J Infect Dis, 1989 Feb, 159(2), 205 - 10 Expression of pertussis toxin correlates with pathogenesis in Bordetella species; Monack D et al.; Pertussis toxin is a principal determinant of virulence produced by Bordetella pertussis in the disease whooping cough and is the primary toxinogenic component of the pertussis vaccine . This toxin is not produced by the closely related species Bordetella parapertussis . Toxinogenic strains of B . parapertussis were constructed by the conjugative introduction of cloned pertussis toxin genes . These and analogous toxinogenic and nontoxinogenic strains of B . pertussis were assayed for their toxicity and reactogenic activities . Expression of active toxin by either Bordetella sp . correlated with the induction of leukocytosis, anaphylaxis, and histamine sensitivity. J Bacteriol, 1989 Feb, 171(2), 643 - 9 SecA protein autogenously represses its own translation during normal protein secretion in Escherichia coli; Schmidt MG et al.; The Escherichia coli secA gene, whose expression is responsive to the protein secretion status of the cell, is the second gene in an operon . We found that both the basal and induced levels of SecA biosynthesis are dependent on prior translation of the upstream gene, gene X, and identified two large gene X-secA transcripts . The 10-fold derepression of secA expression by protein export defects was at the translational level since no further increases in gene X or secA mRNA levels were detected during this period, and a secA-lacZ protein fusion but not an operon fusion was appropriately derepressed . Furthermore, overexpression of the SecA protein severely reduced expression of only the secA-lacZ protein fusion, indicating that SecA autogenously represses its own translation. J Immunol, 1989 Feb 1, 142(3), 808 - 12 IL-6 modulates the synthesis of a specific set of acute phase plasma proteins in vivo; Marinkovic S et al.; Human rIL-6, produced either in COS cells or Escherichia coli, similarly stimulates the production of acute phase plasma proteins in cultured human and rat hepatoma cells . This anabolic effect in hepatoma cells suggested a potential in vivo role of the cytokine in mediating the hepatic response to inflammation . Injection of IL-6 into adult male rats elicited a cytokine-specific change in the liver expression of acute phase proteins . As predicted from in vitro studies, glucocorticoids were needed to achieve a maximal IL-6 response in vivo . Optimal conditions were found to be two i.p . injections of 35 to 120 micrograms IL-6 and 65 micrograms dexamethasone per kg body weight administered at 12-h intervals . Within 24 h, the plasma concentrations for alpha 2-macroglobulin, fibrinogen, thiostatin, and hemopexin were increased to levels approximating those observed in acute phase animals . These results support the notion that direct interaction of IL-6 with the liver is an essential part in initiating the hepatic acute phase reaction. Exp Hematol, 1989 Feb, 17(2), 191 - 7 Stimulation of human hematopoietic progenitor cell proliferation and differentiation by recombinant human interleukin 3 . Comparison and interactions with recombinant human granulocyte-macrophage and granulocyte colony-stimulating factors; Ottmann OG et al.; Recombinant human interleukin 3 (IL3) produced in Escherichia coli was purified and its activities examined in cultures of highly enriched human bone marrow progenitor cells . Human IL3 stimulated multipotential (CFU-GEMM) and erythroid (BFU-E) progenitor cells, generating 95% more BFU-E than recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF) . No further enhancement of BFU-E or CFU-GEMM occurred when IL3 and GM-CSF were used in combination . Human IL3 was more effective than GM-CSF in stimulating granulocyte-macrophage colony-forming cells (CFU-GM) in short-term suspension cultures, but did not induce an increase of CFU-GM, BFU-E, or CFU-GEMM above input levels . IL3 was more active on day-14 (d14) than on d7 CFU-GM, similar to GM-CSF, but generated fewer and smaller CFU-GM-derived clones than either GM-CSF or granulocyte CSF (CI-CSF) . The simultaneous addition of plateau levels of IL3 and GM-CSF resulted in an infra-additive augmentation of d7 and d14 CFU-GM-derived clones, whereas IL3 and G-CSF enhanced the number and cellularity predominantly of d14 CFU-GM . In liquid cultures, IL3 induced a greater than 100-fold increase in the number of basophil-mast-like cells and eosinophils and allowed maintenance of these cultures for up to 7 weeks . Human GM-CSF was an almost equally potent, stimulus of eosinophil development but had only a marginal effect on basophilic precursors, whereas G-CSF lacked both activities . Therefore, human IL3 is a multilineage hemopoietic growth factor whose activities appear to encompass and extend beyond those of GM-CSF. J Virol, 1989 Feb, 63(2), 798 - 808 Antigenic determinants and functional domains in core antigen and e antigen from hepatitis B virus; Salfeld J et al.; The precore/core gene of hepatitis B virus directs the synthesis of two polypeptides, the 21-kilodalton subunit (p21c) forming the viral nucleocapsid (serologically defined as core antigen {HBcAg}) and a secreted processed protein (p17e, serologically defined as HBe antigen {HBeAg}) . Although most of their primary amino acid sequences are identical, HBcAg and HBeAg display different antigenic properties that are widely used in hepatitis B virus diagnosis . To locate and to characterize the corresponding determinants, segments of the core gene were expressed in Escherichia coli and probed with a panel of polyclonal or monoclonal antibodies in radioimmunoassays or enzyme-linked immunosorbent assays, Western blots, and competition assays . Three distinct major determinants were characterized . The single conformational determinant responsible for HBc antigenicity in the assembled core (HBc) and a linear HBe-related determinant (HBe1) were both mapped to an overlapping hydrophilic sequence around amino acid 80; a second HBe determinant (HBe2) was assigned to a location in the vicinity of amino acid 138 but found to require for its antigenicity the intramolecular participation of the extended sequence between amino acids 10 and 140 . It is postulated that HBcAg and HBeAg share common basic three-dimensional structure exposing the common linear determinant HBe1 but that they differ in the presentation of two conformational determinants that are either introduced (HBc) or masked (HBe2) in the assembled core . The simultaneous presentation of HBe1 and HBc, two distinctly different antigenic determinants with overlapping amino acid sequences, is interpreted to indicate the presence of slightly differently folded, stable conformational states of p21c in the hepatitis B virus nucleocapsid. Biochem Biophys Res Commun, 1989 Jan 31, 158(2), 489 - 96 Amino acid sequence of the bacterioferritin (cytochrome b1) of Escherichia coli-K12; Andrews SC et al.; The complete amino acid sequence of bacterioferritin (cytochrome b1) from Escherichia coli-K12 has been derived from the nucleotide sequence of the cloned gene . It comprises 158 amino acid residues giving an Mr of 18,495 . The identity of the gene product was confirmed by an 87 residue N-terminal sequence obtained from the purified protein, but it differs significantly from much of the previously published partial amino acid sequence (1) . Secondary structure prediction indicates a high alpha-helical content consistent with a 4-helix-bundle conformation . The fully assembled bacterioferritin molecule comprising 24 identical subunits and 12 haem moieties is a tetracosamer with an Mr of approximately 452,000. Biochem Biophys Res Commun, 1989 Jan 31, 158(2), 595 - 602 Expression of an enzymatically active murine retroviral reverse transcriptase in human cells; Jean-Jean O et al.; The region of the pol gene of the Moloney murine leukemia virus (M-MuLV) encoding the reverse transcriptase and RNase H activities was inserted in an eukaryotic expression vector and transiently expressed in human cultured cells . This results in the expression of high levels of reverse transcriptase activity . This enzyme, partially purified, also carries a RNase H activity, has the biochemical requirements of the viral enzyme and is recognized and inhibited by antibodies directed against a M-MuLV reverse transcriptase expressed in Escherichia coli. Gene, 1989 Jan 30, 75(1), 185 - 91 The role of a static bend in the DNA of the aroF regulatory region of Escherichia coli; Cobbett C et al.; DNA fragments containing the regulatory regions of two genes repressed by the tyrR gene product exhibit retarded electrophoretic mobility in polyacrylamide gels indicating the presence of static bends in the DNA . In the aroF gene this bend has been localized to a region containing two TyrR protein-binding sites . A point mutation between these two sites reduced the degree of bending observed but did not reduce the level of repression, indicating the static bend may not be an important component of the repression mechanism. Gene, 1989 Jan 30, 75(1), 177 - 84 The structure of the Escherichia coli hemB gene; Li JM et al.; The Escherichia coli hemB gene, which encodes 5-aminolevulinic acid dehydratase, and was cloned into pTZ18U, a multicopy plasmid, was sequenced . The hemB insert was double-digested with restriction enzymes and recloned back into pTZ18U and pTZ19U to allow for sequencing in two directions . In a second procedure, used to fill in gaps and to confirm the sequence derived from the first procedure, the whole insert was cloned into M13 phages . A nested set of deletions was constructed and recloned into M13 . Both the double-digested fragments cloned into plasmids pTZ18U and pTZ19U and the overlapping fragments contained in M13 phages were sequenced using the dideoxy procedure with {35S}dATP . Computer software was used to identify coding regions and the correct reading frame . Two promoter regions, two Shine-Dalgarno sequences and two possible start sites were identified . Extensive homologies with yeast (36%), human liver (40%) and rat liver (40%) amino-acid (aa) sequences were observed, especially in the 16-aa Zn-binding region (75%) and the 4 aa surrounding the essential lysine at the active site (100% for rat and human proteins) . Computer analysis of promoter strength and two independent analyses of codon usage indicated that the hemB gene is moderately expressed. Gene, 1989 Jan 30, 75(1), 167 - 75 Characterization of the Escherichia coli protein-export gene secB; Kumamoto CA et al.; The Escherichia coli secB gene product is required for normal export of envelope proteins out of the cell cytoplasm . In this report, we present the identification and nucleotide sequence of the secB coding sequence . The secB structural gene overlaps almost completely with a predicted open reading frame (ORF) that is encoded on the opposite strand . To establish the identity of the secB ORF, we characterized a secB mutation that caused total loss of secB function, based upon its phenotype . This mutation resulted from a nucleotide change that caused an ochre mutation in one ORF (the secB gene) and a silent (no amino acid change) codon change in the opposite ORF. Gene, 1989 Jan 30, 75(1), 157 - 65 Nucleotide sequence of the F plasmid transfer gene, traH: identification of a new gene and a promoter within the transfer operon; Ham LM et al.; The nucleotide sequence of the F plasmid transfer gene traH, which is involved in F-pilus assembly in Escherichia coli K-12, has been determined . From the sequence data, it would appear that traH encodes a 38,897-dalton precursor polypeptide which is processed to give a periplasmic protein . Furthermore, a new gene, trbF, has been located immediately upstream of traH and shown to be expressed by means of a translational fusion to lacZ . Using galK fusion and S1 nuclease protection studies, a weak traJ-dependent promoter, P trbF, has been mapped upstream and adjacent to trbF . Transcription of trbF and traH from P trbF may well serve to complement transcription from the major tra operon promoter PY located some 16 kb upstream of these genes. FEBS Lett, 1989 Jan 30, 243(2), 366 - 70 Purification and crystallization of ferric enterobactin receptor protein, FepA, from the outer membranes of Escherichia coli UT5600/pBB2; Jalal MA et al.; The ferric enterobactin receptor protein, FepA, was isolated and purified from the outer membranes of a genetically transformed strain of Escherichia coli (UT5600/pBB2) using anion-exchange chromatography, chromatofocusing and gel filtration . The purified protein was found to crystallize from 25 mM sodium phosphate buffer in the presence of 0.8% beta-D-octylglucoside under a range of conditions . The protein formed mostly small rods and needle-shaped crystals in the hanging drop method. FEBS Lett, 1989 Jan 30, 243(2), 193 - 8 Molecular cloning and sequencing of the glycogen phosphorylase gene from Escherichia coli; Choi YL et al.; The glgP gene, which codes for glycogen phosphorylase, was cloned from a genomic library of Escherichia coli . The nucleotide sequence of the glgP gene contained a single open reading frame encoding a protein consisting of 790 amino acid residues . The glgP gene product, a polypeptide of Mr 87,000, was confirmed by SDS-polyacrylamide gel electrophoresis . The deduced amino acid sequence showed that homology between glgP of E . coli and rabbit glgP, human glgP, potato glgP, and E . coli malP was 48.6, 48.6, 