62 Escherichia coli isolates from pigs with diarrhea were blotted onto nitrocellulose and incubated with anti-STb serum . The chemiluminescence produced by the action of horseradish peroxidase with luminol and H2O2 was recorded by exposure of X-ray film . Over 90% correlation was observed between the rat or pig intestinal ligated loop assay and a radioactive DNA probe and the ECL immunodot assay for the detection of STb . In addition, using this new and sensitive technique, we could detect STb in the feces of a newborn pig inoculated with an STb-producing E . coli strain . Detection of STb-producing E . coli in pigs with diarrhea will be greatly facilitated by the use of this convenient and rapid diagnostic assay.

Exp Mol Pathol, 1991 Oct, 55(2), 196 - 202
Endotoxin hepatotoxicity augmented by ethanol; Shibayama Y et al.; To determine whether alcohol increases endotoxin hepatotoxicity, we administered ethanol (4.8 g/kg body wt in 4 ml of water) to rats through a gastric tube, then immediately injected endotoxin (2, 2.5, or 3 mg/kg body wt) . In the rats pretreated with ethanol, the injection of 2 mg/kg body wt of endotoxin induced a slight rise of serum transaminase . However, when 2.5 mg/kg body wt of endotoxin was given, there were no significant histopathological or biochemical differences between the rats pretreated with ethanol and those pretreated with water . Moreover, there was no significant difference in mortality rates between the rats pretreated with ethanol and the controls when 3 mg/kg body wt (LD50) of endotoxin was injected . These results suggest that acute administration of alcohol enhances endotoxin hepatotoxicity when the dose of endotoxin is small, but that the effect of alcohol is masked when larger doses of endotoxin are given.

Carcinogenesis, 1991 Oct, 12(10), 1971 - 3
Release of N2,3-ethanoguanine from haloethylnitrosourea-treated DNA by Escherichia coli 3-methyladenine DNA glycosylase II; Habraken Y et al.; 3-Methyladenine DNA glycosylase II (Gly II), purified from Escherichia coli cells which carry the plasmid PYN1000, has been tested for its ability to release N2,3-ethanoguanine from DNA modified by the antitumor agent N-{2-chloroethyl-1,2-14C}-N'-cyclohexyl-N-nitrosourea ({14C}CCNU) . Gly II has been shown to release N2,3-ethanoguanine in a protein- and time-dependent manner at a rate that exceeds the rate at which this enzyme releases other alkylated bases from {14C}CCNU-modified DNA . This finding widens the known substrate specificity for Gly II to include a modified base which bears an exocyclic ring structure, a class of modifications caused by a variety of chemical carcinogens.

Am J Physiol, 1991 Oct, 261(4 Pt 2), H1317 - 23
Neutrophil passage through isolated perfused rabbit lungs; Schutte H et al.; Kinetics of polymorphonuclear leukocyte (PMN) lung passage were investigated in ex vivo isolated and ventilated left rabbit lungs, perfused with buffer solution at physiological flow rate . 111In-labeled PMNs of rabbit or human origin were injected into the pulmonary artery, and the first fraction of PMNs that rapidly passed the lung together with coinjected erythrocytes, was collected separately for external radioactivity counting . Washout of initially retained PMNs from the lung was monitored by use of a sodium-iodide detector . Recirculation of cells was avoided by insertion of a filter in the perfusion circuit . A fraction of 16.6 +/- 1.3% of rabbit PMNs rapidly passed the lung vasculature, followed by an exponential washout of initially retained PMNs {half-time (t50) of lung transit 8.1 +/- 0.6 min} . Slightly higher t50 (12.2 +/- 1.0 min) was obtained upon use of human PMNs . Reduction in flow by 50% caused a marked prolongation of PMN transit (t50 = 27.8 +/- 5.1 min), whereas increase in flow to 150% only insignificantly decreased t50 . Rise in pulmonary venous pressure to 5 and 8 mmHg caused retardation of PMN lung transit (t50 = 15.3 +/- 0.6 and 31.6 +/- 3.6 min) . Preincubation of PMNs with 2 ng/ml endotoxin for 1 h induced marked delay in PMN washout (t50 = 26.1 +/- 2.8 min) . In conclusion, single-pass PMN kinetics in isolated lungs correspond to in vivo studies previously reported, thus allowing elucidation of PMN-endothelial interactions in an intact lung vasculature under standardized conditions.

Proc Natl Acad Sci U S A, 1991 Oct 1, 88(19), 8769 - 73
Engineering the substrate specificity of glutathione reductase toward that of trypanothione reduction; Henderson GB et al.; Glutathione reductase (EC 1.6.4.2; CAS registry number 9001-48-3) and trypanothione reductase (CAS registry number 102210-35-5), which are related flavoprotein disulfide oxidoreductases, have marked specificities for glutathione and trypanothione, respectively . A combination of primary sequence alignments and molecular modeling, together with the high-resolution crystal structure of human glutathione reductase, identified certain residues as potentially being responsible for substrate discrimination . Site-directed mutagenesis of Escherichia coli glutathione reductase was used to test these predictions . The mutation of Asn-21 to Arg demonstrated that this single change was insufficient to generate the greater discrimination against trypanothione shown by human glutathione reductase compared with the E . coli enzyme . However, the mutation of Ala-18, Asn-21, and Arg-22 to the amino acid residues (Glu, Trp, and Asn, respectively) in corresponding positions in Trypanosoma congolense trypanothione reductase confirmed that this region of polypeptide chain is intimately involved in substrate recognition . It led to a mutant form of E . coli glutathione reductase that possessed essentially no activity with glutathione but that was able to catalyze trypanothione reduction with a kcat/Km value that was 10% of that measured for natural trypanothione reductases . These results should be of considerable importance in the design of trypanocidal drugs targeted at the differences between glutathione and trypanothione metabolism in trypanosomatids and their hosts.

Proc Natl Acad Sci U S A, 1991 Oct 1, 88(19), 8646 - 50
A soluble, single-chain T-cell receptor fragment endowed with antigen-combining properties; Novotny J et al.; A strategy for the production of small, soluble, single-chain T-cell receptor (scTCR) fragments that carry an intact TCR antigen-combining site is presented . The rationale is based on structural similarity between TCR and antibody molecules and use of computer modeling methods to derive a model structure of a human scTCR variable (V)-domain dimer . A gene encoding the RFL3.8 TCR protein, specific for the hapten fluorescein in the context of major histocompatibility complex class II and composed of one V alpha and one V beta domain joined via a flexible peptide linker, was assembled in an Escherichia coli plasmid . Subsequently, the protein was produced in a bacterial expression system, purified, refolded, and found to be poorly soluble at neutral pH in aqueous buffers . An inspection of the computer-generated V alpha-V beta domain model showed several surface exposed hydrophobic residues . When these were replaced by water-soluble side chains via site-directed mutagenesis of the corresponding gene, a soluble protein resulted and was shown to have antigen-binding properties equivalent to those of the intact TCR of the RFL3.8 T-cell clone . These results demonstrate the feasibility of obtaining TCR fragments endowed with antigen-combining properties by protein engineering in E . coli.

Proc Natl Acad Sci U S A, 1991 Oct 1, 88(19), 8460 - 4
A human nuclear uracil DNA glycosylase is the 37-kDa subunit of glyceraldehyde-3-phosphate dehydrogenase; Meyer-Siegler K et al.; We have isolated and characterized a plasmid (pChug 20.1) that contains the cDNA of a nuclear uracil DNA glycosylase (UDG) gene isolated from normal human placenta . This cDNA directed the synthesis of a fusion protein (Mr 66,000) that exhibited UDG activity . The enzymatic activity was specific for a uracil-containing polynucleotide substrate and was inhibited by a glycosylase antibody or a beta-galactosidase antibody . Sequence analysis demonstrated an open reading frame that encoded a protein of 335 amino acids of calculated Mr 36,050 and pI 8.7, corresponding to the Mr 37,000 and pI 8.1 of purified human placental UDG . No homology was seen between this cDNA and the UDG of herpes simplex virus, Escherichia coli, and yeast; nor was there homology with the putative human mitochondrial UDG cDNA or with a second human nuclear UDG cDNA . Surprisingly, a search of the GenBank data base revealed that the cDNA of UDG was completely homologous with the 37-kDa subunit of human glyceraldehyde-3-phosphate dehydrogenase . Human erythrocyte glyceraldehyde-3-phosphate dehydrogenase was obtained commercially in its tetrameric form . A 37-kDa subunit was isolated from it and shown to possess UDG activity equivalent to that seen for the purified human placental UDG . The multiple functions of this 37-kDa protein as here and previously reported indicate that it possesses a series of activities, depending on its oligomeric state . Accordingly, mutation(s) in the gene of this multifunctional protein may conceivably result in the diverse cellular phenotypes of Bloom syndrome.

Proc Natl Acad Sci U S A, 1991 Oct 1, 88(19), 8450 - 4
Catalytic properties of an Escherichia coli formate dehydrogenase mutant in which sulfur replaces selenium; Axley MJ et al.; Formate dehydrogenase H of Escherichia coli contains selenocysteine as an integral amino acid . We have purified a mutant form of the enzyme in which cysteine replaces selenocysteine . To elucidate the essential catalytic role of selenocysteine, kinetic and physical properties of the mutant enzyme were compared with those of wild type . The mutant and wild-type enzymes displayed similar pH dependencies with respect to activity and stability, although the mutant enzyme profiles were slightly shifted to more alkaline pH . Both enzymes were inactivated by reaction with iodoacetamide; however, addition of the substrate, formate, was necessary to render the enzymes susceptible to alkylation . Alkylation-induced inactivation was highly dependent on pH, with each enzyme displaying an alkylation vs . pH profile suggestive of an essential selenol or thiol . Both forms of the enzyme use a ping-pong bi-bi kinetic mechanism . The mutant enzyme binds formate with greater affinity than does the wild-type enzyme, as shown by reduced values of Km and Kd . However, the mutant enzyme has a turnover number which is more than two orders of magnitude lower than that of the native selenium-containing enzyme . The lower turnover number results from a diminished reaction rate for the initial step of the overall reaction, as found in kinetic analyses that employed the alternative substrate deuterioformate . These results indicate that the selenium of formate dehydrogenase H is directly involved in formate oxidation . The observed differences in kinetic properties may help explain the evolutionary conservation of selenocysteine at the enzyme's active site.

