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 g