42.3, and 46.1%, respectively . Within this homologous region, the active site, glycogen storage site, and pyridoxal-5'-phosphate binding site are well conserved . The enzyme activity of glycogen phosphorylase increased after introduction on a multicopy of the glgP gene. Biochim Biophys Acta, 1989 Jan 30, 978(2), 293 - 8 Protonophoric effects of antimalarial drugs and alkylamines in Escherichia coli membranes; Nissani E et al.; Inside-out vesicles of Escherichia coli whose lumen was acidified by substrate oxidation, were used to study the mode of pH gradient dissipation by quinoline-containing antimalarial drugs and alkylamines . The pH was dissipated by micromolar drug concentrations, the dibasic chloroquine being most potent, followed by the monobasic mefloquine, quinine and the dibasic 7H-quinoline . The time dependence of pH dissipation as a function of membrane potential suggests that the monoprotonated forms of the drugs are able to cross the bacterial membrane . Alkylamines were able to dissipate the pH gradient in the 0.01-5 mM range, their rank order of potency being related to their hydrophobicity . Tertiary amines were less effective than less hydrophobic primary primary amines, implying an effect of molecular volume of their diffusion across the membrane . Both sets of results suggest that amphiphilic weak bases can cross membranes in their free-base form, become protonated in an acid environment and diffuse in this form along their concentration gradient and aided by the membrane potential, thereby dissipating the pH gradient. Biochim Biophys Acta, 1989 Jan 30, 978(2), 299 - 304 The F1F0-ATPase of Escherichia coli . The substitution of alanine by tyrosine at position 25 in the c-subunit affects function but not assembly; Fimmel AL et al.; A site-directed mutation in the gene which codes for the c-subunit of the F1F0-ATPase, resulting in the substitution of Ala-25 by Tyr, has been constructed and characterized . A plasmid carrying the mutation was used to transform strain AN943 (uncE429) . The resulting strain is unable to grow on succinate as sole carbon source and possesses an uncoupled growth yield . Membranes prepared from the mutant possess low levels of ATPase activity and are proton-impermeable . The F1-ATPase activity was found to be inhibited by 80% when bound to the membrane . When carried on a plasmid, the mutation is dominant in complementation tests with all mutant unc alleles tested and when transformed into wild-type strain AN346, the mutation results in an uncoupled phenotype . A mutant which overcomes this dominance was isolated and found to possess an 11-amino-acid deletion extending from Ile-55 to Met-65 within the c-subunit . These results are discussed in relation to the previously isolated Ala-25 to Thr mutant (Fimmel, A.L., Jans, D.A., Hatch, L., James, L.B., Gibson, F . and Cox, G.B . (1985) Biochim . Biophys . Acta 808, 252-258) and in relation to a previously proposed model for the F0 (Cox, G.B., Fimmel, A.L., Gibson, F . and Hatch, L . (1986) Biochim . Biophys . Acta 849, 62-69). Gene, 1989 Jan 30, 75(1), 21 - 30 Expression of synthetic genes encoding bovine and human basic fibroblast growth factors (bFGFs) in Escherichia coli; Knoerzer W et al.; Synthetic genes encoding bovine and human basic fibroblast growth factors (bFGFs) were assembled and cloned using established Escherichia coli expression plasmids . Transformed E . coli cells were able to synthesize either a fusion protein, comprising the first seven amino acids of beta-galactosidase, a linker fragment and bovine FGF, or genomic human bFGF . The two growth factors were purified from E . coli lysates by cation exchange and heparin-Sepharose affinity chromatography . The purified recombinant proteins were biologically active as monitored by their mitogenic activity for bovine aortic endothelial cells and their angiogenic capacity in the rabbit cornea. FEBS Lett, 1989 Jan 30, 243(2), 199 - 204 A novel lectin-independent interaction of P fimbriae of Escherichia coli with immobilized fibronectin; Westerlund B et al.; Binding of P fimbriae of uropathogenic Escherichia coli to purified human fibronectin and human placental type IV collagen was studied . In an enzyme immunoassay, purified P fimbriae bound strongly to immobilized intact fibronectin and to the aminoterminal 30-kDa fragment and the 120-140-kDa carboxyterminal fragments of fibronectin . Binding to the gelatin-binding 40-kDa fragment of fibronectin was considerably weaker . No binding to immobilized type IV collagen was seen . The interaction between P fimbriae and immobilized fibronectin was not inhibited by alpha-D-Gal-(1-4)-beta-D-Gal-1-O-Me, a receptor analog of P fimbriae . Moreover, a mutated P fimbria lacking the lectin activity behaved similarly in the adherence assays . Recombinant strains expressing the corresponding cloned fimbriae genes bound to immobilized fibronectin, but no binding to soluble 125I-labelled fibronectin was found . The results suggest that P fimbriae interact with immobilized fibronectin and that the binding mechanism does not involve the lectin activity of the fimbriae. Biochim Biophys Acta, 1989 Jan 27, 990(1), 15 - 7 Biosynthesis of tetrahydrobiopterin: a sensitive assay of 6-pyruvoyltetrahydropterin synthase using {2'-3H}dihydroneopterin 3'-triphosphate as substrate; Kerler F et al.; Pyruvoyltetrahydropterin synthase catalyzes the release of tritiated water from {2'-3H}dihydroneopterin 3'-triphosphate . The tritiated water formed during the enzymatic reaction is separated from substrate by adsorption of the latter to activated charcoal . The sensitivity and specificity of the assay allows the determination of the enzyme in crude cell extract. Nucleic Acids Res, 1989 Jan 25, 17(2), 617 - 29 Complex structural behavior of oligopurine-oligopyrimidine sequence cloned within the supercoiled plasmid; Parniewski P et al.; Synthetic sequence GATCC(AG)7ATCG(AT)4CG(AG)7 was cloned into plasmid and its structural behavior under the influence of supercoiling was analysed by chemical modification at variety of experimental conditions . It was found that this sequence adopts at least two different non-B conformations depending on -delta and pH values . Moreover, 12 nucleotide long non-pur.pyr spacer region separating two identical (AG)7 blocks does not provide a significant energy barrier protecting against unusual structures formation. J Biol Chem, 1989 Jan 25, 264(3), 1882 - 6 Export of prepro-alpha-factor from Escherichia coli; Lecker S et al.; Yeast prepro-alpha-factor translocates posttranslationally into yeast microsomes in vitro . This process is strongly influenced by the extreme carboxyl-terminal region of the protein . These features contrast with the properties of most eucaryotic proteins which are translocated into the endoplasmic reticulum . We have extended these studies by introducing the gene for the wild-type and several mutant forms of prepro-alpha-factor into Escherichia coli . Prepro-alpha-factor is secreted into the periplasm and processed to pro-alpha-factor . Its translocation across the plasma membrane requires the membrane potential and the secY gene product . Deletion mutant analysis showed that features of the pro-segment were essential for secretion of prepro-alpha-factor in E . coli, while the carboxyl-terminal region, which is required in yeast, is dispensible in E . coli . Neither size nor the presence of a unique topogenic sequence was sufficient to explain the requirement for the pro-segment. J Biol Chem, 1989 Jan 25, 264(3), 1689 - 93 Purification and characterization of human recombinant precursor interleukin 1 beta; Hazuda D et al.; We have purified the 31-kDa precursor of human interleukin 1 beta (proIL1 beta) from recombinant Escherichia coli expressing the protein . The recombinant precursor was characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, spectroscopy, Western blot, and for biological and receptor binding activity . The protein migrates at the expected molecular weight on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and analytical gel filtration columns . The specific activity of the recombinant precursor is less than 10(2) units/mg in the EL4 thymoma assay compared with 5 x 10(8) units/mg for the recombinant 17-kDa mature protein . The inactivity of the precursor is attributable to the inability of the protein to bind the IL1 receptor on EL4 cells as shown by receptor competition studies using 125I-labeled 17-kDa IL1 beta . Inactivity of the IL1 beta precursor is not due to degradation of the protein in either the bioactivity or receptor binding assays . The inactive IL1 beta precursor is converted to an active form following proteolysis with chymotrypsin which generates a carboxyl-terminal fragment of 17 kDa that is 6 orders of magnitude more active than the starting IL1 beta precursor . Removal of the first 114 amino acids from proIL1 beta generates a fully active molecule . In contrast, removal of the first 77 amino acids by treatment with trypsin only partially restores activity . The resultant 22-kDa protein exhibits a 600-fold increase in both biological and receptor binding activity, demonstrating a direct correlation between the ability of sequences within the pro-region to inhibit biological activity and inhibit binding to the IL1 receptor . Far-UV circular dichroism spectroscopy indicates that proIL1 beta is similar in secondary structure to mature IL1 beta; both proteins are nonhelical beta sheet proteins. J Biol Chem, 1989 Jan 25, 264(3), 1457 - 60 Site-directed mutagenesis of Escherichia coli succinyl-CoA synthetase . beta-Cys325 is a nonessential active site residue; Mann CJ et al.; Chemical modification experiments have shown that sulfhydryl groups play an important role in the mechanism of action of Escherichia coli succinyl-CoA synthetase . One of these sulfhydryl groups has been localized in the beta-subunit of the enzyme using the coenzyme A affinity analog, CoA disulfide-S,S-dioxide (Collier, G . E., and Nishimura, J . S . (1978) J . Biol . Chem . 253, 4938-4943) . Recently, it has been shown that the reactive sulfhydryl group resides in Cys325 (Nishimura, J . S., Mitchell, T., Ybarra, J., and Matula, J . M., submitted to Eur . J . Biochem . for publication) . In the present study, we have changed Cys325 to a glycine residue using the technique of site-directed mutagenesis and have purified the mutant enzyme to homogeneity . The resulting mutant enzyme is 83% as active as wild type enzyme . In contrast to wild type succinyl-CoA synthetase, the mutant is refractory to chemical modification by CoA disulfide-S,S-dioxide and methyl methanethiolsulfonate . It is also less reactive with N-ethylmaleimide . Thus, beta-Cys325 is a nonessential active site residue. J Biol Chem, 1989 Jan 25, 264(3), 1887 - 93 Ubiquitin function studied by disulfide engineering; Ecker DJ et al.; Disulfide engineering was used to probe the role of conformational mobility in ubiquitin-mediated proteolysis . Six genes that encode cysteine-containing mutants of ubiquitin were constructed, expressed in Escherichia coli and the proteins purified . Single cysteine-containing mutants and a 4/14 disulfide were active in degradation of a substrate protein in vitro, while the 4/66 disulfide, which cross-links the NH2- and COOH-terminal strands of the protein, was only 20-30% active . The solution structure of the 4/66 mutant was solved: the disulfide is left-handed with no perturbations in the backbone from that of wild type ubiquitin . The results suggest that conformational mobility is required for the activity of ubiquitin in signaling proteolysis. J Biol Chem, 1989 Jan 25, 264(3), 1723 - 8 Proton motive force-dependent and -independent protein translocation revealed by an efficient in vitro assay system of Escherichia coli; Yamada H et al.; Inverted membrane vesicles prepared from Escherichia coli spheroplasts were fractionated by means of sucrose gradient centrifugation, and a vesicle preparation exhibiting efficient and quantitative translocation of secretory proteins was obtained . The translocation of OmpA and an uncleavable model protein, uncleavable OmpF-Lpp, took place almost completely in 2-3 min, whereas that of OmpF-Lpp, a chimeric secretory protein, required 20 min for completion . The requirement of the proton motive force (delta muH+) for in vitro translocation was then examined with these three proteins . The translocation of all these proteins was significantly inhibited by the addition of carbonyl cyanide m-chlorophenylhydrazone (CCCP) or when stripped membrane vesicles lacking F1-ATPase were used, suggesting that delta muH+ generally participates in the translocation reaction . The inhibition was complete with OmpF-Lpp, whereas significant amounts of uncleavable OmpF-Lpp and OmpA were translocated at a slower rate even with the stripped membrane vesicles in the presence of a high concentration of carbonyl cyanide m-chlorophenylhydrazone . The delta muH+-independent translocation was inhibited by a nonhydrolyzable ATP analogue . These results indicate that although translocation of OmpF-Lpp obligatory requires delta muH+, the latter two proteins can be translocated in not only a delta muH+-dependent manner but also a delta mu H+-independent manner. Nucleic Acids Res, 1989 Jan 25, 17(2), 587 - 600 Multiple DNA repair activities for 3'-deoxyribose fragments in Escherichia coli; Bernelot-Moens C et al.; Escherichia coli contains multiple enzymes that hydrolyze deoxyribose fragments (phosphoglycolaldehyde, PGA) from the 3' termini of a synthetic DNA substrate . The major such activities are the main bacterial apurinic endonucleases, exonuclease III and endonuclease IV . In a double mutant deficient in both of these oxidation repair enzymes, Mg++-dependent 3'-PGA diesterase was detected at 3% the level found in wild-type bacteria . Gel filtration fractionated this residual diesterase activity into two peaks of Mr 40,000-52,000 (Pool A) and Mr 22,000-30,000 (Pool B) with differing abilities to remove 3'-phosphates from DNA . These multiple repair activities were resolved in 3'-PGA diesterase activity gels . The exonuclease III and endonuclease IV bands were identified using the purified proteins and by their specific absence from strains defective for the respective structural genes . Gel filtration Pool B yielded two activity bands of apparent Mr 25,000 and 28,000, but Pool A did not form a new band in the activity gels . Incubation of activity gels in different transition metals or boiling of the samples before electrophoresis also served to distinguish the various activities . The possible identities of the novel E . coli 3'-PGA diesterases and the importance of multiple repair enzymes for 3' damages are discussed. Biochemistry, 1989 Jan 24, 28(2), 685 - 91 Low-temperature unfolding of a mutant of phage T4 lysozyme . 