Proc Natl Acad Sci U S A, 1991 Oct 1, 88(19), 8283 - 7
Mechanism of synergistic transcriptional transactivation by the human glucocorticoid receptor; Wright AP et al.; Induction of transcription from a promoter with two upstream glucocorticoid response elements is 10- to 20-fold greater than that from a similar promoter with only one response element . We have shown that interactions involving the major transactivation domain of the glucocorticoid receptor (tau 1) are the sole determinant of such synergistic transactivation by the receptor . The other transactivation domain of the receptor (tau 2) did not mediate synergistic transactivation, and therefore the ability to synergize is operationally distinct from the transactivation function per se . The level of synergistic transactivation observed in vivo can be accounted for by the level of cooperative DNA binding seen in vitro for a glucocorticoid receptor derivative containing only the tau 1 and DNA-binding domains . Cooperative DNA binding was also observed using a tau 1-DNA-binding domain protein, which was expressed in Escherichia coli and extensively purified . Therefore, it is likely that direct protein-protein interactions between tau 1 domains mediate the cooperative DNA binding . The role of cooperative DNA binding for synergistic transactivation in vivo is discussed in relation to other possible mechanisms.

Oncogene, 1991 Oct, 6(10), 1899 - 902
Arg encodes a widely expressed 145 kDa protein-tyrosine kinase; Perego R et al.; Arg encodes a protein highly related to the c-abl gene product with regard to overall structural architecture as well as the amino acid sequences of their tyrosine kinase, and src-homologous 2 and 3 domains . The two genes form a distinct subfamily of non-receptor tyrosine kinases and share a common homolog in Drosophila . In this study we characterized the arg protein product by expression of its coding sequence in bacteria . The recombinant arg protein was detected in bacterial lysates by immunoblotting and exhibited a molecular mass of 145 kDa . Phosphoamino acid analysis of the arg gene product following an immune complex autokinase reaction revealed tyrosine phosphorylation and established that it possesses tyrosine kinase activity . High-titer antibody capable of detecting the cellular arg gene product was generated by expressing a carboxy-terminal segment of arg in bacteria and using the recombinant protein as an immunogen . The arg gene product was identified in cultured human cells as a 145 kDa protein that exhibited autokinase activity . Analysis of arg expression in murine tissues revealed that arg, like c-abl, is widely expressed, further extending the similarities between the two genes, and suggesting that arg probably functions in signaling pathways fundamental to many cell types.

Mol Cell Biol, 1991 Oct, 11(10), 5090 - 100
Activation of skeletal alpha-actin gene transcription: the cooperative formation of serum response factor-binding complexes over positive cis-acting promoter serum response elements displaces a negative-acting nuclear factor enriched in replicating myoblasts and nonmyogenic cells; Lee TC et al.; Three upstream CBAR cis-acting promoter elements, containing the inner core CC(A/T)6GG of the serum response element (SRE), are required for myogenic cell type-restricted expression of the avian skeletal alpha-actin gene (K.L . Chow and R.J . Schwartz, Mol . Cell . Biol . 10:528-538, 1990) . These actin SRE elements display differential binding properties with two distinct nuclear proteins, serum response factor (SRF) and another factor described here as F-ACT1 . SRF is able to bind to all actin SREs with various affinities . This multisite interaction is marked by cooperative binding events in that the two high-affinity proximal and distal SREs facilitate the weak central-site interaction with SRF, leading to the formation of a higher-order SRF-promoter complex . Functional analyses reveal that undisrupted multiple SRF-DNA interactions are absolutely essential for promoter activity in myogenic cells . F-ACT1, present at higher levels in nonmyogenic cells and replicating myoblasts than in myotubes, binds solely to the proximal SRE, and its binding is mutually exclusive with that of SRF owing to their overlapping base contacts . The cooperative promoter binding by SRF, however, can effectively displace prebound F-ACT1 . In addition, an intact F-ACT1 binding site acts as a negative promoter element by restricting developmentally timed expression in myoblasts . F-ACT1 may therefore act as a repressor of skeletal alpha-actin gene transcription . This interplay between F-ACT1 and SRF may constitute a developmental as well as a physiologically regulated mechanism which modulates sarcomeric actin gene expression.

Mol Cell Biol, 1991 Oct, 11(10), 5005 - 15
Differential DNA binding by monomeric, homodimeric, and potentially heteromeric forms of the thyroid hormone receptor; Lazar MA et al.; Binding of the thyroid hormone receptor (TR) to thyroid hormone-responsive elements (TREs) is crucial for regulation of gene expression by thyroid hormone . The TR binds to each half-site of a palindromic TRE separately, as a monomer, or simultaneously, as a homodimer . In addition, the TR monomer interacts with a 42-kDa protein that may be responsible for an increase in the apparent size and stability of the TR-TRE complex after incubation with liver nuclear extract . The multiple DNA-binding forms of the TR contact the TRE differently but compete for binding in a dynamic equilibrium which is highly dependent on the relative concentrations of TR and nuclear protein . Thus, protein-protein interactions are likely to determine the context in which the TR binds to target genes and regulates the transcriptional response to thyroid hormone.

Mol Gen Genet, 1991 Oct, 229(2), 319 - 23
Molecular analysis of the aidD6::Mu d1 (bla lac) fusion mutation of Escherichia coli K12; Volkert MR et al.; In this report we present genetic and biochemical evidence indicating that the aidD6::Mu d1 (bla lac) fusion is an insertion of Mu d1 (bla lac) into the alkB coding sequence . We describe the phenotypic effects resulting from this mutation and compare them with the effects of alkB22, alkA and ada mutations . We also constructed an alkA alkB double mutant and compared its phenotype with that of the single mutant strains . The observation that the methyl methanesulfonate (MMS) and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) resistance of the double mutant is approximately at the level predicted from the additive sensitivity of each of the single mutants suggests that these two gene products act in different pathways of DNA repair.

Mol Gen Genet, 1991 Oct, 229(2), 301 - 6
Construction of a fusion gene comprising the Taka-amylase A promoter and the Escherichia coli beta-glucuronidase gene and analysis of its expression in Aspergillus oryzae; Tada S et al.; Northern blot analysis of glucose-grown and starch-grown mycelia of Aspergillus oryzae RIB40 was conducted using the cloned Taka-amylase A (TAA) gene as a probe . The amount of mRNA homologous to the TAA gene was increased when this fungus was grown with starch as a sole carbon source . In order to analyze the induction mechanism, we inserted the Escherichia coli uidA gene encoding beta-glucuronidase (GUS) down-stream of the TAA promoter and introduced the resultant fusion gene into the A . oryzae genome . Production of a functional GUS protein was induced by starch, but not by glucose . When the effects of various sugars on expression of the fusion gene were examined, the results suggested that the expression of the fusion gene was under control of the TAA gene promoter.

Mol Gen Genet, 1991 Oct, 229(2), 238 - 44
A 2.6 kb intron separates the signal peptide coding sequence of an anther-specific protein from the rest of the gene in sunflower; Domon C et al.; We have isolated and sequenced an anther-specific gene from sunflower which encodes an 800-nucleotide transcript detectable in the peripheral anther cells . It contains an intron of 2615 bp, which separates the first exon (77 bp) coding for a putative signal peptide of 21 amino acids, from the second exon (563 bp) coding for a 100 amino acid polypeptide . The 5' and 3' untranslated regions comprise respectively 13 and 264 bp . The SF2 gene is present in the sunflower genome in several copies, all or most of which contain a closely related intron.

Mol Gen Genet, 1991 Oct, 229(2), 181 - 8
The tobacco transcription activator TGA1a binds to a sequence in the 5' upstream region of a gene encoding a TGA1a-related protein; Fromm H et al.; We have isolated and characterized a tobacco gene, designated G13, encoding a leucine zipper DNA-binding protein related to the transcription activator TGA1a . The G13 coding region is divided into eight exons and the amino acid sequence of the encoded protein (PG13) shows 76% homology to TGA1a . Their putative DNA-contacting regions (basic domains) are identical and they both bind to the same target sequences in vitro . By contrast, some differences are apparent between these proteins at the carboxyl end of the dimerization region (leucine zipper) . The basic and leucine zipper domains are encoded on separate small exons . Analysis by DNAse I footprinting, gel shift and competition experiments revealed that TGA1a and PG13 synthesized in Escherichia coli, and the tobacco nuclear factor ASF-1 all bind to at least one site in the 5' upstream region of G13 . The presence of a TGA1a binding site in the upstream region of a TGA1a-related gene suggests that transcription of this gene is autoregulated.

Mol Gen Genet, 1991 Oct, 229(2), 175 - 80
Studies on the alteration of chromosome copy number and cell division potential in a dnaA mutant of Escherichia coli; Fralick JA; The dnaA167 mutant of Escherichia coli, N167, maintains, on the average, two replicating chromosomes per cell at the permissive growth temperature of 30 degrees C and only one per cell at the higher permissive growth temperature of 38 degrees C . When the growth temperature of this mutant is changed from 30 degrees to 38 degrees C the cells rapidly readjust their chromosome copy number from two to one . I have examined the kinetics of this transition with reference to DNA replication and cell division . My results indicate that this mutant uncouples cell division from chromosome duplication to achieve the appropriate copy number, suggesting that the dnaA gene product may be involved in the coordination between these two cellular events.

J Gen Virol, 1991 Oct, 72 ( Pt 10), 2445 - 56
Fine mapping of canine parvovirus B cell epitopes; Lopez de Turiso JA et al.; In this report we describe the topological mapping of neutralizing domains of canine parvovirus (CPV) . We obtained 11 CPV-specific monoclonal antibodies (MAbs), six of which are neutralizing . The reactivities were as determined by ELISA and Western blot (immunoblot) analysis . VP2, the most abundant protein of the CPV capsid, seemed to contain all the neutralization sites . Also, an almost full-length genomic clone of CPV was constructed in the bacterial plasmid pUC18 to enable expression of CPV proteins . All the neutralizing MAbs recognized recombinant VP2 when it was expressed as a free protein in Escherichia coli but not when expressed as a fusion protein with glutathione-S-transferase . When two large fragments containing about 85% and 67% of the C terminus of VP2 were expressed, no neutralization sites were detected . When fusion proteins containing the N terminus were expressed, two linear determinants were mapped, one between residues 1 to 10 of VP2, and the other between amino acids 11 and 23 . The peptide 11 GQPAVRNERATGS 23, recognized by MAb 3C9, was synthesized chemically and checked for immunogenicity, not being able to induce neutralizing activity . Although the antibody response in rabbits to all the fusion proteins was uniformly high, the anti-CPV response was very variable . Protein from pCPVEx11, which contains a T cell epitope (peptide PKIFINLAKKKKAG) present in the VP1-specific region as well as the B cell epitopes, seemed to be the most effective in inducing virus neutralization.