1 . Equilibrium studies; Chen BL et al.; The mutant protein I3C-C97/C54T of phage T4 lysozyme is free of sulfhydryl groups and has a genetically engineered disulfide bridge between positions 3 and 97 (Perry & Wetzel, 1986) . This protein has a maximum stability at 12 degrees C in 3 M guanidinium chloride and undergoes reversible high- and low-temperature melting at 28 and -3 degrees C, respectively, in this medium . The free energy of stabilization of the protein has been studied over a range of temperature that includes both melting transitions . The stability curve fits a constant delta Cp model over the entire range, permitting an unusually complete determination of the thermodynamic parameters of the protein and demonstrating that the low-temperature unfolded form of the protein may be interpreted as an extrapolation with constant delta Cp of the high-temperature unfolded form . The free energy of unfolding is a linear function of guanidinium concentration within experimental error which permits a rough estimate of the stability of the protein at low temperatures and of the differential interaction of the unfolded protein with guanidinium chloride . These equilibrium studies provide a basis for the interpretation of the kinetic studies reported in the following paper. Biochemistry, 1989 Jan 24, 28(2), 486 - 93 Mechanism of ketol acid reductoisomerase--steady-state analysis and metal ion requirement; Chunduru SK et al.; Ketol acid reductoisomerase is an enzyme of the branched-chain amino acid biosynthetic pathway . It catalyzes two separate reactions: an acetoin rearrangement and a reduction . This paper reports on the purification of the enzyme from a recombinant Escherichia coli and on the steady-state kinetics of the enzyme . The kinetics of the reaction were determined for the forward and reverse reaction by using the appropriate chiral substrates . At saturating metal ion concentrations the mechanism follows an ordered pathway where NADPH binds before acetolactate . The product of the rearrangement of acetolactate, 3-hydroxy-3-methyl-2-oxobutyrate, is shown to be kinetically competent as an intermediate in the enzyme-catalyzed reaction . Starting with acetolactate, Mg2+ is the only divalent metal ion that will support enzyme catalysis . For the reduction of 3-hydroxy-3-methyl-2-oxobutyrate, Mn2+ is catalytically active . Product and dead-end inhibition studies indicate that the binding of metal ion and NADPH occurs randomly . In the forward reaction direction, the deuterium kinetic isotope effect on V/K is 1.07 when acetolactate is the substrate and 1.39 when 3-hydroxy-3-methyl-2-oxobutyrate is the substrate. Eur J Pharmacol, 1989 Jan 24, 160(1), 125 - 32 Angiotensin converting enzyme inhibitors and expression of des-Arg9-BK (kinin B1) receptors in vivo; Nwator IA et al.; The effect of the selective kinin B1 receptor agonist des-Arg9-BK was studied on blood pressure and on the in vitro aorta of rabbits pretreated 18 h earlier with lipopolysaccharide from E . coli, an infusion of bradykinin or with one of three angiotensin converting enzyme inhibitors captopril, enalapril or teprotide . The hypotensive response in vivo and contractile response seen on the in vitro aorta was selectively increased to des-Arg9-BK in all pretreated groups compared to controls, effects which were blocked by the selective competitive kinin B1 receptor antagonist des-Arg9-{Leu8}BK . Dexamethasone given to lipopolysaccharide pretreated rabbits had no effect on the increased hypotensive response seen with des-Arg9-BK . The skin vascular permeability response to des-Arg9-BK, bradykinin and histamine remained unchanged in the groups pretreated with lipopolysaccharide or captopril compared to controls . The possible mechanism(s) whereby angiotensin converting enzyme inhibitors produce this effect and the possible relevance to the inflammatory side-effects seen with this group of drugs is discussed. Biochemistry, 1989 Jan 24, 28(2), 893 - 9 Evidence for a tRNA/rRNA interaction site within the peptidyltransferase center of the Escherichia coli ribosome; Marconi RT et al.; A nine-base oligodeoxyribonucleotide complementary to bases 2497-2505 of 23S rRNA was hybridized to both 50S subunits and 70S ribosomes . The binding of the probe to the ribosome or ribosomal subunits was assayed by nitrocellulose filtration and by sucrose gradient centrifugation techniques . The location of the hybridization site was determined by digestion of the rRNA/cDNA heteroduplex with ribonuclease H and gel electrophoresis of the digestion products, followed by the isolation and sequencing of the smaller digestion fragment . The cDNA probe was found to interact specifically with its rRNA target site . The effects on probe hybridization to both 50S and 70S ribosomes as a result of binding deacylated tRNA(Phe) were investigated . The binding of deacylated tRNA(Phe), either with or without the addition of poly(uridylic acid), caused attenuation of probe binding to both 50S and 70S ribosomes . Probe hybridization to 23S rRNA was decreased by about 75% in both 50S subunits and 70S ribosomes . These results suggest that bases within the 2497-2505 site may participate in a deacylated tRNA/rRNA interaction. Biochemistry, 1989 Jan 24, 28(2), 449 - 55 Fragmentation of an endogenous inhibitor upon complex formation with high- and low-Ca2+-requiring forms of calcium-activated neutral proteases; Nakamura M et al.; The interaction of an endogenous inhibitor for the calcium-activated neutral protease (CANP or calpain EC 3.4.22.17) with CANP was examined by SDS-polyacrylamide gel electrophoresis, immunoblot analysis, and gel filtration . Fragmentation of the inhibitor (Mr 110K) by mCANP, a high-Ca2+-requiring form, was shown only in the presence of Ca2+ ions of millimolar order, with decreased inhibitor activity recovered from gel extracts in the 110-kDa area . This fragmentation took place even when the inhibitor could completely inhibit the caseinolytic activity of mCANP . The fragmented inhibitor retained considerable inhibitor activity after the CANP-inhibitor complex was dissociated by the addition of EDTA, and 69% of the initial activity was recovered from the mixture reacted with excess mCANP lacking the 110-kDa band . A C-terminal fragment of CANP inhibitor produced in Escherichia coli (Mr 40K) was also hydrolyzed by mCANP in the presence of Ca2+ . The interaction of both forms of the inhibitor with mu CANP, a low-Ca2+-requiring form, led to the same phenomena in the presence of micromolar levels of Ca2+ . CANP inhibitor could not completely inhibit the autolysis of mCANP and mu CANP, indicating that these were intramolecular events . Gel filtration analysis revealed that the mass of the smallest fragment with inhibitor activity was about 15,000 daltons . These results suggest that CANP inhibitor may act in the manner of a suicide substrate. Biochemistry, 1989 Jan 24, 28(2), 611 - 6 G protein beta gamma subunits from bovine brain and retina: equivalent catalytic support of ADP-ribosylation of alpha subunits by pertussis toxin but differential interactions with Gs alpha; Casey PJ et al.; We have examined the ability of the beta gamma subunits of guanine nucleotide binding regulatory proteins (G proteins) to support the pertussis toxin (PT) catalyzed ADP-ribosylation of G protein alpha subunits . Substoichiometric amounts of the beta gamma complex purified from either bovine brain G proteins or the bovine retinal G protein, Gt, are sufficient to support the ADP-ribosylation of the alpha subunits of Gi (the G protein that mediates inhibition of adenylyl cyclase) and Go (a G protein of unknown function) by PT . This observation indicates that ADP-ribosylated G protein oligomers can dissociate into their respective alpha and beta gamma subunits in the absence of activating regulatory ligands, i.e., nonhydrolyzable GTP analogues or fluoride . Additionally, the catalytic support of ADP-ribosylation by bovine brain beta gamma does not require Mg2+ . Although the beta gamma subunit complexes purified from bovine brain G proteins and the beta gamma complex of Gt support equally the ADP-ribosylation of alpha subunits by PT, there is a marked difference in their abilities to interact with Gs alpha . The enhancement of deactivation of fluoride-activated Gs alpha requires 25-fold more beta gamma from Gt than from brain G proteins to produce a similar response . This difference in potency of beta gamma complexes from the two sources was also observed in the ability of beta gamma to produce an increase in the activity of recombinant Gs alpha produced in Escherichia coli. Biochemistry, 1989 Jan 24, 28(2), 478 - 86 Probing the functional role of threonine-113 of Escherichia coli dihydrofolate reductase for its effect on turnover efficiency, catalysis, and binding; Fierke CA et al.; The role of Thr-113 of Escherichia coli dihydrofolate reductase in binding and catalysis was probed by amino acid substitution . Thr-113, a strictly conserved residue that forms a hydrogen bond to the active-site Asp-27 and to the amino group of methotrexate through a fixed water molecule, was replaced by valine . The kinetic scheme is identical in form with the wild-type scheme, although many of the rate constants vary, including a decrease in the association rate constants and an increase in the dissociation rate constants for folate ligands, a decrease in the hydride-transfer rate constant in both directions, and an increase in the intrinsic pKa of Asp-27 . Overall, replacement of Thr-113 by Val decreases the binding of folate substrates by approximately 2.3 kcal/mol . These multiple complex changes on various ground and transition states underscore the optimal properties of a strictly conserved residue in the evolution of catalytic function. Biochim Biophys Acta, 1989 Jan 23, 1007(1), 73 - 9 Ethidium bromide-mediated renaturation of denatured closed circular DNAs: mechanistic aspects and fractionation of DNA on a molecular weight basis; Lau PP et al.; When closed circular duplex DNAs are exposed to alkali in the presence of ethidium bromide, from 0 to 100% of the DNA can be recovered as the fully base-paired duplex (native) form upon neutralization of the solutions . The fraction of native DNA depends on the concentration of ethidium bromide, time of incubation, ionic strength and temperature of the solutions before neutralization as well as the molecular weight and superhelix density of the DNA . Limiting ethidium concentrations exist below and above which 0 and 100% of the DNA, respectively, is recovered as native material under a given set of incubation conditions regardless of the length of time of incubation before neutralization . The strong molecular weight dependence of the fraction of DNA recovered in the native form after a given time of pre-neutralization incubation at ethidium concentrations between the limiting values noted above allows larger DNAs to remain fully denatured upon neutralization while smaller DNAs in the same mixture are fully renatured . This permits the rapid fractionation of mixtures of closed duplex DNAs on the basis of molecular weight when a technique for the separation of denatured from fully base-paired DNA is applied to such mixtures . Such a separation has been demonstrated through the marked enrichment of plasmid cloning vector DNA containing cloned inserts in the fractions that remain denatured after neutralization of alkaline solutions of these DNAs containing ethidium bromide. Biochim Biophys Acta, 1989 Jan 23, 1007(1), 127 - 9 RNA transfection of Escherichia coli by electroporation; Taketo A; Cells of Escherichia coli were efficiently transfected with Q beta phage RNA by electroporation . A single voltage shock at 6.25 kV/cm with a 25 microF capacitor resulted in an infectivity yield considerably higher than that attained by a lysozyme-EDTA spheroplast method or a CaCl2 procedure . A linear relationship was found between concentration of the input RNA and yield of the transfectants, over a wide range . Efficiency of the electroporation-mediated transfection (electrotransfection) was increased by addition of certain sodium salts but decreased by preincubation in a Tris buffer containing sucrose. J Mol Biol, 1989 Jan 20, 205(2), 465 - 7 Molecular size and symmetry of the bacterioferritin of Escherichia coli . X-ray crystallographic characterization of four crystal forms; Smith JM et al.; X-ray crystallographic data from four crystal forms of Escherichia coli bacterioferritin show that the molecule has a diameter in the range 119 to 128 A . Molecules are composed of 24 subunits arranged in 432 symmetry . In both size and symmetry the molecule resembles ferritin from eukaryotes . The four crystal forms are monoclinic, space group P2(1) with unit cell dimensions a = 118.7 A, b = 211.6 A, c = 123.3 A and beta = 119.1 degrees; orthorhombic, C222(1), a = 128.7 A, b = 197.1 A, c = 202.8 A; tetragonal, P4(2)2(1)2, a = b = 210.6 A, c = 145.0 A and cubic, I432, a = 146.9 A. J Mol Biol, 1989 Jan 20, 205(2), 459 - 60 Preliminary crystallographic analysis of glutamine-binding protein from Escherichia coli; Chen P et al.; Glutamine-binding protein from Escherichia coli, an essential component in the active transport of L-glutamine across the cytoplasmic membrane, has been crystallized by vapor diffusion in the presence of ammonium sulfate . The crystals exhibit pseudo-tetragonal symmetry with cell constants a = 77.5 A, b = 78.5 A and c = 90.2 A . Analysis of the diffraction data indicates that the space group is P2(1)2(1)2(1) . There are two molecules per asymmetric unit and the solvent content is estimated to be 53%. J Mol Biol, 1989 Jan 20, 205(2), 449 - 54 Stacked beta-bulges in thymidylate synthase account for a novel right-handed rotation between opposing beta-sheets; Matthews DA et al.; The beta-sandwich in thymidylate synthase comprises two six-stranded mixed beta-sheets, each contributed by one subunit of the dimeric molecule . In contrast to other proteins of known structure in which beta-sheets stack face to face, the central beta-sheets in the thymidylate synthase dimer are related by a right-handed rather than a left-handed twist . Using a highly refined model of an Escherichia coli thymidylate synthase ternary complex, we show that the individual beta-sheets in each subunit are severely distorted by an unusual series of stacked beta-bulges, which partitions each larger sheet into two smaller beta-sheets approximately orthogonal to one another . These stacked beta-bulges are locally stabilized by hydrogen bonding involving eight conserved residues . This extended structure anchors the phosphate of bound dUMP and controls the precise orientation of the catalytically essential active site cysteine . Stereochemical factors associated with the pronounced crease caused by these stacked bulges account for the right-handed twist of opposing beta-sheets. J Mol Biol, 1989 Jan 20, 205(2), 373 - 85 Regulation of expression of the ada gene controlling the adaptive response . Interactions with the ada promoter of the Ada protein and RNA polymerase; Sakumi K et al.; The Ada protein of Escherichia coli catalyzes transfer of methyl groups from methylated DNA to its own molecule, and the methylated form of Ada protein promotes transcription of its own gene, ada . Using an in vitro reconstituted system, we found that both the sigma factor and the methylated Ada protein are required for transcription of the ada gene . To elucidate molecular mechanisms involved in the regulation of the ada transcription, we investigated interactions of the non-methylated and methylated forms of Ada protein and the RNA polymerase holo enzyme (the core enzyme and sigma factor) with a DNA fragment carrying the ada promoter region . Footprinting analyses revealed that the methylated Ada protein binds to a region from positions -63 to -31, which includes the ada regulatory sequence AAAGCGCA . No firm binding was observed with the non-methylated Ada protein, although some DNase I-hypersensitive sites were produced in the promoter by both types of Ada protein . RNA polymerase did bind to the promoter once the methylated Ada protein had bound to the upstream sequence . To correlate these phenomena with the process in vivo, we used the DNAs derived from promoter-defective mutants . No binding of Ada protein nor of RNA polymerase occurred with a mutant DNA having a C to G substitution at position -47 within the ada regulatory sequence . In the case of a -35 box mutant with a T to A change at position -34, the methylated Ada protein did bind to the ada regulatory sequence, yet there was no RNA polymerase binding . Thus, the binding of the methylated Ada protein to the upstream region apparently facilitates binding of the RNA polymerase to the proper region of the promoter . The Ada protein possesses two known methyl acceptor sites, Cys69 and Cys321 . The role of methylation of each cysteine residue was investigated using mutant forms of the Ada protein . The Ada protein with the cysteine residue at position 69 replaced by alanine was incapable of binding to the ada promoter even when the cysteine residue at position 321 of the protein was methylated . When the Ada protein with alanine at position 321 was methylated, it acquired the potential to bind to the ada promoter . These results are compatible with the notion that methylation of the cysteine residue at position 69 causes a conformational change of the Ada protein, thereby facilitating binding of the protein to the upstream regulatory sequence. J Mol Biol, 1989 Jan 20, 205(2), 331 - 41 Early transcribed sequences affect termination efficiency of Escherichia coli RNA polymerase; Goliger JA et al.; We have constructed novel transcription templates in which we have fused the late gene promoters of Escherichia coli phages lambda and 82 upstream from three different rho-independent transcription terminators . Using an in vitro transcription assay and an in vivo galactokinase expression assay, we find that the initial portion of the transcribed region significantly affects the efficiency of some downstream terminators . We have identified, by deletion, substitution and point mutation analysis, sequences responsible for these increased levels of factor-independent readthrough . Since these important sequences occur within about 30 nucleotides of the RNA start site, we suggest that the initial portion of the transcript can affect termination efficiency. J Mol Biol, 1989 Jan 20, 205(2), 291 - 314 Reversibility of nucleotide incorporation by Escherichia coli RNA polymerase, and its effect on fidelity; Kahn JD et al.; During transcription, Escherichia coli RNA polymerase is capable of removing the nucleotide that it has just added to a growing RNA chain, and this removal depends on the presence of small concentrations of pyrophosphate . Chemically, the removal reaction is simply the reversal of the incorporation reaction, and we have observed the generation of free triphosphate as a result . After the removal the enzyme can continue synthesis . To test whether this reaction can provide an error correction mechanism, misincorporation rates were measured at a single position in an RNA transcript by withholding the correct nucleotide for that position, measuring the amount of readthrough transcript, and analyzing the readthrough transcripts with nearest-neighbor analysis and enzymatic RNA sequencing . The removal of pyrophosphate increases the rate of misincorporation . We present a theory that explains how reversible incorporation can increase the available discrimination free energy between correct and incorrect nucleotides and therefore may increase the fidelity of transcription . The formation of a covalent phosphodiester bond allows discrimination on the basis of helical structure as well as base-pairing . We propose that the important discrimination step is the translocation of the enzyme from one site on the DNA template to the next, and that reversible incorporation is necessary in order to take full advantage of the maximum discrimination free energy. FEBS Lett, 1989 Jan 16, 243(1), 8 - 12 The B isozyme of creatine kinase is active as a fusion protein in Escherichia coli: in vivo detection by 31P NMR; Koretsky AP et al.; A cDNA encoding the B isozyme of creatine kinase (CKB) has been expressed in Escherichia coli from a fusion with lacZ carried by lambda gt11 . Western blots indicate that a stable polypeptide with the appropriate mobility for the beta-galactosidase-creatine kinase (beta-gal-CKB) fusion protein cross-reacts with both beta-gal and CKB antiserum . No significant CK activity is detected in control E . coli; however, extracts from cells containing the lambda gt11-CKB construct have a CK activity of 1.54 +/- 0.07 mumol/min per mg protein . The fusion protein appears to provide this activity because immunoprecipitation of protein with beta-gal antiserum leads to a loss of CK activity from extracts . That the enzyme is active in vivo was demonstrated by detection of a phosphocreatine (PCr) peak in the 31P NMR spectrum from E . coli grown on medium supplemented with creatine . As in mammalian brain and muscle, the PCr peak detected was sensitive to the energy status of the E . coli. Biochim Biophys Acta, 1989 Jan 16, 978(1), 112 - 8 Changes in chemical structure and function in Escherichia coli cell membranes caused by freeze-thawing . II . Membrane lipid state and response of cells to dehydration; Souzu H et al.; Glycerol and spermine prevented the freeze-induced alteration of lipid character in membrane fragments derived from Escherichia coli B logarithmic-phase cells, protecting against loss of membrane function and subsequently maintaining higher cell viability . The membrane specimens derived from the stationary-phase cells exhibited high resistance to freezing-induced alteration of the membrane lipid character, and to freezing injury . Freeze-drying of membrane fragment specimens from both logarithmic- and stationary-phase cells gave rise to alterations in lipid state similar to those shown in freeze-thawing of logarithmic-phase cell membrane specimens . Freeze-drying brought about reduction of cell viability in both growth phase specimens . It is suggested, therefore, that the fundamental cause of the injury induced by freezing of living materials is dehydration of lipid-rich systems such as cellular membranes. Biochim Biophys Acta, 1989 Jan 16, 978(1), 105 - 11 Changes in chemical structure and function in Escherichia coli cell membranes caused by freeze-thawing . I . Change of lipid state in bilayer vesicles and in the original membrane fragments depending on rate of freezing; Souzu H; The effect of different rates of freezing on the character of lipids in unilamellar lipid bilayer vesicles and in the original membrane fragments of Escherichia coli B cells was investigated by measuring the temperature-dependent fluorescence polarization ratio changes of cis- and trans-parinaric acids . In lipid bilayer vesicles, both slow and rapid freezing brought about significant alterations in fluorescence polarization ratios in the specimens derived from both logarithmic and stationary-phase cells . In the original membrane fragments derived from logarithmic-phase cells, slow freezing gave rise to a similar alteration in fluorescence polarization ratio change, but no such alteration was found in the case of rapid freezing . Logarithmic-phase cells suffered from a membrane permeability change during slow freezing, which subsequently resulted in low cell viability . The cells suffered only slight impairment in membrane function during rapid freezing, and maintained higher viability . These results suggest that the primary site of damage due to freezing of the cells is the cellular membranes, and this destruction is due to a lipid state change in the membranes brought about by freezing. J Biol Chem, 1989 Jan 15, 264(2), 1310 - 6 Molecular cloning, characterization, and expression of a human 14-kDa lectin; Couraud PO et al.; Full length cDNAs coding for a 14-kDa beta-galactoside binding lectin have been isolated from HL-60 cells and human placenta . Oligonucleotide probes based on a pentapeptide present in several partial sequences of homologous human lectins were used to screen a lambda GT10 HL-60 cDNA library . The HL-60 cDNA clones that were isolated were used to design a synthetic primer representing the 3'-untranslated region of the HL-60 lectin . This primer was then used to synthesize a lambda GT10 human placenta cDNA library, and restriction fragments of the HL-60 cDNA clones were used to screen the library . The cDNA clones for both HL-60 and placenta lectin had identical sequences with short 5'- and 3'-untranslated regions and coded for a 135-amino acid protein which lacks a hydrophobic signal peptide sequence . Biochemical data show that, despite the presence of a possible N-linked glycosylation site, the protein is not glycosylated . Northern and Southern blot analyses indicate that the 14-kDa lectin is encoded for by a single gene . The lectin cDNA was expressed in Escherichia coli and biologically active protein was purified from cell lysates by affinity chromatography. FEMS Microbiol Lett, 1989 Jan 15, 48(2), 247 - 52 Synthetic oligodeoxyribonucleotide probes to detect verocytotoxin-producing Escherichia coli in diseased pigs; Meyer T et al.; Oligonucleotide probes constructed from the sequences published for Shiga-like toxin I (SLT-I) and Shiga-like toxin II (SLT-II) genes and antibody against the purified toxins were used to study the SLT (SLT-IIp) produced