J Immunol, 1991 Oct 1, 147(7), 2229 - 33
Production and characterization of mouse monoclonal antibodies against human monocyte chemoattractant protein-1; Yoshimura T et al.; We developed five different hybridoma cell lines that produced mAb against human monocyte chemoattractant protein-1 (MCP-1) . The subclass of all five antibodies was IgG1 . All five mAb formed complexes with metabolically labeled MCP-1 that could be demonstrated by immunoprecipitation . The antibodies were specific for MCP-1 . They did not cross-react by immunoprecipitation with structurally related host defense cytokines present in metabolically labeled PHA- or LPS-stimulated mononuclear cell culture fluids, nor did they cross-react in a direct ELISA with neutrophil attractant/activation protein-1, with crude platelet lysate proteins, or with pure platelet proteins that have amino acids sequences similar to that of MCP-1 . The mAb also reacted with rMCP-1 expressed in Escherichia coli, suggesting that they recognize protein structure rather than the glycosylated portion of human MCP-1 . When the mAb were mixed with MCP-1, the monocyte chemotactic response to MCP-1 was inhibited . A sandwich ELISA was developed to detect MCP-1 in biologic fluids containing relatively high concentrations of other proteins . The sensitivity was 300 pg/ml, or 30 pg/ELISA well . An anti-MCP-1 mAb column was used in an improved method of MCP-1 purification . Approximately 240 micrograms of MCP-1 were purified from 5 liters of FCS-containing U-105MG cell culture supernatant . The yield was at least 60% . In addition to two forms of MCP-1 reported previously by us, two more forms of MCP-1 were found in a mixture of culture supernatants of PHA- and LPS-stimulated human PBMC.

J Immunol, 1991 Oct 1, 147(7), 2170 - 4
Human eosinophil cytotoxicity-enhancing factor . II . Multiple forms synthesized by U937 cells and their relationship to thioredoxin/adult T cell leukemia-derived factor; Balcewicz-Sablinska MK et al.; Recently, our laboratory reported the purification and partial amino acid sequence of a 10-kDa eosinophil cytotoxicity-enhancing factor (ECEF) polypeptide from the U937 cell source . This cytokine enhanced human eosinophil antibody-dependent cytotoxic function by greater than 200% and was half-maximally effective at a concentration of approximately 1 ng/ml . In this study, we describe the conditions required for ECEF synthesis and the use of rabbit antibody raised to 10-kDa ECEF to investigate the existence of related polypeptide species . Unstimulated U937 cells released an immunoreactive 14-kDA species . Cells stimulated with 7.5 micrograms/ml of LPS also released a 13-kDa species . Cells stimulated with 400 ng/ml of PMA also synthesized a 10-kDa species (equivalent in size to the form we had purified) . This 10-kDa species remained primarily cell associated, but detectable amounts were released into the supernatant by 48 h of culture . In washed cell pellets, the location of the 10-kDa species was found to be in the plasma membrane, externally oriented, as determined by FACS analysis, iodination with the membrane impermeable reagent 125I-sulfosuccinimidyl-3-(4-hydroxyphenyl) propionate, and by its removal with brief trypsin treatment . Partial amino acid sequence data suggested that the 14-, 13-, and 10-kDa species all share the same N-terminal . The 14- and 10-kDa ECEF species were recovered by electroelution from SDS-PAGE gels and tested for activity in the assay of eosinophil cytotoxic function . Because of the amino acid sequence similarities between the ECEF species and thioredoxin (TRX), rTRX (synthesized in Escherichia coli and purified) was also tested for activity . The 14-kDa ECEF and rTRX induced a slight, but consistent and statistically significant enhancement of eosinophil cytotoxic function . By comparison, lower doses of the 10-kDa ECEF induced a major increase in cytotoxic function . Thus the forms of ECEF differ in size, conditions required for synthesis, trafficking by the U937 cell after synthesis, and biologic activity . It is likely that these considerations bear on the involvement of ECEF in the pathophysiology of eosinophilia in vivo.

J Bacteriol, 1991 Oct, 173(20), 6643 - 6
Partitioning of a mini-F plasmid into anucleate cells of the mukB null mutant; Ezaki B et al.; The partition-proficient mini-F plasmid pXX325 was stably maintained in the mukB null mutant, which is defective in chromosome partitioning into the two daughter cells . In the null mutant, the plasmid was partitioned into both nucleate and anucleate daughter cells, independently of host chromosomes.

J Bacteriol, 1991 Oct, 173(20), 6612 - 7
Determination of the nick site at oriT of IncI1 plasmid R64: global similarity of oriT structures of IncI1 and IncP plasmids; Furuya N et al.; The nick site at the origin of transfer, oriT, of IncI1 plasmid R64 was determined . A site-specific and strand-specific cleavage of the phosphodiester bond was introduced during relaxation of the oriT plasmid DNA . Cleavage occurred between 2'-deoxyguanosine and thymidine residues, within the 44-bp oriT core sequence . The nick site was located 8 bp from the 17-bp repeat . A protein appeared to be associated with the cleaved DNA strand at the oriT site following relaxation . This protein was observed to bind to the 5' end of the cleaved strand, since the 5'-phosphate of the cleaved strand was resistant to the phosphate exchange reaction by polynucleotide kinase . In contrast, the 3' end of the cleaved strand appeared free, since it was susceptible to primer extension by DNA polymerase I . The global similarity of the oriT structures of IncI1 and IncP plasmids is discussed.

J Bacteriol, 1991 Oct, 173(20), 6446 - 52
The absence of branched-chain amino acid and growth rate control at the internal ilvEp promoter of the ilvGMEDA operon; Harms EH et al.; The question of whether the promoter ilvEp, located in the coding region of ilvM, the second structural gene in the ilvGMEDA operon, is subject to either amino acid- or growth rate-mediated regulation is examined . The experiments described here were performed with ilvEp-cat and ilvEp-lac fusions carried as single copies on the chromosome . The activity of the ilvEp promoter was found to respond neither to the availability of branched-chain amino acids nor to a wide range of growth rates between 35 to 390 min . In the absence of any known role for the products of the ilvGMEDA operon when repressing levels of branched-chain amino acids are present, there appears to be only a gratuitous role for the transcription at ilvEp.

J Bacteriol, 1991 Oct, 173(20), 6390 - 7
Carbon metabolism regulates expression of the pfl (pyruvate formate-lyase) gene in Escherichia coli; Rasmussen LJ et al.; The anaerobic expression of pfl is reduced both in a strain mutated in the pgi gene and in a pfkA pfkB double mutant strain when cells are grown in medium supplemented with glucose . When cells are grown in medium supplemented with either fructose or pyruvate, no reduction is observed in these strains . The amount of pyruvate in the cells may be responsible for the reduced expression of pfl in the strains mutated in the genes encoding the glycolytic enzymes . Because of the lowered oxygen concentration in the medium, the expression of pfl is induced when an exponentially growing culture enters the stationary phase . This induction is increased when the Casamino Acid concentration is raised 10-fold or when the medium is supplemented with NaCl . Superhelicity of DNA is decreased in a pgi mutant strain grown in medium supplemented with glucose . The superhelicity is also changed, but the opposite way, in a wild-type strain grown in medium supplemented with Casamino Acids at a high concentration or 0.3 M sodium chloride . Our data show that changes in superhelicity do not affect the aerobic expression of pfl but might be important for the anaerobic induction of pfl.

J Bacteriol, 1991 Oct, 173(20), 6347 - 54
Specific DNA binding of the TraM protein to the oriT region of plasmid R100; Abo T et al.; The product of the traM gene of plasmid R100 was purified as the TraM-collagen-beta-galactosidase fusion protein (TraM*) by using a beta-galactosidase-specific affinity column, and the TraM portion of TraM* (TraM') was separated by collagenolysis . Both the TraM* and TraM' proteins were found to bind specifically to a broad region preceding the traM gene . This region (designated sbm) was located within the nonconserved region in oriT among conjugative plasmids related to R100 . The region seems to contain four core binding sites (designated sbmA, sbmB, sbmC, and sbmD), each consisting of a similar number of nucleotides and including a homologous 15-bp sequence . This result, together with the observation that the TraM* protein was located in the membrane fraction, indicates the possibility that the TraM protein has a function in anchoring the oriT region of R100 at the sbm sites to the membrane pore, through which the single-stranded DNA is transferred to the recipient . sbmC and sbmD, each of which contained a characteristic inverted repeat sequence, overlapped with the promoter region for the traM gene . This suggests that the expression of the traM gene may be regulated by its own product.

J Bacteriol, 1991 Oct, 173(19), 6242 - 8
The Escherichia coli ts8 mutation is an allele of fda, the gene encoding fructose-1,6-diphosphate aldolase; Singer M et al.; The ts8 mutant of Escherichia coli has previously been shown to preferentially inhibit stable RNA synthesis when shifted to the nonpermissive temperature . We demonstrate in this report that the ts8 mutation is an allele of fda, the gene that encodes the glycolytic enzyme fructose-1,6-diphosphate aldolase . We show that ts8 and a second fda mutation, h8, isolated and characterized by A . Bock and F . C . Neidhardt, are dominant mutations and that they encode a thermolabile aldolase activity.

J Bacteriol, 1991 Oct, 173(19), 6192 - 8
Indirect stimulation of recombination in Escherichia coli K-12: dependence on recJ, uvrA, and uvrD; Schellhorn HE et al.; Direct and indirect UV-stimulated homologous genetic recombination was investigated in Escherichia coli strains blocked in several host-encoded functions . Genetic recombination was assayed by measuring beta-galactosidase produced after recombination between two noncomplementing lacZ ochre alleles . Both types of stimulation (direct and indirect) were found to be primarily RecF pathway-mediated . In a rec+ background, both direct and indirect stimulation were found to be dependent on uvrD (coding for helicase II) . In a recB21 sbcB15 background, direct and indirect stimulation were uvrD dependent only when the strain was additionally deficient in the UvrABC excision repair pathway . Indirect but not direct stimulation was also dependent on recJ (coding for a 5'-to-3' exonuclease specific for single-stranded DNA) regardless of sbcA or sbcB configuration . The methyl-directed mismatch repair system (mutSLH) also appeared to play an important role in stimulation . On the basis of these findings, we suggest that excision of UV-induced DNA damage is a prelude to UV-mediated stimulation of genetic recombination.