by porcine E . coli O138 and O139 strains . By DNA hybridization assays no homology was observed between SLT-I and SLT-IIp . By contrast the oligonucleotide probe derived from the slt-II A gene detected porcine strains of E . coli producing SLT-IIp and E . coli strains associated with human disease producing SLT-II . Homology of nucleotide sequences between SLT-IIp and SLT-II is reflected by serological cross-reactivity as demonstrated by a dot blot ELISA and neutralization of SLT-IIp with anti-SLT-II . The toxins were distinguishable in their ability to kill HeLa S-3 cells . The oligonucleotide probe and anti-SLT-II can facilitate identification of SLT-IIp producing E . coli to further clarify their role in diseased pigs. FEMS Microbiol Lett, 1989 Jan 15, 48(2), 231 - 6 Variations in cell size and buoyant density of Escherichia coli K12 during glycogen accumulation; Mas J et al.; The effect of glycogen accumulation on buoyant density and volume of Escherichia coli K12 was studied . A procedure consisting of three linear equations is presented . This requires measurement only of three parameters: cell buoyant density, cell volume and specific content of the polymer . Experimental values are then used to calculate intercepts and slopes of the equations by linear regression . From the estimated values of such parameters the in vivo values of several variables of interest can be calculated . These include in vivo density and volume of the glycogen inclusion, as well as density and volume of the structural material in the cell . The results are consistent with the glycogen inclusions being hydrated. FEMS Microbiol Lett, 1989 Jan 15, 48(2), 223 - 6 Development of pLR591, a Streptomyces-Escherichia coli positive selection shuttle vector; Hill RT et al.; The Escherichia coli positive selection vector pEcoR251 was ligated with the broad host range, high copy number Streptomyces plasmid pIJ702 to produce pLR591, a Streptomyces-E . coli positive selection shuttle vector . The EcoRI and thiostrepton resistance genes of pLR591 were expressed in E . coli and Streptomyces lividans respectively . The positive selection shuttle vector pLR591 facilitates the construction in E . coli of genomic libraries which can be screened in Streptomyces strains. Eur J Biochem, 1989 Jan 15, 179(1), 209 - 13 Molecular cloning, sequencing and expression in Escherichia coli of the 25-kDa growth-related protein of Ehrlich ascites tumor and its homology to mammalian stress proteins; Gaestel M et al.; The growth-related 25-kDa protein (p25) of Ehrlich ascites tumor (EAT) has been characterized by molecular cloning and sequencing of cDNA clones detected by hybridization with oligonucleotide probes synthesized according to the amino acid sequence of a tryptic peptide of p25 . Detection of p25 mRNA in EAT of the exponential growth phase and of the stationary phase using cDNA-derived RNA probes demonstrated that the abundance of p25 mRNA is also growth-related . High-level expression of p25 in Escherichia coli has been established by oligonucleotide-directed mutagenesis of cDNA and insertion of the mutated cDNA into a T7-promoter expression vector . Recombinant p25 from the expressed cDNA sequence has been shown to comigrate with EAT p25 in electrophoresis and to react with antibodies against the EAT p25 . On the amino acid level, p25 shows about 80% sequence homology to the human stress protein hsp27 . Furthermore, p25 has similar isoforms of phosphorylation as demonstrated for small mammalian stress proteins from rat and human . From the results obtained, it is concluded that p25 is a mammalian stress protein, the abundance of which is related to growth characteristics of the Ehrlich ascites tumor. Eur J Biochem, 1989 Jan 15, 179(1), 185 - 94 Proteinase K from Tritirachium album Limber . Characterization of the chromosomal gene and expression of the cDNA in Escherichia coli; Gunkel FA et al.; The cDNA and the chromosomal gene encoding proteinase K from Tritirachium album Limber have been cloned in Escherichia coli and the entire nucleotide sequences of the coding region, as well as 5'- and 3'-flanking regions have been determined . The deduced primary translation product consisting of 384 amino acid residues (molecular mass = 40,231 Da) contains an N-terminal region of 105 amino acids not present in the mature protein . By analogy to the evolutionary-related bacterial subtilisins and other serine proteinases it is inferred that the primary secreted product is a zymogen containing a 15-amino-acid signal sequence and a 90-amino-acid propeptide . The propeptide is presumably removed in the later steps of the secretion process or upon secretion into the medium . The nucleotide-sequence analysis of the gene and its flanking regions has revealed that the proteinase-K gene is composed of two exons and one 63-bp-long intron located in the proregion . Furthermore, a putative promoter sequence and a capping site have been identified, suggesting that the transcription-start site is located 103-bp upstream of the ATG initiation codon . To express the proproteinase-K gene in E . coli, proproteinase-K cDNA was cloned in a plasmid vector under control of the tac promoter . The hybrid plasmid pSPPRO, constructed for this purpose, contained the cDNA coding for proproteinase K {from Ala (-91) to the C-terminal Ala (279)} fused to the N-terminal-signal-peptide sequence of the alkaline-phosphatase gene preceded by the tac promoter . E . coli BMH71-18, harbouring this plasmid, exhibited slight proteolytic activity when tested on skimmed-milk plates, suggesting that some fusion proteins were correctly secreted into the periplasm and processed to the mature proteinase K. J Biol Chem, 1989 Jan 15, 264(2), 842 - 7 Molecular cloning and primary structure of cDNA encoding the catalytic domain of rat liver aspartyl-tRNA synthetase; Mirande M et al.; A cDNA clone encoding rat liver aspartyl-tRNA synthetase was isolated by probing a lambda gt11 recombinant cDNA expression library with antibodies directed against the corresponding polypeptide from sheep liver . The 1930-base pairs-long cDNA insert allowed the expression in Escherichia coli of an active enzyme of mammalian origin . The nucleotide sequence of that cDNA, corresponding to the DRS1 gene, was determined . The open reading frame of DRS1 corresponds to a protein of Mr = 57,061, in good agreement with the previously determined molecular weight of the purified enzyme . The deduced amino acid sequence shows extensive homologies with that of yeast cytoplasmic aspartyl-tRNA synthetase, more than 50% of the residues being identical . In rat liver, aspartyl-tRNA synthetase occurs in two distinct forms: a dimeric enzyme and a component of a multienzyme complex comprising the nine aminoacyl-tRNA synthetases specific for arginine, aspartic acid, glutamic acid, glutamine, isoleucine, leucine, lysine, methionine, and proline . The primary structure of the DRS1 gene product is discussed in relation to the occurrence of two distinct forms of that enzyme. J Biol Chem, 1989 Jan 15, 264(2), 838 - 41 Formation of O6-methyldeoxyguanosine at specific sites in a synthetic oligonucleotide designed to resemble a known mutagenic hotspot; Richardson FC et al.; Four synthetic oligodeoxyribonucleotides of the sequence 5'-CCG1TG2G3G4ATATGGGCTG-3' were constructed with a 1',2'-{3H}deoxyguanosine located at one of the four sites indicated (1, 2, 3, or 4) . This sequence was derived from a region of the Escherichia coli xanthine-guanine phosphoribosyltransferase gene where position 4 is a site frequently mutated by N-methyl-N'-nitrosourea as compared to sites 1-3 . These four oligomers were alkylated in both single- and double-stranded form with N-methyl-N'-nitrosourea, and the relative amount of O6-methyldeoxyguanosine (O6-MedGuo) formed at each position was quantitated . Up to a 5-6-fold greater formation of O6-MedGuo was observed at positions 3 and 4 as compared to positions 1 and 2 . This uneven distribution was only observed in oligomers in the double-stranded form, suggesting that secondary structure was an important determinant in generating the uneven distribution of O6-MedGuo . Comparisons between the extent of O6-MedGuo formation and mutation frequency at the four positions suggest that a difference in the formation of promutagenic adducts at specific sites is just one of the factors involved in the generation of mutagenic "hotspots." The novel method developed was applied to the study of formation of O6-MedGuo at specific sites; however, it should be suitable for studying the formation and repair of DNA adducts generated by a variety of chemicals in a wide variety of DNA sequences. J Biol Chem, 1989 Jan 15, 264(2), 767 - 75 Conformational flexibility and folding of synthetic peptides representing an interdomain segment of polypeptide chain in the pyruvate dehydrogenase multienzyme complex of Escherichia coli; Radford SE et al.; Synthetic peptides (32 residues in length) were synthesized with amino acid sequences identical to, or related to, the long (alanine + proline)-rich region of polypeptide chain that links the innermost lipoyl domain to the dihydrolipoamide dehydrogenase-binding domain in the dihydrolipoyl acetyltransferase component of the pyruvate dehydrogenase multienzyme complex of Escherichia coli . The 400-MHz 1H NMR spectra of the peptide (Mr approximately 2800) closely resembled the sharp resonances in the spectrum of the intact complex (Mr approximately 5 x 10(6}, and the apparent pKa (6.4) of the side chain of a histidine residue in one of the peptides was found to be identical to that previously observed for a histidine residue inserted by site-directed mutagenesis into the corresponding position in the same (alanine + proline)-rich region of a genetically reconstructed enzyme complex . These results strongly support the view that the three long (alanine + proline)-rich regions of the dihydrolipoyl acetyltransferase chains are exposed to solvent and enjoy substantial conformational flexibility in the enzyme complex . More detailed analysis of the peptides by circular dichroism and by 1H and 13C NMR spectroscopy revealed that they were disordered in structure but were not random coils . In particular, all the Ala-Pro peptide bonds were greater than 95% in the trans configuration, consistent with a stiffening of the peptide structure . Differences in the sequences of the three long (alanine + proline)-rich segments may reflect structural tuning of these segments to optimize lipoyl domain movement in enzyme catalysis. J Biol Chem, 1989 Jan 15, 264(2), 1041 - 5 Primary structure of rat brain prostaglandin D synthetase deduced from cDNA sequence; Urade Y et al.; The amino acid sequence of rat brain prostaglandin D synthetase (Urade, Y., Fujimoto, N., and Hayaishi, O . (1985) J . Biol . Chem . 260, 12410-12415) was determined by a combination of cDNA and protein sequencing . cDNA clones specific for this enzyme were isolated from a lambda gt11 rat brain cDNA expression library . Nucleotide sequence analyses of cloned cDNA inserts revealed that this enzyme consisted of a 564- or 549-base pair open reading frame coding for a 188- or 183-amino acid polypeptide with a Mr of 21,232 or 20,749 starting at the first or second ATG . About 60% of the deduced amino acid sequence was confirmed by partial amino acid sequencing of tryptic peptides of the purified enzyme . The recognition sequence for N-glycosylation was seen at two positions of amino acid residues 51-53 (-Asn-Ser-Ser-) and 78-80 (-Asn-Leu-Thr-) counted from the first Met . Both sites were considered to be glycosylated with carbohydrate chains of Mr 3,000, since two smaller proteins with Mr 23,000 and 20,000 were found during deglycosylation of the purified enzyme (Mr 26,000) with N-glycanase . The prostaglandin D synthetase activity was detected in fusion proteins obtained from lysogens with recombinants coding from 34 and 19 nucleotides upstream and 47 and 77 downstream from the first ATG, indicating that the glycosyl chain and about 20 amino acid residues of N terminus were not essential for the enzyme activity . The amino acid composition of the purified enzyme indicated that about 20 residues of hydrophobic amino acids of the N terminus are post-translationally deleted, probably as a signal peptide . These results, together with the immunocytochemical localization of this enzyme to rough-surfaced endoplasmic reticulum and other nuclear membrane of oligodendrocytes (Urade, Y., Fujimoto, N., Kaneko, T., Konishi, A., Mizuno, N., and Hayaishi, O . (1987) J . Biol . Chem . 