J Bacteriol, 1991 Oct, 173(19), 6184 - 91
Control of glucose metabolism by enzyme IIGlc of the phosphoenolpyruvate-dependent phosphotransferase system in Escherichia coli; Ruyter GJ et al.; The quantitative effects of variations in the amount of enzyme IIGlc of the phosphoenolpyruvate:glucose phosphotransferase system (PTS) on glucose metabolism in Escherichia coli were studied . The level of enzyme IIGlc could be adjusted in vivo to between 20 and 600% of the wild-type chromosomal level by using the expression vector pTSG11 . On this plasmid, expression of the structural gene for enzyme IIGlc, ptsG, is controlled by the tac promoter . As expected, the control coefficient (i.e., the relative increase in pathway flux, divided by the relative increase in amount of enzyme) of enzyme IIGlc decreased in magnitude if a more extensive pathway was considered . Thus, at the wild-type level of enzyme IIGlc activity, the control coefficient of this enzyme on the growth rate on glucose and on the rate of glucose oxidation was low, while the control coefficient on uptake and phosphorylation of methyl alpha-glucopyranoside (an enzyme IIGlc-specific, nonmetabolizable glucose analog) was relatively high (0.55 to 0.65) . The implications of our findings for PTS-mediated regulation, i.e., inhibition of growth on non-PTS compounds by glucose, are discussed.

J Bacteriol, 1991 Oct, 173(19), 6162 - 7
Selection, expression, and nucleotide sequencing of the glutamate dehydrogenase gene of Peptostreptococcus asaccharolyticus; Snedecor B et al.; The gene for the catabolic NAD-linked glutamate dehydrogenase of Peptostreptococcus asaccharolyticus was cloned by selection of Escherichia coli for complementation of a biosynthetic defect . Cloned fragments containing the gene and the P . asaccharolyticus transcription and translation signals are very highly expressed in E . coli . The nucleotide sequence of the cloned gene was determined . It codes for a polypeptide of 421 amino acids, the sequence of which is similar to those of the NADP-accepting glutamate dehydrogenases . The sequence similarity of this protein to the mammalian glutamate dehydrogenases, which accept both NADP and NAD, is greater than its similarity to the bacterial NADP-specific dehydrogenases, suggesting that this NAD-specific bacterial glutamate dehydrogenase and the NADP-specific bacterial dehydrogenases diverged separately from the line leading to the dual-specificity mammalian glutamate dehydrogenases.

J Bacteriol, 1991 Oct, 173(19), 6018 - 24
Molecular cloning and sequence of the thdF gene, which is involved in thiophene and furan oxidation by Escherichia coli; Alam KY et al.; Our previous work resulted in the isolation of mutant strains of Escherichia coli K-12 which were able to oxidize furans and thiophenes as a result of mutations in several novel genes . Some of the genes involved in thiophene oxidation were cloned into the multicopy vector pUC19 . The plasmid pKA10 carries a 3.8-kb chromosomal fragment which encodes a previously undiscovered gene involved in thiophene oxidation . Three proteins with approximate molecular sizes of 48, 30, and 26 kDa were overproduced by cells carrying pKA10 . Maxicell experiments and DNA sequence analysis indicated that the 48- and 26-kDa proteins are encoded by pKA10, whereas the 30-kDa protein is apparently chromosomally derived . A cassette specifying kanamycin resistance was inserted into various sites on pKA10 . An insertion which abolished the 48-kDa protein also abolished thiophene oxidation . Chromosomal integration of pKA10::Kan allowed us to locate the chromosomal insert of pKA10 at 84 min on the E . coli genetic map by transduction . Since no previously identified genes involved in thiophene metabolism are located in this region, we designated the gene for the 48-kDa protein as thdF . Sequencing of the 3.8-kb insert revealed an overlap of several hundred bases with the regulatory and structural regions of the tnaA gene, which is also located at 84 min . The 26-kDa protein is probably truncated tnaA protein . An open reading frame corresponding to the 48-kDa thdF protein was located next to the tnaA gene, which encodes tryptophanase, but was transcribed in the opposite sense.

J Bacteriol, 1991 Oct, 173(19), 5944 - 53
The tdh and serA operons of Escherichia coli: mutational analysis of the regulatory elements of leucine-responsive genes; Rex JH et al.; The tdh promoter of Escherichia coli is induced seven- to eightfold when cells are grown in the presence of exogenous leucine . A scheme was devised to select mutants that exhibited high constitutive expression of the tdh promoter . The mutations in these strains were shown to lie within a previously identified gene (lrp) that encodes Lrp (leucine-responsive regulatory protein) . By deletion analysis, the site of action of Lrp was localized to a 25-bp region between coordinates -69 and -44 of the tdh promoter . Disruption of a 12-bp presumptive target sequence found in this region of tdh resulted in constitutively derepressed expression from the tdh promoter . Similar DNA segments (consensus, TTTATTCtNaAT) were also identified in a number of other promoters, including each of the Lrp-regulated promoters whose nucleotide sequence is known . The sequence of the promoter region of serA, an Lrp-regulated gene, was determined . No Lrp consensus target sequence was present upstream of serA, suggesting that Lrp acts indirectly on the serA promoter . A previously described mutation in a leucine-responsive trans-acting factor, LivR (J . J . Anderson, S . C . Quay, and D . L . Oxender, J . Bacteriol . 126:80-90, 1976), resulted in constitutively repressed expression from the tdh promoter and constitutively induced expression from the serA promoter . The possibility that LivR and Lrp are allelic is discussed.

J Bacteriol, 1991 Oct, 173(19), 5935 - 43
Dimethyl sulfoxide reductase of Escherichia coli: an investigation of function and assembly by use of in vivo complementation; Sambasivarao D et al.; Dimethyl sulfoxide (DMSO) reductase of Escherichia coli is a membrane-bound, terminal anaerobic electron transfer enzyme composed of three nonidentical subunits . The DmsAB subunits are hydrophilic and are localized on the cytoplasmic side of the plasma membrane . DmsC is the membrane-intrinsic polypeptide, proposed to anchor the extrinsic subunits . We have constructed a number of strains lacking portions of the chromosomal dmsABC operon . These mutant strains failed to grow anaerobically on glycerol minimal medium with DMSO as the sole terminal oxidant but exhibited normal growth with nitrate, fumarate, and trimethylamine N-oxide, indicating that DMSO reductase is solely responsible for growth on DMSO . In vivo complementation of the mutant with plasmids carrying various dms genes, singly or in combination, revealed that the expression of all three subunits is essential to restore anaerobic growth . Expression of the DmsAB subunits without DmsC results in accumulation of the catalytically active dimer in the cytoplasm . The dimer is thermolabile and catalyzes the reduction of various substrates in the presence of artificial electron donors . Dimethylnaphthoquinol (an analog of the physiological electron donor menaquinone) was oxidized only by the holoenzyme . These results suggest that the membrane-intrinsic subunit is necessary for anchoring, stability, and electron transport . The C-terminal region of DmsB appears to interact with the anchor peptide and facilitates the membrane assembly of the catalytic dimer.

Invest Ophthalmol Vis Sci, 1991 Oct, 32(11), 2906 - 11
Sustained-release endotoxin . A model for inducing corneal neovascularization; Li WW et al.; The rabbit corneal pocket assay is one of the most frequently used systems for the study of angiogenesis . This model particularly is useful to identify stimulators of new blood vessel formation . More recently, however, interest in inhibitors of angiogenesis has grown, and several antiangiogenic agents have been identified . Investigations of angiogenesis inhibitors require a reliable model for the stimulation of neovascularization . One method was modified to produce corneal neovascularization by implanting into the rabbit cornea a sustained-release polymer containing endotoxin (Escherichia coli lipopolysaccharide) . The implant was prepared by mixing weighed quantities of endotoxin with ethylene vinyl acetate copolymer (Elvax) and forming 1-mm3 pellets containing 1%, 7.5%, 15%, 20%, 30%, and 40% (w/w) of endotoxin . Pure Elvax pellets were implanted as controls . Intrastromal corneal pockets were created in 92 eyes of male, albino New Zealand rabbits (n = 80), and sterilized endotoxin-copolymer implants were introduced . The growth rate of new vessels was measured by slit-lamp biomicroscopy . Endotoxin loads of 15% (n = 40) produced a strong neovascularization response with minimal stromal edema, with a mean growth rate of 0.21 +/- 0.12 mm/day . Loads of 1%, 7.5%, and 20% yielded 0.1 +/- 0.03 mm/day, 0.27 +/- 0.05 mm/day, 0.30 +/- 0.06 mm/day, respectively (n = 8, each group) . Higher loads (30% and 40%; n = 8, each group) produced intense neovascularization, accompanied by severe corneal edema that obscured accurate measurement of the vessels . Corneal pockets that did not contain polymer implants were avascular . When endotoxin-Elvax pellets were removed, the new vessels regressed within 2 weeks.(ABSTRACT TRUNCATED AT 250 WORDS)

Eur J Biochem, 1991 Oct 1, 201(1), 199 - 202
Crystal structure of the Tyr45Trp mutant of ribonuclease T1 in a complex with 2'-adenylic acid; Koellner G et al.; The recombinant Tyr45Trp mutant of Lys25-ribonuclease T1 was overexpressed and purified from an Escherichia coli strain . The mutant enzyme, which shows reduced activity towards GpA and increased activity towards pGpC, pApC and pUpC compared with wild-type RNase T1, was co-crystallized with 2'-adenylic acid by microdialysis . The space group is P212121 with unit cell dimenions a = 4.932(2), b = 4.661(2), c = 4.092(1) nm . The crystal structure was solved using the coordinates of the isomorphous complex of wild-type RNase T1 with 2'-AMP . The refinement was based on Fhkl of 7726 reflexions with Fo greater than or equal to 1 sigma (Fo) in the resolution range of 2.0-0.19 nm and converged with an R factor of 0.179 . The adenosine of 2'-AMP is not bound to the guanosine binding site, as could be expected from the mutation of Tyr45Trp, but is stacked on the Gly74 carbonyl group and the His92 imidazole group which form a subsite for substrate binding, as already observed in the wild-type 2'-AMP complex . The point mutation of Tyr45Trp does not perturb the backbone conformation and the Trp-indole side chain is in a comparable position to the phenolic Tyr45 of the wild-type enzyme.