262, 15132-15136) suggest that this enzyme is a membrane-associated protein. FEMS Microbiol Lett, 1989 Jan 15, 48(2), 155 - 9 Transport of nutrients into the renal brush border membrane vesicles as marker in evaluating the role of antipili antibodies in modulation of ascending pyelonephritis in rats; Garg UC et al.; The uptake of D-glucose, L-aspartate, L-lysine and L-proline was investigated in renal brush border membrane (BBM) vesicles prepared from control, infected or passively-immunized-infected rats . Except L-aspartate, a progressive decrease in the uptake of these nutrients in both infected and immunized-infected groups during the course of infection was observed, but the changes were less apparent in immunized-infected rats than in non-immunized ones . The uptake of L-aspartate was increased in vesicles from early stages of infection but decreased in those from later stages . Also in L-aspartate uptake, the changes were smaller in immunized animals . The uptake of nutrients was detectable earlier than were histopathological alterations of both kidneys . The observations demonstrated that uptake of D-glucose and amino acids in the kidneys is disturbed prior to appearance of histopathological lesions and thus can be used for early detection of the disease . The data also demonstrate that antipili antibodies afford partial protection against ascending pyelonephritis. Biochem J, 1989 Jan 15, 257(2), 321 - 9 A truncated v-abl-derived tyrosine-specific tyrosine kinase expressed in Escherichia coli; Pritchard ML et al.; Several biochemical properties of a 43 kDa v-abl-encoded tyrosine-specific protein kinase (p43v-abl) expressed in Escherichia coli were examined . p43v-abl is a fragment of a 60 kDa v-abl-encoded precursor, p60v-abl, and could be generated by limited proteolysis of a purified p60v-abl with trypsin . Tryptic cleavage of p60v-abl was prevented in the presence of ATP . These results suggest that the catalytic kinase domain of v-abl-derived protein can be separated from other (regulatory) domains by limited proteolysis . p43v-abl readily phosphorylated tyrosine residues on several different protein and peptide substrates, including peptides containing only two amino acid residues . However, the local sequence of the tyrosine-containing peptide substrate significantly affected its rate of phosphorylation . Thus the primary structure and local conformation at the tyrosine acceptor site can play an important role in determining the substrate specificity of v-abl-derived kinase . Phosphorylation by p43v-abl requires Mn2+, Co2+ or Mg2+ and exhibits a strong preference for ATP as phosphate donor . Analogues of ATP and the thiol-reactive reagent N-ethylmaleimide inhibited p43v-abl kinase activity . Purified p43v-abl is intrinsically thermolabile (t1/2 = 5 min at 40 degrees C) and phosphorylates glycerol inefficiently (Km = 1.4 M). J Biol Chem, 1989 Jan 15, 264(2), 990 - 4 Site-directed mutagenesis of Pro-17 located in the glycine-rich region of adenylate kinase; Tagaya M et al.; Proline 17 in the glycine-rich region of adenylate kinase was replaced by Gly (the Gly-mutant) or Val (the Val-mutant) by site-directed mutagenesis . The mutant enzymes were purified to homogeneous states on sodium dodecyl sulfate-gel electrophoresis after solubilization of the proteins from the pellets of cell lysates of Escherichia coli . The apparent Km values of the Gly- and the Val-mutants for AMP increased approximately 7- and 24-fold, respectively, as compared with that of the wild-type enzyme . The apparent Km values for ATP also increased 7- and 42-fold in the Gly- and Val-mutants, respectively . In contrast, Vmax values of both mutant enzymes were comparable to that of the wild-type enzyme . These results suggest that Pro-17 plays an important role for the binding of substrates, but not for catalytic efficiency, although it does not directly interact with substrates . Adenosine diphosphopyridoxal, which specifically modifies Lys-21 in adenylate kinase (Tagaya, M., Yagami, T., and Fukui, T . (1987) J . Biol . Chem . 262, 8257-8261), inactivated the wild-type and mutant enzymes at almost the same rates . Interestingly, both mutant enzymes showed higher specificities for adenine nucleotides than the wild-type enzyme . Both mutant enzymes were less resistant than the wild-type enzyme against inactivation at elevated temperatures or by treatment with trypsin . It would appear that most of the properties of the mutant enzymes may be explained on the basis of a need for conformational flexibility of the loop which includes Pro-17 for substrate binding. J Biol Chem, 1989 Jan 15, 264(2), 1336 - 43 Characterization of the helicase activity of the Escherichia coli UvrAB protein complex; Oh EY et al.; The requirement for nucleotide hydrolysis in the DNA repair mechanism of the Escherichia coli UvrABC protein complex has been analyzed . The DNA-activated UvrAB ATPase activity is part of a helicase activity exhibited by the UvrAB protein complex . The helicase acts only on short duplexes and, therefore, is unlike other helicases such as those involved in DNA replication that unwind long duplexes . The strand displacement activity occurs in the 5'----3' direction and requires either ATP or dATP . The helicase activity is inhibited by UV photoproducts . The absence of this activity in a complex formed with proteolyzed UvrB (UvrB*), a complex also deficient in the endonuclease activity, suggests that this activity is important in the repair mechanism . The UvrAB protein complex may remain bound to a damaged site and by coupling the energy derived from ATP hydrolysis, alter the DNA conformation around the damage site to one that is permissive for endonucleolytic events . The conformational changes may take the form of DNA unwinding. J Biol Chem, 1989 Jan 15, 264(2), 1000 - 4 Isolation and characterization of the Escherichia coli mutL gene product; Grilley M et al.; The Escherichia coli mutL gene product has been purified to near homogeneity from an overproducing clone . The mutL locus encodes a polypeptide of 70,000 daltons as determined by denaturing gel electrophoresis . The native molecular weight of MutL protein as calculated from the sedimentation coefficient of 5.5 S and Stokes radius of 61 A is 139,000 daltons, indicating that MutL exists as a dimer in solution . In addition to its ability to complement methyl-directed DNA mismatch repair in mutL-deficient cell-free extracts, DNase I protection experiments demonstrate that the purified MutL protein interacts with the MutS-heteroduplex DNA complex in the presence of ATP. Eur J Biochem, 1989 Jan 15, 179(1), 155 - 60 A comparison of an ATPase from the archaebacterium Halobacterium saccharovorum with the F1 moiety from the Escherichia coli ATP synthase; Stan-Lotter H et al.; A purified ATPase associated with membranes from Halobacterium saccharovorum was compared with the F1 moiety from the Escherichia coli ATP synthase . The halobacterial enzyme was composed of two major (I and II) and two minor subunits (III and IV), whose molecular masses were 87 kDa, 60 kDa, 29 kDa and 20 kDa, respectively . The isoelectric points of these subunits ranged from 4.1 to 4.8, which in the case of the subunits I and II was consistent with the presence of an excess of acidic amino acids (20-22 mol/100 mol) . Peptide mapping of subunits I and II denatured with sodium dodecyl sulfate showed no relationship between the primary structures of the individual halobacterial subunits or similarities to the subunits of the F1 ATPase from E . coli . Trypsin inactivation of the halobacterial ATPase was accompanied by the partial degradation of the major subunits . This observation, taken in conjunction with molecular masses of the subunits and the native enzyme, was consistent with the previously proposed stoichiometry of 2:2:1:1 . These results suggest that H . saccharovorum, and possibly, halobacteria in general, possess an ATPase which is unlike the ubiquitous F0F1 ATP synthase. Biochem J, 1989 Jan 15, 257(2), 355 - 9 Investigation of alpha-deuterium kinetic isotope effects on the purine nucleoside phosphorylase reaction by the equilibrium-perturbation technique; Lehikoinen PK et al.; 1 . alpha-Deuterium kinetic isotope effects on the phosphorolysis of inosine catalysed by Escherichia coli purine nucleoside phosphorylase were measured by the equilibrium-perturbation technique, by using the change in absorbance at 250 nm (approx . 20%) . 2 . Values of 2H(V/K) of 1.13(9) at pH 5.0, 1.10(5) at pH 6.1, 1.09(4) at pH 7.3, 1.08 at pH 8.4 and 1.16(4) at pH 9.4 were obtained . 3 . These are compared with literature alpha-deuterium kinetic isotope effects for this and related reactions . 4 . The equilibrium constant, defined as {inosine}.{H2PO4-}/{hypoxanthine} {alpha-Rib f OPO3H-}, is 46 at 25 degrees C . 5 . N-3-beta-D-Ribofuranosylhypoxanthine, an impurity in chemically synthesized inosine, is a substrate. Cell, 1989 Jan 13, 56(1), 119 - 29 The MerR heavy metal receptor mediates positive activation in a topologically novel transcription complex; O'Halloran TV et al.; Several physical and chemical signals from the extracellular environment are known to be transduced into changes in gene expression through multiple step pathways; however, mechanisms for triggering cellular responses to heavy metal stress have yet to be elucidated . We demonstrate here one such mechanism that employs a single heavy metal receptor protein, MerR, to directly activate transcription of the bacterial mercuric ion resistance operon . The mercuric ion-MerR complex and E . coli RNA polymerase holoenzyme synergistically bind to the metal responsive promoter in an unprecedented spatial relationship to form transcriptionally competent complexes . The activator binds adjacent to and overlaps with the polymerase molecule between the consensus -35 and -10 promoter regions . Our results support a model for transcriptional activation that includes both effector-induced protein-protein interactions and activator-induced alteration in DNA structure. Science, 1989 Jan 13, 243(4888), 206 - 10 Correct folding of circularly permuted variants of a beta alpha barrel enzyme in vivo; Luger K et al.; An important question in protein folding is whether the natural amino and carboxyl termini and the given order of secondary structure segments are critical to the stability and to the folding pathway of proteins . Here it is shown that two circularly permuted versions of the gene of a single-domain beta alpha barrel enzyme can be expressed in Escherichia coli . The variants are enzymically active and are practically indistinguishable from the original enzyme by several structural and spectroscopic criteria, despite the creation of new termini and the cleavage of a surface loop . This novel genetic approach should be useful for protein folding studies both in vitro and in vivo. Cell, 1989 Jan 13, 56(1), 103 - 9 Conservation of complex DNA recognition domains between families of restriction enzymes; Cowan GM et al.; One polypeptide, designated S, confers sequence-specificity to the multisubunit type I restriction enzymes . Two families of such enzymes, K and A, include members that recognize diverse, bipartite, target sequences . The S polypeptides of the K family, while having areas of near identity, also contain two extensive regions of variable sequence . We now show that one of these, comprising the N-terminal 150 amino acids, specifies recognition of one component of the bipartite target sequence . We have determined the sequence recognized by EcoE, a member of the A family . This sequence, 5'GAG(N7)ATGC, has the trinucleotide GAG in common with EcoA and with StySB of the K family . We determined the nucleotide sequences of the S genes of EcoA and EcoE, and compared their predicted amino acid sequences with each other and with those of the five members of the K family . There is no general sequence similarity between families, but the domain of the S polypeptide of StySB, which specifies GAG, shows nearly 50 per cent identity with the amino variable region of the S polypeptides of EcoA and EcoE . A complex domain that recognizes and directs methylation of GAG is therefore common to enzymes of generally dissimilar amino acid sequence. Nucleic Acids Res, 1989 Jan 11, 17(1), 19 - 29 Nuclear transcriptional activity of the tobacco plastid psbA promoter; Cornelissen M et al.; The plastid psbA promoter of tobacco was used with the aim to construct plastid specific marker genes . Upon transfer to the tobacco nuclear genome the plastid promoter fragment appeared to specify a messenger RNA . Placed 5' to the bar or nptII coding sequences the level of expression is sufficient to obtain a selectable phenotype . The transcription start site in the nucleus is site specific and is located 4 nucleotides downstream relative to the start site used in plastids . Translational fusions of the psbA coding sequence with the nptII and bar coding sequences revealed that the psbA leader sequence and the psbA translation start codon, being the second ATG codon, are recognized by the plant cytoplasmic translation apparatus . A promoter cassette utilisable in both E . coli and tobacco, was obtained by placing the CaMV 35S enhancer 5' to the psbA promoter. Nucleic Acids Res, 1989 Jan 11, 17(1), 135 - 45 Mutational analysis of the nucleotide sequence at the FNR-dependent nirB promoter in Escherichia coli; Jayaraman PS et al.; During anaerobic growth of E . coli, the FNR protein activates transcription initiation at the nirB promoter . After chemical synthesis using deliberately contaminated nucleotides, we isolated a series of recombinant plasmids with single point mutations or one base pair deletions in the nirB promoter . The effects of these alterations on the anaerobic induction of promoter activity were measured . Mutations that abolish anaerobic induction identify the -10 hexamer sequence whilst changes that allow reduced induction suggest positions involved in FNR binding . Comparison of the nucleotide sequence of the nirB promoter with other promoters that are regulated by FNR show clear homologies, suggesting consensus sequences for FNR binding sites, and confirming that some of the point mutations described here do indeed act by weakening FNR binding. J Chromatogr, 1989 Jan 6, 461, 45 - 61 Production scale purification of biosynthetic human insulin by