EMBO J, 1991 Oct, 10(10), 3113 - 22
A single base change in the acceptor stem of tRNA(3Leu) confers resistance upon Escherichia coli to the calmodulin inhibitor, 48/80; Chen MX et al.; We have isolated several classes of spontaneous mutants resistant to the calmodulin inhibitor 48/80 which inhibits cell division in Escherichia coli K12 . Several mutants were also temperature sensitive for growth and this property was exploited to clone a DNA fragment from an E . coli gene library restoring growth at 42 degrees C and drug sensitivity at 30 degrees C in one such mutant . Physical and genetic mapping confirmed that both the mutation and the cloned DNA were located at 15.5 min on the E . coli chromosome at a locus designated feeB . By subcloning, complementation analysis and sequencing, the feeB locus was identified as identical to the tRNA(CUALEU) gene . When the mutant locus was isolated and sequenced, the mutation was confirmed as a single base change, C to A, at position 77 in the acceptor stem of this rare Leu tRNA . In other studies we obtained evidence that this mutant tRNA, recognizing the rare Leu codon, CUA, was defective in translation at both permissive and non-permissive temperatures . The feeB1 mutant is defective in division and shows a reduced growth rate at non-permissive temperature . We discuss the possibility that the mutant tRNA(3Leu) is limiting for the synthesis of a polypeptide(s), requiring several CUA codons for translation which in turn regulates in some way the level or activity of the drug target, a putative cell cycle protein.

EMBO J, 1991 Oct, 10(10), 3061 - 6
Expression of the replication protein Arp of phasyl shows dual regulation by an antisense promoter; Gielow A et al.; Phasyl is the smallest naturally occurring replicon found so far in Escherichia coli . It encodes a protein which is essential for autonomous replication (Arp) . The transcriptional start of the arp gene was mapped . A strong antisense promoter was found in close proximity to the arp promoter . The inactivation of this promoter led in cis to a strong increase of the transcription of the arp gene and to the inactivation of autonomous replication of phasyl . The product of the antisense promoter is an 83 nt RNA molecule, which is not translated . The antisense RNA led in trans to the inhibition of the translation of the arp mRNA, presumably mediated by the formation of an RNA-RNA hybrid in which the Shine-Dalgarno sequence of the arp transcript is sequestered . The expression of the arp gene is thus controlled by two negatively acting mechanisms: it is subject to a transcriptional control in cis exerted by the antisense promoter and to a translational control in trans mediated by the antisense RNA . Inactivation of one mechanism of control cannot be compensated by the remaining one.

EMBO J, 1991 Oct, 10(10), 2941 - 7
Poliovirus proteinase 3C converts an active form of transcription factor IIIC to an inactive form: a mechanism for inhibition of host cell polymerase III transcription by poliovirus; Clark ME et al.; In HeLa cells, RNA polymerase III (pol III)-mediated transcription is severely inhibited by poliovirus infection . This is due primarily to a reduction in the transcriptional activity of TFIIIC, a transcription factor which binds in a sequence specific manner to the internal promoter of pol III genes . Using gel retardation assays, we have shown previously that inhibition of pol III transcription by poliovirus is correlated with disappearance of a transcriptionally active form of TFIIIC (complex I) concomitant with the appearance of a faster mobility, transcriptionally inactive form of TFIIIC (complex III) . We show here that a poliovirus with a point mutation in the proteinase 3C (3Cpro) region failed to produce complex III and is limited in its ability to inhibit pol III transcription compared with the wild-type virus . Incubation of purified 3Cpro, expressed in Escherichia coli, with transcriptionally active TFIIIC (complex I) in vitro resulted in generation of the transcriptionally inactive complex III form of TFIIIC . In an in vitro transcription assay, treatment of the complex I form of TFIIIC with 3Cpro almost completely inhibited pol III transcription . Finally expression of the 3Cpro gene in transfected HeLa cells resulted in significant inhibition of pol III-mediated transcription . The results presented here suggest that proteolysis of the transcriptionally active form of TFIIIC by poliovirus 3Cpro is a mechanism by which poliovirus inhibits host cell RNA pol III transcription.

EMBO J, 1991 Oct, 10(10), 2773 - 82
Decoding signals for membrane protein assembly using alkaline phosphatase fusions; McGovern K et al.; We have used genetic methods to investigate the role of the different domains of a bacterial cytoplasmic membrane protein, MalF, in determining its topology . This was done by analyzing the effects of MalF topology of deleting various domains of the protein using MalF-alkaline phosphatase fusion proteins . Our results show that the cytoplasmic domains of the protein are the pre-eminent topogenic signals . These domains contain information that determines their cytoplasmic location and, thus, the orientation of the membrane spanning segments surrounding them . Periplasmic domains do not appear to have equivalent information specifying their location and membrane spanning segments do not contain information defining their orientation in the membrane . The strength of cytoplasmic domains as topogenic signals varies, correlated with the density of positively charged amino acids within them.

Endocrinology, 1991 Oct, 129(4), 1779 - 83
Human growth hormone variant produces insulin-like and lipolytic responses in rat adipose tissue; Goodman HM et al.; Genes for normal human pituitary GH (hGH-N) and the GH variant (hGH-V) were expressed in stably transfected mouse mammary cells . The biological properties of hGH-N and hGH-V secreted into the medium were examined using rat adipocytes or epididymal fat segments . Methionyl-hGH produced in E . coli served as a reference standard . The three preparations were quite similar in their ability to bind specifically to intact fat cells and were virtually indistinguishable in their ability to increase glucose oxidation (an insulin-like response), induce refractoriness to insulin-like stimulation, and induce lipolysis in the presence of glucocorticoid . We conclude that placentally expressed hGH-V has a spectrum of metabolic activity comparable to pituitary hGH-N and may contribute to regulation of carbohydrate and lipid metabolism during pregnancy.

Crit Care Med, 1991 Oct, 19(10), 1294 - 302
Inhibition of thromboxane synthesis reduces endotoxin-induced right ventricular failure in sheep; Redl G et al.; BACKGROUND AND METHODS: There is a marked decrease of the right ventricular ejection fraction after the administration of a bolus of endotoxin to sheep . This hemodynamic response may be the result of thromboxane-mediated pulmonary hypertension . Right ventricular function was studied in an ovine model after the administration of endotoxin (1 microgram/kg Escherichia coli) with and without pretreatment with OKY-046, a selective thromboxane synthetase inhibitor . RESULTS: OKY-046 attenuated the endotoxin-induced increase in pulmonary arterial pressure and prevented the early decreases in right ventricular ejection fraction and cardiac output . However, thromboxane synthetase inhibition failed to prevent endotoxin-induced hypoxemia . The marked increase in plasma thromboxane concentrations, which is usually seen after the administration of endotoxin, was prevented by pretreating the animals with OKY-046 . On the other hand, increased plasma prostacyclin concentrations were observed in sheep treated with the thromboxane synthetase inhibitor . CONCLUSION: This series of experiments shows that the early endotoxin-induced decrease in right ventricular ejection fraction can be alleviated by the application of OKY-046.

Clin Pharmacol Ther, 1991 Oct, 50(4), 429 - 36
Dose-ranging study of the novel recombinant plasminogen activator BM 06.022 in healthy volunteers; Martin U et al.; The novel recombinant plasminogen activator BM 06.022 consists of the kringle 2 and protease domains of human tissue-type plasminogen activator and is unglycosylated because of its expression in Escherichia coli cells . Pharmacokinetics for activity and hemostatic effects of BM 06.022 were studied in 18 healthy male volunteers after an intravenous bolus injection over 2 minutes . BM 06.022 was administered successively at doses of 0.1125, 0.55, 2.2, 3.3, 4.4, and 5.5 MU to three volunteers . Plasma fibrinogen was unchanged; effects of BM 06.022 were observed on plasminogen only at higher doses, and dose-dependent effects were seen on alpha 2-antiplasmin and fibrin D-dimers . The concentration of plasminogen and alpha 2-antiplasmin was 87% +/- 3% and 79% +/- 3%, respectively, of baseline 2 hours after injection of 5.5 MU of BM 06.022 . Fibrin D-dimers were highest with 1147 +/- 380 ng/ml at 5.5 MU of BM 06.022 . The area under the activity concentration-time curve (AUC) increased dose-dependently and linearly . At 5.5 MU of BM 06.022, the AUC was 313 +/- 47 IU.hr.ml-1, the total plasma clearance was 306 +/- 40 ml/min, and the half-life was 14.4 +/- 1.1 minutes.

Plant Mol Biol, 1991 Oct, 17(4), 895 - 909
cDNA cloning and expression of Brassica napus enoyl-acyl carrier protein reductase in Escherichia coli; Kater MM et al.; The onset of storage lipid biosynthesis during seed development in the oilseed crop Brassica napus (rape seed) coincides with a drastic qualitative and quantitative change in fatty acid composition . During this phase of storage lipid biosynthesis, the enzyme activities of the individual components of the fatty acid synthase system increase rapidly . We describe a rapid and simple purification procedure for the plastid-localized NADH-dependent enoyl-acyl carrier protein reductase from developing B . napus seed, based on its affinity towards the acyl carrier protein (ACP) . The purified protein was N-terminally sequenced and used to raise a potent antibody preparation . Immuno-screening of a seed-specific lambda gt11 cDNA expression library resulted in the isolation of enoyl-ACP reductase cDNA clones . DNA sequence analysis of an apparently full-length cDNA clone revealed that the enoyl-ACP reductase mRNA is translated into a precursor protein with a putative 73 amino acid leader sequence which is removed during the translocation of the protein through the plastid membrane . Expression studies in Escherichia coli demonstrated that the full-length cDNA clone encodes the authentic B . napus NADH-dependent enoyl-ACP reductase . Characterization of the enoyl-ACP reductase genes by Southern blotting shows that the allo-tetraploid B . napus contains two pairs of related enoyl-ACP reductase genes derived from the two distinct genes found in both its ancestors, Brassica oleracea and B . campestris . Northern blot analysis of enoyl-ACP reductase mRNA steady-state levels during seed development suggests that the increase in enzyme activity during the phase of storage lipid accumulation is regulated at the level of gene expression.