reversed-phase high-performance liquid chromatography; Kroeff EP et al.; A process based on reversed-phase high-performance liquid chromatography (RP-HPLC) has been developed for the purification of biosynthetic human insulin (BHI) . The RP-HPLC procedure has been successfully integrated into the multimodal chromatographic production process used to purify kilogram quantities of BHI . Axial compression column technology was used in the scale-up process . The RP-HPLC procedure yields an insulin product having high chemical purity and full biological activity. J Mol Biol, 1989 Jan 5, 205(1), 71 - 83 Temporal regulation and overlap organization of two Caulobacter flagellar genes; Kaplan JB et al.; The biogenesis of the bacterial flagellum and chemotaxis apparatus in both Escherichia coli and Caulobacter crescentus requires the ordered expression of over 40 genes whose expression is controlled by a trans-acting regulatory hierarchy . In C . crescentus, additional control mechanisms ensure that the transcription of these genes is initiated at the correct time in the cell cycle . We demonstrate here that two flagellar genes, flaE and flaY, whose products function in trans to modulate the level of transcription of other flagellar genes, are themselves temporally controlled . DNA sequence analysis of the 3413 base-pairs encompassing the flaE and flaY coding sequences and the 5' regulatory region showed that flaE encodes a protein of 16,000 Mr and flaY a protein of 17,000 Mr . Evidence that flaE and flaY are transcribed as a polycistronic message includes (1) the polar effect of Tn5 insertions; (2) deletion analysis showing that the flaE promoter is essential for complementation of both flaE and flaY alleles; and (3) nuclease S1 assays showing protection of a transcript spanning both genes . The transcript start site in front of flaE was determined and the -10 region conforms to the E . coli sigma 28 promoter consensus sequence . Nuclease S1 analysis also revealed a protected fragment whose size was consistent with a transcript initiating in vivo at a consensus "nif" promoter sequence in front of the flaY gene . The entire promoter region and an upstream consensus sequence that might be a regulatory element for the flaY gene lies within the carboxyl-terminal coding sequence of the flaE gene. J Mol Biol, 1989 Jan 5, 205(1), 269 - 73 Expression of native rabbit light meromyosin in Escherichia coli . Observation of a powerful internal translation initiation site; Maeda K et al.; The cDNA-sequence coding for rabbit skeletal muscle light meromyosin (LMM) was placed under the control of the lambda promoter (PL) of an Escherichia coli expression vector . The resulting plasmid pEXLMM74 expressed non-fused rabbit skeletal muscle LMM with yields ranging from 1 to 5% of the total proteins of E . coli . This LMM was specifically recognized by polyclonal antibodies raised against chicken pectoralis muscle myosin . It could be highly enriched from E . coli extracts by using two cycles of high and low ionic strength buffer . The partially purified protein contained a major side-product, with a calculated molecular mass of 59 kilodaltons, that is produced by translation initiation from a site in the coding region of LMM . After deletion of the translation initiation site derived from the expression plasmid, only the 59 kilodalton protein is expressed in E . coli from the resulting plasmid pEXLMM59 . Both the 74 and 59 kilodalton proteins were shown to form paracrystals . They were studied by electron microscopy using negative staining and were found to show characteristic striations with an axial periodicity of about 43 nm . By circular dichroism measurement we showed that the purified 59 kilodalton protein is folded mostly as an alpha-helix. J Mol Biol, 1989 Jan 5, 205(1), 179 - 88 Structure of phage 434 Cro protein at 2.35 A resolution; Mondragon A et al.; The crystal structure of phage 434 Cro protein has been determined and refined against 2.35 A data to an R-factor of 19.5% . The protein comprises five alpha-helices and shows the helix-turn-helix motif found in other repressor proteins. J Mol Biol, 1989 Jan 5, 205(1), 103 - 13 Comparison of ultraviolet irradiation-induced mutagenesis of the lacI gene in Escherichia coli and in human 293 cells; Hsia HC et al.; We report the sequence changes in the Escherichia coli lacI gene in 133 mutants detected after passage of an ultraviolet-irradiated shuttle vector human 293 cells . The results are compared with our previous studies of the lacI gene after ultraviolet light treatment in E . coli . In human cells, base substitutions predominate, and frameshifts are found much less frequently than in bacteria . The most frequent base change is the G.C to A.T transition . Overall, 110 to 112 transitions were G.C to A.T . Some of the hotspots seen in lacI in bacteria are prominent also in human 293 cells, suggesting that the same lesions are targeting mutations in both systems . Transitions are found almost exclusively at sequences at which pyrimidine-pyrimidine photoproducts can form . The data are consistent with the notion that a significant fraction of ultraviolet irradiation-induced mutagenesis in mammalian systems occurs by adding an A across from a photolesion . Double mutations are significantly more frequent in human cells than in bacteria . Reasons for this difference are discussed. J Biol Chem, 1989 Jan 5, 264(1), 596 - 601 Shiga toxin, Shiga-like toxin II variant, and ricin are all single-site RNA N-glycosidases of 28 S RNA when microinjected into Xenopus oocytes; Saxena SK et al.; Ricin, Shiga toxin, and Shiga-like toxin II (SLT-II, Vero toxin 2) exhibit an RNA N-glycosidase activity which specifically removes a single base near the 3' end of 28 S rRNA in isolated rat liver ribosomes and deproteinized 28 S rRNA (Endo Y., Mitsui, K., Motizuki, M., & Tsurugi, K . (1987) J . Biol . Chem . 262, 5908-5912; Endo Y . & Tsurugi, K . (1987) J . Biol . Chem . 262, 8128-8130, Endo, Y., Tsurugi, K., Yutsudo, T., Takeda, Y., Ogasawara, K . & Igarashi, K . (1988) Eur . J . Biochem . 171, 45-50) . These workers identified the single base removed, A-4324, by examining a 28 S rRNA degradation product which was generated by contaminating ribonucleases associated with the ribosomes . To determine whether this N-glycosidase activity applies in living cells, we microinjected ricin into Xenopus oocytes . We also microinjected Shiga toxin and a variant of Shiga-like toxin II (SLT-IIv) . All three toxins specifically removed A-3732, located 378 nucleotides from the 3' end of 28 S rRNA . This base is analogous to the site observed in rat 28 S rRNA for ricin, Shiga toxin, and SLT-II . Purified, glycosylated, ricin A chain contains this RNA N-glycosidase activity in oocytes . We also demonstrated that the nonglycosylated A subunit of recombinant ricin exhibits this RNA N-glycosidase activity when injected into Xenopus oocytes . Ricin, Shiga toxin, and SLT-IIv also caused a rapid decline in oocyte protein synthesis for nonsecretory proteins. J Biol Chem, 1989 Jan 5, 264(1), 540 - 5 An RNA secondary structure switch between the inactive and active conformations of the Escherichia coli 30 S ribosomal subunit; Ericson G et al.; Psoralen cross-linking was used to produce intramolecular cross-links in the Escherichia coli 16 S ribosomal RNA in the inactive and active forms of the 30 S subunit . A number of psoralen cross-links were made in the inactive form that were not made in the active form . The most frequent of these cross-links was sequenced by a series of techniques and identified as C-924 to U-1532 . In this region, a three-base complementary, (921-923).(1532-1534), forms a site where psoralen can stack and produce a cross-link between C-924 and U-1532 . When psoralen monoadducts were placed on inactive subunits and the conformation was switched to the active form before cross-linking, a new cross-link involving U-1393 was detected . U-1393 is part of the complementarity, (923-925).(1391-1393), that has previously been proposed as being an element of the functional secondary structure on the basis of sequence comparison . The complementarity between (921-923).(1532-1534) occurs in most nonmitochondrial small subunit RNAs; however, there are several counter examples in which it does not occur . This suggests that this alternate secondary structure interaction is not necessary for the function of the 30 S subunit. J Biol Chem, 1989 Jan 5, 264(1), 531 - 9 Biosynthesis and incorporation into protein of norleucine by Escherichia coli; Bogosian G et al.; The methionine analog norleucine was produced during the synthesis of bovine somatotropin by Escherichia coli strain W3110G containing the recombinant plasmid pBGH1 . Norleucine was generated by the leucine biosynthetic pathway from pyruvate or alpha-ketobutyrate in place of alpha-ketoisovalerate as the initial substrate . The intracellular level of norleucine was high enough to permit the analog to compete successfully with methionine for incorporation into protein . Two ways were found to prevent either the formation of norleucine or its incorporation into protein . The endogenous synthesis of norleucine was eliminated by deleting the leucine operon . The addition of sufficient methionine or 2-hydroxy-4-methylthiobutanoic acid, a precursor of methionine, to the culture medium prevented any norleucine from being incorporated into protein. J Biol Chem . 1989 Jan 5;264(1):5. Crystallization of the bifunctional biotin operon repressor; Brennan RG et al.; The bifunctional birA gene product, BirA, which represses the biotin biosynthetic bio operon and also activates biotin in Escherichia coli, has been crystallized . The crystals have the tetragonal space group P4(1)2(1)2, or its enantiomorph, with unit cell dimensions a = b = 114.0 A and c = 60.2 A and diffract to at least 2.3 A resolution . The crystal packing requires that the monomers of the birA protein be arranged as dimers with two-fold symmetry . BirA is the first protein to be crystallized that is both a transcriptional regulator and an enzyme. J Biol Chem, 1989 Jan 5, 264(1), 342 - 6 Fast measurement of galactoside transport by lactose permease; Dornmair K et al.; Lactose permease of Escherichia coli was reconstituted into vesicles of dimyristoylphosphatidylcholine, and the rate of galactoside counterflow was measured in the millisecond time range . The turnover number and the half-saturation constant for transport agree with the values known for cells . This result demonstrates that lactose permease is the sole protein necessary for galactoside transport . Furthermore, lactose permease seems not to require a high level of negatively charged lipids or a certain degree of unsaturation of the lipid hydrocarbon chains . However, the lipids must be in the fluid state, because the transport rate drastically decreases below the lipid ordered fluid phase transition. J Biol Chem, 1989 Jan 5, 264(1), 220 - 4 A 37-base pair element in the far upstream spacer region can enhance transcription of rat rDNA in vitro and can bind to the core promoter-binding factor(s); Garg LC et al.; Previous studies in this laboratory have demonstrated that a 174-base pair (bp) rat rDNA spacer region located more than 2 kilobase pairs upstream of the initiation site, can enhance rat rDNA transcription in vitro independent of its orientation or distance or when inserted downstream of the initiation site . Further dissection of this region showed that transcription of a rDNA fragment containing just 37 bp of the spacer sequence, located between -2.183 and -2.219 kilobase pairs upstream of the initiation site, is 8-fold greater than that of the rDNA fragment devoid of the spacer element . Electrophoretic mobility shift assay demonstrated specific interaction of the 37-bp DNA fragment with a cellular protein(s) . The spacer DNA competed for essential transcription factors as demonstrated by the absence of transcription following preincubation of the extract with the 37-bp fragment . Similar competition was also observed when a 58-bp PolI promoter was substituted for the enhancer fragment . The binding of the factor(s) to the enhancer element was not altered when coding and noncoding strands of the 37-bp oligodeoxynucleotide were used separately in the competition assay . Since the 37-bp enhancer region and the core promoter do not exhibit any significant sequence homology, the factor(s) appears to interact with these cis-acting elements in a sequence-independent manner. J Biol Chem, 1989 Jan 5, 264(1), 465 - 71 Recombinant alpha i-3 subunit of G protein activates Gk-gated K+ channels; Mattera R et al.; G proteins, particularly those sensitive to pertussis toxin, are difficult to separate biochemically, creating uncertainty in functional assignments . For this reason the cDNAs encoding G alpha i-3 and two of the G alpha s splice variants were expressed as fusion proteins in Escherichia coli using a T7 promoter-based expression system . These proteins were denoted r alpha i-3 and r alpha s (short and long) and accumulated in bacteria to as much as 5-10% of total cellular protein, of which 5-10% was soluble in lysates . Soluble r alpha subunits were tested for stimulation of K+ channel activity in inside-out atrial membrane patches and for reconstitution of cyc- adenylyl cyclase activity . r alpha i-3, activated either by guanosine 5'-(3-thio)triphosphate (GTP gamma S) or AlF-4, stimulated in a concentration-dependent manner single channel K+ currents in isolated atrial membrane patches of three species: guinea pigs, neonatal rats, and embryonic chick . In contrast, GTP gamma S-activated r alpha s did not . In agreement with a similar study by Graziano et al . (Graziano, M . P., Casey, P . J . and Gilman, A . G . (1987) J . Biol . Chem . 