Plant Mol Biol, 1991 Oct, 17(4), 653 - 67
Stress responses in alfalfa (Medicago sativa L.) 11 . Molecular cloning and expression of alfalfa isoflavone reductase, a key enzyme of isoflavonoid phytoalexin biosynthesis; Paiva NL et al.; The major phytoalexin in alfalfa is the isoflavonoid (-)-medicarpin (or 6aR, 11aR)-medicarpin . Isoflavone reductase (IFR), the penultimate enzyme in medicarpin biosynthesis, is responsible for introducing one of two chiral centers in (-)-medicarpin . We have isolated a 1.18 kb alfalfa cDNA (pIFRalf1) which, when expressed in Escherichia coli, converts 2'-hydroxyformononetin stereospecifically to (3R)-vestitone, as would be predicted for IFR from alfalfa . The calculated molecular weight of the polypeptide (35,400) derived from the 954 bp open reading frame compares favorably to estimated Mrs determined for IFR proteins purified from other legumes . The transcript (1.4 kb) is highly induced in elicited alfalfa cell cultures . The kinetics of induction are consistent with the appearance of IFR activity, the accumulation of medicarpin, and the observed induction of other enzymes in the pathway . Low levels of IFR transcripts were found in healthy plant parts (roots and nodules) which accumulate low levels of a medicarpin glucoside . IFR appears to be encoded by a single gene in alfalfa . The cloning of IFR opens up the possibility of genetic manipulation of phytoalexin biosynthesis in alfalfa by altering isoflavonoid stereochemistry.

Plant Mol Biol, 1991 Oct, 17(4), 581 - 90
Isolation and sequence analysis of a cDNA clone for a carrot calcium-dependent protein kinase: homology to calcium/calmodulin-dependent protein kinases and to calmodulin; Suen KL et al.; Recently, a novel type of calcium-dependent protein kinase (CDPK) that requires neither calmodulin nor phospholipids for activation, has been described in plants . We have isolated a cDNA clone for carrot CDPK by probing a library of somatic embryo cDNAs with oligonucleotides corresponding to highly conserved regions of protein kinases . The product of this gene overexpressed in Escherichia coli reacted strongly with monoclonal antibodies to soybean CDPK . The deduced amino acid sequence of carrot CDPK reveals two major functional domains . An N-terminal catalytic domain with greatest homology to calcium/calmodulin-dependent protein kinase type II from rat brain is coupled to a C-terminal calcium-binding domain resembling calmodulin . These features of the primary sequence explain how CDPK binds calcium and suggest a model for CDPK regulation based on similarities to animal calcium/calmodulin-dependent protein kinases.

Biochemistry, 1991 Oct 1, 30(39), 9478 - 85
Cytoplasmic phosphorylating domain of the mannitol-specific transport protein of the phosphoenolpyruvate-dependent phosphotransferase system in Escherichia coli: overexpression, purification, and functional complementation with the mannitol binding domain; van Weeghel RP et al.; The cytoplasmic C-terminal domain, residues 348-637, and the membrane-bound N-terminal domain, residues 1-347, of EIImtl have been subcloned and expressed in Escherichia coli . The N-terminal domain, IICmtl, contains the mannitol binding site, and the C-terminal domain, IIBAmtl, contains the activity-linked phosphorylation sites, His-554 and Cys-384 . Overexpression of the BA domain was achieved by a translational in-frame fusion of the gene with the cro ATG start codon, downstream of the strong PR promoter of phage lambda . The domain has been purified and characterized in in vitro complementation assays . It possessed no mannitol phosphorylation activity itself but was able to restore the phosphoenolpyruvate-dependent phosphorylation activity of two EIImtl phosphorylation site mutants, lacking His-554 or Cys-384 . The complementary N-terminal domain was also expressed . Membranes possessing IICmtl were unable to phosphorylate mannitol at the expense of phosphoenolpyruvate . However, when the membranes were combined with the purified C-terminal domain, mannitol phosphorylation activity was restored . Mannitol transport and phosphorylation were also restored in vivo when the two plasmids encoding the N- and C-terminal domains were expressed in the same cell . These data demonstrate the existence of structurally and functionally distinct domains in EIImtl: a cytoplasmic domain with phosphorylating activity and a membrane-bound N-terminal domain which, in the presence of the cytoplasmic domain, is able to actively transport and phosphorylate mannitol . The ability to separate, overproduce, and purify structurally stable, enzymatically active domains opens the way for 3D structural studies as well as complete kinetic analysis of the activities of the individual domains and their interactions.

Am J Hum Genet, 1991 Oct, 49(4), 860 - 7
Molecular characterization of two galactosemia mutations: correlation of mutations with highly conserved domains in galactose-1-phosphate uridyl transferase; Reichardt JK et al.; Galactosemia is an autosomal recessive disorder of human galactose metabolism caused by deficiency of the enzyme galactose-1-phosphate uridyl transferase (GALT) . The molecular basis of this disorder is at present not well understood . We report here two missense mutations which result in low or undetectable enzymatic activity . First, we identified at nucleotide 591 a transition which substitutes glutamine 188 by arginine . The mutated glutamine is not only highly conserved in evolution (conserved also in Escherichia coli and Saccharomyces cerevisiae), but is also two amino acid residues downstream from the active site histidine-proline-histidine triad and results in about 10% of normal enzymatic activity . The arginine 188 mutation is the most common galactosemia mutation characterized to date . It accounts for one-fourth of the galactosemia alleles studied . Second, we report the substitution of arginine 333 by tryptophan, caused by a transition at nucleotide 1025 . The area surrounding this missense mutation is the most highly conserved domain in the homologous enzymes from E . coli, yeast, and humans, and this mutation results in undetectable enzymatic activity, suggesting that this is a severe mutation . This second mutation appears to be rare, since it was found only in the patient we sequenced . Our data provide further evidence for the heterogeneity of galactosemia at the molecular level, heterogeneity which might be related to the variable clinical outcome observed in this disorder.

J Virol, 1991 Oct, 65(10), 5624 - 30
Substrate specificity of recombinant human immunodeficiency virus integrase protein; LaFemina RL et al.; Recombinant human immunodeficiency virus type 1 (HIV-1) integrase (IN) produced in Escherichia coli efficiently cleaves two nucleotides from the 3' end of synthetic oligonucleotide substrates which mimic the termini of HIV-1 proviral DNA . Efficient cleavage was restricted to HIV-1 substrates and did not occur with substrates derived from other retroviruses . Mutagenesis of the U5 long terminal repeat (LTR) terminus revealed only moderate effects of mutations outside the terminal four bases of the U5 LTR and highlighted the critical nature of the conserved CA dinucleotide motif shared by all retroviral termini . Integration of the endonuclease cleavage products occurs subsequent to cleavage, and evidence that the cleavage and integration reactions may be uncoupled is presented . Competition cleavage reactions demonstrated that IN-mediated processing of an LTR substrate could be inhibited by competition with LTR and non-LTR oligonucleotides.

J Infect Dis, 1991 Oct, 164(4), 796 - 9
Heterogeneity of immunotypes of heat-labile enterotoxins of enterotoxigenic Escherichia coli of human origin; Qu ZH et al.; A new technique, checkerboard immunoblotting (CBIB), has been applied to detect and to differentiate heat-labile enterotoxins, (LTs), from enterotoxigenic strains of Escherichia coli of human origin using polyclonal and monoclonal antibodies . Optimal conditions of production and release of LTs were defined using CBIB . LT release was enhanced when E . coli cells were treated with 8 M urea . LT production was highest when E . coli strains were incubated with shaking (200 rpm) at 37 degrees C for 12 h in CAYE-2 medium . Two hundred and five strains of E . coli, isolated from patients with diarrhea in Japan, Thailand, the United States, Mexico, and Brazil, were examined for LT . Of 133 LT-positive strains, 4 (3%) produced an LT that reacted like H-LT-1 (originally isolated from E . coli strain H-74-114) while 126 strains (94.7%) produced LT that reacted like H-LT-2 (originally isolated from strain H-10407) or H-LT-3 (from strain H-240-3) . Three strains of human origin (2.3%) produced an LT that reacted like P-LT (produced by E . coli strains of porcine origin) . This study shows that CBIB, a simple, efficient, and practical assay, might be useful for epidemiologic surveys and for evaluation of serologic responses to LTs and antitoxic vaccines.

Infect Immun, 1991 Oct, 59(10), 3796 - 800
Scanning and transmission electron microscopic study of adherence of Escherichia coli O103 enteropathogenic and/or enterohemorrhagic strain GV in enteric infection in rabbits; Licois D et al.; The GV strain (serotype O103:H2:K-), originally isolated from a diarrheic rabbit, is an enteropathogenic Escherichia coli strain that produces diarrhea without synthesizing the classical enterotoxins and that is not invasive . This strain is characterized by a 117-kb plasmid (pREC-1) . Histological study of the gut by scanning electron microscopy and transmission electron microscopy was performed on the GV strain, on a derivative strain cured of pREC-1, and on transconjugants obtained by transfer of pREC-1 to nonpathogenic strains E . coli K-12 and 6100, not belonging to the O103 serogroup . The GV strain adhered to the epithelial cells of the ileum and large intestine, whereas the cured GV strain did not . Transfer of plasmid pREC-1 to E . coli K-12 or 6100 allowed the bacteria to attach to the intestinal mucosa in the same manner as that of the wild-type GV strain . Thus, pREC-1 seems to play an important role in attachment to and colonization of the intestinal tract of rabbits by E . coli serogroup O103 . Scanning electron microscopy showed numerous bacteria attached together and closely associated with intestinal villi . Transmission electron microscopy revealed effacing lesions characteristic of enteropathogenic E . coli strains: effacing of microvilli and cuplike projections (pedestal formations) associated with an acute inflammatory and hemorrhagic response . In contrast with the results reported for rabbit pathogenic O15 strains, it appeared that the Peyer's patches were not involved in the early stages of infection with the O103 GV strain . This strain may represent a model for the study of the virulence and pathogenic effects of enteropathogenic and enterohemorrhagic E . coli strains.

Infect Immun, 1991 Oct, 59(10), 3708 - 14
Efficacy of enteric-coated protease in preventing attachment of enterotoxigenic Escherichia coli and diarrheal disease in the RITARD model; Mynott TL et al.; In this study, we report on a novel approach based on modification of the intestinal surface to prevent diarrhea caused by enterotoxigenic Escherichia coli (ETEC) . The removable intestinal tie adult rabbit diarrhea (RITARD) model was used to test the efficacy of an enteric-coated protease preparation (Detach; Enzacor Technology Pty . Ltd.) in the prevention of bacterial attachment and diarrheal disease caused by colonization factor antigen I-positive (CFA/I+) E . coli H10407 . Protease was administered orally to rabbits 18 h prior to challenge with 10(11) bacteria . Four groups of rabbits were inoculated with different ETEC strains which produced different combinations of adhesin and enterotoxin or with sterile phosphate-buffered saline . Occurrence of diarrhea during the subsequent 24-h incubation period was recorded . Oral administration of protease was successful in reducing diarrhea and diarrhea-induced death in six of seven (86%) rabbits infected with CFA/I+, heat-stable and heat-labile toxin-positive E . coli (H10407) . Seven of eight (87%) rabbits not protected by protease treatment died or developed severe diarrhea . Quantitative analysis of bacterial cultures obtained from the small intestine of rabbits showed a significant (P less than 0.001) 2,000-fold reduction in CFU per centimeter of intestine following treatment with protease . The efficacy of protease treatment was 99.5%, with very wide confidence limits (greater than 0 to 99.9%) . The data indicate that the use of protease to prevent ETEC diarrheal disease has considerable potential.

Infect Immun, 1991 Oct, 59(10), 3536 - 46
Characterization of the 35-kilodalton Treponema pallidum subsp . pallidum recombinant lipoprotein TmpC and antibody response to lipidated and nonlipidated T . pallidum antigens; Schouls LM et al.; The gene encoding the 35-kDa immunogenic Treponema pallidium subsp . pallidum (T . pallidum) membrane protein C, TmpC, was cloned, sequenced, and expressed in Escherichia coli . The deduced amino acid sequence carries on N-terminal signal sequence with a four-amino-acid motif, which is characteristic for bacterial lipoproteins . Metabolic labeling with radioactive palmitic acid of E . coli expressing TmpC revealed incorporation of the fatty acid into the antigen . The antigen was overproduced, purified to near homogeneity and used in an enzyme-linked immunosorbent assay (ELISA) to evaluate its potential for the serodiagnosis of syphilis . Although all sera from untreated secondary syphilis patients were reactive in this TmpC ELISA, only a minority of the serum samples from untreated patients in the primary or early latent stage of the disease contained significant anti-TmpC antibodies . To study the influence of the lipid moiety on the antigenic properties of the TmpC, TmpA, and TpD lipoproteins, plasmids encoding nonlipidated forms of these antigens were constructed . In addition, a plasmid expressing a lipidated form of the otherwise non-lipid-modified antigen TmpB was constructed . Immunization and absorption experiments with these lipidated and nonlipidated antigens showed that antibodies against the lipid moiety of lipoproteins could not be detected on immunoblots, neither in sera from infected rabbits nor in sera from animals immunized with the lipoproteins . In addition, we were unable to demonstrate cross-reactivity between antibodies against the T . pallidum lipoproteins and those reactive to the Venereal Diseases Research Laboratories test, suggesting that antibodies reactive to the Venereal Diseases Research Laboratories test are unrelated to antilipoprotein antibodies.

Infect Immun, 1991 Oct, 59(10), 3531 - 5
Molecular characterization of the humoral response to the 41-kilodalton flagellar antigen of Borrelia burgdorferi, the Lyme disease agent; Berland R et al.; The earliest humoral response in patients infected with Borrelia burgdorferi, the agent of Lyme disease, is directed against the spirochete's 41-kDa flagellar antigen . In order to map the epitopes recognized on this antigen, 11 overlapping fragments spanning the flagellin gene were cloned by polymerase chain reaction and inserted into an Escherichia coli expression vector which directed their expression as fusion proteins containing glutathione S-transferase at the N terminus and a flagellin fragment at the C terminus . Affinity-purified fusion proteins were assayed for reactivity on Western blots (immunoblots) with sera from patients with late-stage Lyme disease . The same immunodominant domain was bound by sera from 17 of 18 patients . This domain (comprising amino acids 197 to 241) does not share significant homology with other bacterial flagellins and therefore may be useful in serological testing for Lyme disease.

Glycoconj J, 1991 Oct, 8(5), 424 - 33
Blood group type glycosphingolipids of human kidneys . Structural characterization of extended globo-series compounds; Holgersson J et al.; Blood group type glycosphingolipids present in kidneys of blood group A and B human individuals have been isolated and structurally characterized by mass spectrometry, proton NMR spectroscopy, degradation studies and by their reactivity with various monoclonal antibodies and Escherichia coli bacteria . The two major complex glycolipids present in the blood group A and B kidneys were globopentaosylceramide (IV3Gal beta-Gb4Cer) and the X pentaglycosylceramide (III3Fuc alpha-nLc4Cer) . The major blood group A glycolipid in the blood group A kidneys was based on the type 4 chain (globo-series) . There were also small amounts of the type 2 chain and trace amounts of the type 1 and type 3 chain based A glycolipids . In addition, the blood group H type 4 chain structure was present together with Le(a) and Le(b) compounds . In the blood group B kidneys, the major B glycolipids were monofucosylated hexa- and octaglycosylceramides, where the former were based on the type 2 carbohydrate chain . The blood group B type 4 chain heptaglycosylceramide was found to be a minor component making up only about 1% of the total blood group B structures.

Infect Immun, 1991 Oct, 59(10), 3836 - 40
Nucleotide sequence analysis of the Legionella micdadei mip gene, encoding a 30-kilodalton analog of the Legionella pneumophila Mip protein; Bangsborg JM et al.; After the demonstration of analogs of the Legionella pneumophila macrophage infectivity potentiator (Mip) protein in other Legionella species, the Legionella micdadei mip gene was cloned and expressed in Escherichia coli . DNA sequence analysis of the L . micdadei mip gene contained in the plasmid pBA6004 revealed a high degree of homology (71%) to the L . pneumophila mip gene, with the predicted secondary structures of the two Mip proteins following the same pattern . Southern hybridization experiments, with the plasmid pBA6004 as the probe, suggested that the mip gene of L . micdadei has extensive homology with the mip-like genes of several Legionella species . Furthermore, amino acid sequence comparisons revealed significant homology to two eukaryotic proteins with isomerase activity (FK506-binding proteins).

Plant Cell, 1991 Oct, 3(10), 1121 - 30
Expression of a maize sucrose phosphate synthase in tomato alters leaf carbohydrate partitioning; Worrell AC et al.; We isolated a complementary DNA sequence for the enzyme sucrose phosphate synthase (SPS) from maize utilizing a limited amino acid sequence . The 3509-bp cDNA encodes a 1068-amino acid polypeptide . The identity of the cDNA was confirmed by the ability of the cloned sequence to direct sucrose phosphate synthesis in Escherichia coli . Because no plant-specific factors were necessary for enzymatic activity, we can conclude that SPS enzyme activity is conferred by a single gene product . Sequence comparisons showed that SPS is distantly related to the enzyme sucrose synthase . When expressed from a ribulose bisphosphate carboxylase small subunit promoter in transgenic tomatoes, total SPS activity was boosted up to sixfold in leaves and appeared to be physiologically uncoupled from the tomato regulation mechanism . The elevated SPS activity caused a reduction of starch and increase of sucrose in the tomato leaves . This result clearly demonstrates that SPS is involved in the regulation of carbon partitioning in the leaves.

Mol Microbiol, 1991 Oct, 5(10), 2447 - 58
Differential gene expression from the Escherichia coli atp operon mediated by segmental differences in mRNA stability; McCarthy JE et al.; The atp operon of Escherichia coli directs synthesis rates of protein subunits that are well matched to the requirements of assembly of the membrane-bound H(+)-ATPase (alpha 3 beta 3 gamma 1 delta 1 epsilon 1a1b2c10-15) . Segmental differences in mRNA stability are shown to contribute to the differential control of atp gene expression . The first two genes of the operon, atpl and atpB, are rapidly inactivated at the mRNA level . The remaining seven genes are more stable . It has previously been established that the translational efficiencies of the atp genes vary greatly . Thus differential expression from this operon is achieved via post-transcriptional control exerted at two levels . Neither enhancement of translational efficiency nor insertion of repetitive extragenic palindromic (REP) sequences into the atplB intercistronic region stabilized atpl . We discuss the implications of these results in terms of the pathway of mRNA degradation and of the role of mRNA stability in the control of gene expression.

J Biochem (Tokyo), 1991 Oct, 110(4), 501 - 7
Total synthesis of a gene for octopus rhodopsin and its preliminary expression; Harada Y et al.; To carry out systematic structure-function studies of octopus rhodopsin, photoreceptor protein of octopus visual cells, by means of specific amino-acid replacements, we have totally synthesized a DNA duplex of 1,365 base pairs that encodes the entire octopus rhodopsin of 455 amino acids {Ovchinnikov et al . (1988) FEBS Lett . 232, 69-72} by introducing codons preferred in Escherichia coli . Total synthesis simplifies site-specific mutagenesis in all parts of the gene by replacement of short restriction fragments by their newly synthesized counterparts containing the required nucleotide alterations . Thirty unique restriction sites were introduced in the octopus rhodopsin gene, which was assembled on a plasmid in two steps . Five cartridge genes of 344, 296, 320, 212, and 317 base pairs capable of being expressed independently were first constructed by using 48 synthetic oligonucleotides ranging in size from 54 to 73 nucleotides . The entire gene was constructed by consecutive linkage of cartridge genes . These cartridge genes were designed to correspond to the transmembrane helical unit of octopus rhodopsin, resulting in easy construction of various chimeric rhodopsins . The nucleotide sequences were confirmed by sequencing the cartridges as well as the entire gene . These synthetic genes were cloned into an expression vector carrying the trp promoter of E . coli, and were preliminarily expressed in vitro and in vivo.

Proc Natl Acad Sci U S A, 1991 Oct 1, 88(19), 8445 - 9
SOS-inducible DNA repair proteins, RuvA and RuvB, of Escherichia coli: functional interactions between RuvA and RuvB for ATP hydrolysis and renaturation of the cruciform structure in supercoiled DNA; Shiba T et al.; The ruv operon is induced by treatments that damage DNA and is regulated by the LexA repressor . It encodes two proteins, RuvA and RuvB, that are involved in DNA repair, recombination in RecE and RecF pathways, and mutagenesis . RuvB protein was previously purified and has ATP-binding activity and weak ATPase activity . To study the biochemical properties of RuvA and its interaction with RuvB, we purified RuvA protein to near homogeneity from an over-producing strain . RuvA bound more efficiently to single-stranded DNA than to double-stranded DNA . RuvA bound to DNA greatly enhanced the ATPase activity of RuvB; the enhancing effect of various forms of DNA was in the order of supercoiled DNA greater than single-stranded DNA greater than linear double-stranded DNA . UV irradiation further enhanced the ATPase stimulatory effect of supercoiled DNA dose dependently . The RuvA-RuvB complex has an activity that renatures the cruciform structure in supercoiled DNA . From these experiments and previous work, we infer that the RuvA-RuvB complex may promote branch migration in recombination and may correct irregular structures in DNA, such as cruciforms and hairpins, to facilitate DNA repair using ATP as the energy source.

Mol Gen Genet, 1991 Oct, 229(2), 206 - 12
A single amino acid substitution changes the substrate specificity of quinoprotein glucose dehydrogenase in Gluconobacter oxydans; Cleton-Jansen AM et al.; Gluconobacter oxydans contains pyrroloquinoline quinone-dependent glucose dehydrogenase (GDH) . Two isogenic G . oxydans strains, P1 and P2, which differ in their substrate specificity with respect to oxidation of sugars have been analysed . P1 can oxidize only D-glucose, whereas P2 is also capable of the oxidation of the disaccharide maltose . To investigate the nature of this maltose-oxidizing property we cloned the gene encoding GDH from P2 . Expression of P2 gdh in P1 enables the latter strain to oxidize maltose, indicating that a mutation in the P2 gdh gene is responsible for the change in substrate specificity . This mutation could be ascribed to a 1 bp substitution resulting in the replacement of His 787 by Asn.

Mol Gen Genet, 1991 Oct, 229(2), 197 - 205
Identification of Escherichia coli genes whose expression increases as a function of external pH; Heyde M et al.; Using transposon TnphoA and a plate screening method, we have isolated a set of Escherichia coli strains carrying phoA fusions with genes whose expression is modulated as a function of external pH . Besides fusions with the ompF gene and the malB locus, thirteen independent fusions were analysed whose expression is maximal during growth at pHs ranging from 7.0 to 8.5 and minimal during growth at pH 5.0 . Six different genetic loci, called phmA, phmB, phmC, phmD, phmE and phmF (for pH modulated) were characterized and localized on the E . coli chromosome at approx . 12, 18, 41, 45, 75 and 84 min, respectively . Expression of phmA::phoA fusions is also influenced when internal pH or environmental conditions such as osmolarity or anaerobiosis are modified . EnvZ protein is not involved in the regulation of phm::phoA fusions.

J Bacteriol, 1991 Oct, 173(20), 6605 - 11
Regulation of Escherichia coli secA mRNA translation by a secretion-responsive element; Schmidt MG et al.; The Escherichia coli secA gene, whose translation is responsive to the proficiency of protein export within the cell, is the second gene in a three-gene operon and is flanked by gene X and mutT . By using gene fusion and oligonucleotide-directed mutagenesis techniques, we have localized this translationally regulated site to a region at the end of gene X and the beginning of secA . This region has been shown to bind SecA protein in vitro . These studies open the way for a direct investigation of the mechanism of secA regulation and its coupling to the protein secretion capability of the cell.

J Bacteriol, 1991 Oct, 173(19), 6307 - 10
Membrane localization of the HflA regulatory protease of Escherichia coli by immunoelectron microscopy; Zorick TS et al.; The hflA locus of Escherichia coli specifies a multisubunit protease that selectively degrades the cII transcriptional activator of phage lambda . The regulated turnover of cII is critical for the choice between the lytic and lysogenic pathways of viral development . Previous cell fractionation work has indicated that HflA is associated with the inner membrane fraction . We have sought to demonstrate that the HflA protease is localized in the cell membrane of intact cells . To achieve this goal, we have combined electron microscopy of thin-sectioned E . coli cells with antibody tagging by a colloidal gold label . Using antibody to purified HflA protein, we have found preferential membrane labeling for hflA+ cells but not for hflA mutant cells . We conclude that HflA protease is localized in the cell membrane . The membrane location for HflA protein may serve as a component of a targeting mechanism to limit the action of the regulatory protease to selected cytoplasmic proteins.

Plant Mol Biol, 1991 Oct, 17(4), 641 - 52
The atp1 and atp2 operons of the cyanobacterium Synechocystis sp . PCC 6803; Lill H et al.; The two operons atp1 and atp2, encoding the subunits of the F0F1 ATP-synthase, have been cloned and sequenced from the cyanobacterium Synechocystis sp . PCC 6803 . The organization of the different genes in the operons have been found to resemble that of the cyanobacteria Synechococcus sp . PCC 6301 and Anabaena sp . PCC 7120 . The Synechocystis F0F1 ATP-synthase has nine subunits . A tenth open reading frame with unknown function was detected at the 5' end of atp1, coding for a putative gene product similar to uncI in Escherichia coli . A promoter structure was inferred for the Synechocystis atp operons and compared to other known promoters of cyanobacteria . Even though the operon structure of atp1 and atp2 in Synechocystis resembles the corresponding operons of Synechococcus, the amino acid sequences of individual gene products show marked differences . Genetic distances between cyanobacterial genes and genes for ATP-synthase subunits from other species have been calculated and compiled into evolutionary trees.

Protein Expr Purif, 1991 Oct-Dec, 2(5-6), 432 - 41
Expression in Escherichia coli: purification and properties of the yeast general transcription factor TFIID; Burton N et al.; A T7 RNA polymerase expression system has been used for the efficient expression of the yeast RNA polymerase general transcription factor TFIID (TFIIDY), the TATA-box factor (previously called BTF1) in Escherichia coli . Expression of the gene was performed at 25 degrees C instead of 37 degrees C to increase the total amount of soluble TFIIDY . Soluble TFIIDY was purified in three chromatographic steps and was eluted from the final column, a heparin-5PW HPLC column, in two peaks at 0.38 M (peak I) and 0.42 M (peak II) KCl in which this protein was 52% and greater than 95% pure, respectively . The protein in both peaks was active in an in vitro transcription assay . However, while TFIIDY from peak II was essentially indistinguishable from the material isolated from yeast, the protein of peak I differed in a number of biochemical characteristics, having a lower specific activity in an in vitro transcription assay and displaying an altered pattern of bands in a DNA band shift assay . Despite these differences, the proteins in both peaks have identical molecular weights on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, have indistinguishable N-terminal amino acid sequences, and apparently exist as monomers under the conditions used for the heparin-5PW chromatography.

Protein Expr Purif, 1991 Oct-Dec, 2(5-6), 394 - 401
High-level expression and secretion of a lysine-containing analog of Escherichia coli heat-stable enterotoxin; Greenberg RN et al.; The mechanism of action of the heat-stable enterotoxin STa secreted from enterotoxigenic forms of Escherichia coli has remained elusive, in part due to a tedious, low-yield purification procedure . We report here a method for obtaining large amounts of a biologically active lysine-containing analog of STa . Initial attempts to express the toxin using an expression vector that did not encode a signal sequence resulted in no biologically active material being recovered either from lysed cells or as a secretory product . However, use of the secretion vector pJAL36, which contains the STII enterotoxin signal sequence, allowed large amounts of an STa derivative containing the additional sequence Ser-Thr-Lys at the amino terminus of the mature enterotoxin to be readily purified from culture supernatants . This enterotoxin analog, known as KSTa-1, was equal in biological and receptor binding activity to the native toxin STa . The lysine residue present in KSTa-1 promises to be useful as a reactive amino acid that is readily derivatized to allow coupling of the enterotoxin to supports for affinity chromatography and antigenic conjugates . Additionally, the insertion of the lysine residue carboxy terminal to the Ser-Thr sequence adds a reversible "handle" to the toxin sequence in that the Ser-Thr-Lys segment can be removed by treatment with trypsin, releasing the native form of STa.

Protein Expr Purif, 1991 Oct-Dec, 2(5-6), 372 - 8
BPTI and N-terminal extended analogues generated by factor Xa cleavage and cathepsin C trimming of a fusion protein expressed in Escherichia coli; Lauritzen C et al.; A recombinant gene for BPTI (bovine pancreatic trypsin inhibitor) is expressed in Escherichia coli using a MBP (maltose-binding protein) fusion vector . BPTI is fused through an FXa (blood coagulation factor Xa protease) target sequence (Ile-Glu-Gly-Arg) to the C-terminus of MBP . The MBP moiety of the hybrid protein enables purification in one step utilizing MBP's affinity to cross-linked amylose, and the FXa target sequence allows specific cleavage of the hybrid protein . Effective FXa cleavage is achieved by spacing the FXa target sequence and Arg-1 of the BPTI sequence with four residues (Met-Glu-Ala-Glu) . The resulting N-terminal extended BPTI is readily converted to the wild-type sequence by trimming with cathepsin C exopeptidase, for the activity of which the spacing tetrapeptide is optimized . FXa cleavage is prohibited when the target sequence is placed next to Arg-1 . In this construction, off-target cleavage at a somewhat homologous sequence (Val-Pro-Gly-Arg) results in five- or six-residue extended BPTI, indicating new details of the FXa specificity . The yield of highly purified recombinant BPTI is 3-6 mg/liter of culture, making the MBP-BPTI expression system convenient for the production of sufficient amounts of protein for NMR studies . 1H NMR is used to analyze the N-extended BPTI analogues.

Protein Expr Purif, 1991 Oct-Dec, 2(5-6), 350 - 4
Purification and characterization of recombinant Pneumocystis carinii thymidylate synthase; Santi DV et al.; Catalytically active Pneumocystis carinii thymidylate synthase is expressed to the extent of about 4% of the soluble protein in Escherichia coli chi 2913 harboring plasmid pUETS-1.8 (U . Edman, J . C . Edman, B . Lundgren, and D . V . Santi, Proc . Natl . Acad . Sci . USA 86, 6503-6507, 1989) . Ion-exchange, affinity, hydrophobic, and reactive dye agarose chromatography steps were explored to devise a large-scale purification protocol for P . carinii thymidylate synthase . Sequential DE52, Q-Sepharose, phenyl-Sepharose, and Cibacron Blue F3GA chromatography yielded enzyme that was homogeneous by SDS-PAGE in a yield of over 50% . The sequence of the first 10 amino acid residues of the purified protein was in accord with that predicted from the DNA sequence . Isoelectric focusing gave a pI of 6.2 . Kinetic analysis of the purified enzyme revealed that the Km values were 4.7 +/- 1.3 microM for dUMP and 15.7 +/- 4.3 microM for 5,10-methylenetetrahydrofolate, similar to those of many other thymidylate synthases; the kcat of the most active preparation was 0.8 s-1 . The enzyme is stable for at least 2 months when stored at -80 degrees C in the presence of 40% glycerol, Tris-HCl, and thiol.

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