262, 11375-11381), both r alpha s forms reconstituted GTP gamma S-stimulated cyc- adenylyl cyclase activity, albeit at concentrations 50-100 times higher than those needed with native Gs . The concentrations of r alpha i-3 needed to stimulate the K+ channels were also higher than needed with native human erythrocyte Gk, in this case 30-50 times . Single K+ channel currents stimulated by r alpha i-3 were indistinguishable from those stimulated by the natural effector acetylcholine . Thus, bacterial expression of G alpha subunits provided the means to demonstrate unequivocally that Gi-3 has intrinsic Gk activity. J Biol Chem, 1989 Jan 5, 264(1), 437 - 42 Export of Escherichia coli alkaline phosphatase attached to an integral membrane protein, SecY; Akiyama Y et al.; The SecY protein is a membrane-bound factor required for bacterial protein export and embedded in the cytoplasmic membrane by its 10 transmembrane segments . We previously proposed a topology model for this protein by adapting the Manoil-Beckwith TnphoA approach, a genetic method to assign local disposition of a membrane protein from the enzymatic activity of the alkaline phosphatase (PhoA) mature sequence attached to the various regions . SecY-PhoA hybrid proteins with the PhoA domain exported to the periplasmic side of the membrane have been obtained at the five putative periplasmic domains of the SecY sequence . We now extended this method to apply it to follow export of the newly synthesized PhoA domain . Trypsin treatment of detergent-solubilized cell extracts digested the internalized (unfolded) PhoA domain but not those exported and correctly folded . One of the hybrid proteins was cleaved in vivo after export to the periplasm, providing a convenient indication for the export . Results of these analyses indicate that export of the PhoA domain attached to different periplasmic regions of SecY occurs rapidly and requires the normal functioning of the secY gene supplied in trans . Thus, this membrane protein with multiple transmembrane segments contains multiple export signals which can promote rapid and secY-dependent export of the PhoA mature sequence attached to the carboxyl-terminal sides. J Mol Biol, 1989 Jan 5, 205(1), 149 - 64 Lambda cro repressor complex with OR3 operator DNA . 19F nuclear magnetic resonance observations; Metzler WJ et al.; The interaction of lambda cro repressor with DNA is probed using synthetic 17 base-pair OR3 operators in which 5-fluorodeoxyuridine has been systematically incorporated at each of the nine positions normally occupied by a thymidine residue . By monitoring changes in chemical shift of the fluorine resonances upon cro repressor binding in aqueous buffers of varying 2H2O content, we have examined the specific cro repressor-OR3 DNA complex in detail . The results are interpreted in the context of the popular model for cro repressor-OR3 complex derived from the three-dimensional structure of the cro repressor in the absence of DNA . The results presented here not originally predicted by the model are: (1) there is an asymmetry in the environment at the two ends of the operator, although the base-pairs involved and the cro repressor dimer are symmetric; (2) there appears to be distortion of the DNA helix at two distinct positions; (3) changes of the DNA environment in the middle of the helix suggest additional DNA distortion not near the contact areas proposed in the model. J Biol Chem, 1989 Jan 5, 264(1), 305 - 11 Mutations in the conserved proline 43 residue of the uncE protein (subunit c) of Escherichia coli F1F0-ATPase alter the coupling of F1 to F0; Miller MJ et al.; The conserved Pro43 residue of the uncE protein (subunit c) of the Escherichia coli F1F0-ATPase was changed to Ser or Ala by oligonucleotide-directed mutagenesis, and the mutations were incorporated into the chromosome . The resultant mutant strains were capable of oxidative phosphorylation as indicated by their ability to grow on succinate and had growth yields on glucose that were 80-90% of wild type . Membrane vesicles from the mutants were slightly less efficient than wild type vesicles in ATP-driven proton pumping as indicated by ATP-dependent quenching of quinacrine fluorescence . The decreased quenching response was not due to increased H+ leakiness of the mutant membranes or to loss of F1-ATPase activity from the membrane . These results indicate that the mutant F1F0-ATPases are defective in coupling ATP hydrolysis to H+ translocation . The membrane ATPase activity of the mutants was inhibited less by dicyclohexylcarbodiimide than that of wild type . The decrease in sensitivity to inhibition by dicyclohexylcarbodiimide was caused primarily by dissociation of the F1-ATPase from the mutant F0 in the ATPase assay mixture . These results support the idea that Pro43, and neighboring conserved polar residues play an important role in the binding and functional coupling of F1 to F0 . Although a Pro residue is found at position 43 in all species of subunit c studied, surprisingly, it is not absolutely essential to function. J Biol Chem, 1989 Jan 5, 264(1), 409 - 18 Expression of Gs alpha in Escherichia coli . Purification and properties of two forms of the protein; Graziano MP et al.; Cloning of complementary DNAs that encode either of two forms of the alpha subunit of the guanine nucleotide-binding regulatory protein (Gs) that stimulates adenylyl cyclase into appropriate plasmid vectors has allowed these proteins to be synthesized in Escherichia coli (Graziano, M.P., Casey, P.J., and Gilman, A.G . (1987) J . Biol . Chem . 262, 11375-11381) . A rapid procedure for purification of milligram quantities of these proteins is described . As expressed in E . coli, both forms of Gs alpha (apparent molecular weights of 45,000 and 52,000) bind guanosine 5'-(3-O-thio)triphosphate stoichiometrically . The proteins also hydrolyze GTP, although at different rates (i.e . 0.13.min-1 and 0.34.min-1 at 20 degrees C for the 45- and the 52-kDa forms, respectively) . These rates reflect differences in the rate of dissociation of GDP from the two proteins . Both forms of recombinant Gs alpha have essentially the same kcat for GTP hydrolysis, approximately 4.min-1 . Recombinant Gs alpha interacts functionally with G protein beta gamma subunits and with beta-adrenergic receptors . The proteins can also be ADP-ribosylated stoichiometrically by cholera toxin . This reaction requires the addition of beta gamma subunits . Both forms of recombinant Gs alpha can reconstitute GTP-, isoproterenol + GTP-, guanosine 5'-(3-O-thio)triphosphate-, and fluoride-stimulated adenylyl cyclase activity in S49 cyc- membranes to maximal levels, although their specific activities for this reaction are lower than that observed for Gs purified from rabbit liver . Experiments with purified bovine brain adenylyl cyclase indicate that the affinity of recombinant Gs alpha for adenylyl cyclase is 5-10 times lower than that of liver Gs under these assay conditions; however, the intrinsic capacity of the recombinant protein to activate adenylyl cyclase is normal . These findings suggest that Gs alpha, when synthesized in E . coli, may fail to undergo a posttranslational modification that is crucial for high affinity interaction of the G protein with adenylyl cyclase. J Biol Chem, 1989 Jan 5, 264(1), 10 - 3 Identification of distinct cytoplasmic targets for ras/R-ras and rho regulatory proteins; Garrett MD et al.; The protein products of the mammalian ras genes, p21ras, are regulatory guanine nucleotide binding proteins that are involved in the control of cell proliferation, though the exact biochemical processes regulated are unknown . Recently a cytoplasmic protein has been identified that interacts with and increases the GTPase activity of p21ras . It has been shown that this GTPase-activating protein, or GAP, interacts with the effector domain of ras, leading us and others to propose that GAP may be the target for regulation by p21ras . It has become apparent that ras is part of a much larger family of proteins, and at least 15 ras-related genes have now been identified in the mammalian genome . Each encodes a small (about 21 kDa) guanine nucleotide binding protein, but the functions of none of these regulatory molecules are known . We report here that mammalian cytoplasmic extracts contain GAP-like activity toward the products of two other ras-related genes, R-ras and rho . It appears that p23R-ras interacts with the same 125-kDa GAP protein as p21ras whereas p21rho interacts with a distinct 29-kDa protein, rho GAP. J Biol Chem, 1989 Jan 5, 264(1), 119 - 23 The fidelity of DNA polymerases during in vitro replication of a template containing 5-bromouracil at a specific site; Heinrich M et al.; The miscoding properties of a 5-bromodeoxyuridine (dB) containing DNA template during in vitro replication have been investigated . 5-bromodeoxyuridine was introduced site-specifically into the amber 16 codon of a 25-mer oligodeoxynucleotide representing part of the sequence of phi x174am16(+)DNA . The dB containing oligodeoxynucleotide served as a template for in vitro replication by DNA polymerase alpha, DNA polymerase I (Escherichia coli) and AMV reverse transcriptase . The amber 16 revertant assay was used to detect the presence of misincorporated bases in the replication products . For all three DNA polymerases, the presence of dB does not constitute a significant barrier to replication . Errors at the position of dB substitution were found to originate exclusively from dGTP:dB mispairing during in vitro replication thus inducing A-T----G-C transitions . The dGTP:dB mismatches are formed at a 2-4-fold higher frequency as compared to dGTP:T mismatches . Our results indicate that the miscoding potential of dB-substituted DNA templates during replication is only weak at the specific site observed. FEBS Lett, 1989 Jan 2, 242(2), 218 - 24 Improved strategies for the determination of protein structures from NMR data: the solution structure of acyl carrier protein; Holak TA et al.; The hybrid method that combines the early stages of a distance geometry program with simulated annealing in the presence of NMR constraints was optimized to obtain structures fully consistent with the observed NMR data . This was achieved by using more restrictive bounds of the NOE constraints than those usually used in the literature and by grouping the NOEs into classes dependent on the quality of the experimental NOE data . The 'floating' stereospecific assignment introduced at the simulated annealing stage of the calculations further improved the definition of the local conformation . An improved sampling and convergence property of the hybrid method was obtained by means of fitting the substructure obtained from the distance geometry program to different conformations . Compared to the standard hybrid methods, this procedure gave superior structures for a 77 amino acid protein, acyl carrier protein from Escherichia coli. FEBS Lett, 1989 Jan 2, 242(2), 431 - 4 SecA protein is directly involved in protein secretion in Escherichia coli; Kawasaki H et al.; A high-expression plasmid for the secA gene was constructed . The SecA protein was then overproduced in E . coli and purified . The purified SecA stimulated the in vitro translocation of a model secretory protein into inverted membrane vesicles pretreated with 4 M urea . Membrane vesicles from a secAts mutant exhibited lower translocation activity, which was enhanced by SecA . These results indicate that SecA is directly involved in protein secretion across the cytoplasmic membrane. FEBS Lett, 1989 Jan 2, 242(2), 319 - 24 Reconstitution of apo-porphobilinogen deaminase: structural changes induced by cofactor binding; Scott AI et al.; Expression of porphobilinogen deaminase in a hemB- strain of E . coli has permitted the isolation of the apoenzyme, i.e . deaminase lacking the porphobilinogen-derived dipyrromethane cofactor . Incubation of purified apoenzyme with porphobilinogen resulted in reconstitution of the covalently attached dipyrromethane cofactor, indicating no additional cofactors or enzymes are required for biosynthesis of holoenzyme . Electrophoretic and 13C-NMR spectroscopic analyses demonstrate that the apoenzyme exists in a conformationally unstable form which is converted to a highly stable tertiary structure on covalent attachment of the dipyrromethane cofactor. Eur J Biochem, 1989 Jan 2, 178(3), 741 - 9 In vitro synthesis of colominic acid by membrane-bound sialyltransferase of Escherichia coli K-235 . Kinetic properties of this enzyme and inhibition by CMP and other cytidine nucleotides; Ortiz AI et al.; The membrane-bound sialyltransferase obtained from Escherichia coli K-235 grown in a chemically defined medium (ideal for colominic acid production) was studied . The in vivo half-life calculated for this enzyme was 20 h . Kinetic tests revealed (at 33 degrees C and pH 8.3) hyperbolic behaviour with respect to CMP-Neu5Ac (Km250 microM) and a transition temperature at 31.3 degrees C . The enzyme was inhibited by NH4+, some divalent cations and by several agents that react with thiol groups . Detergents and fatty acids also inhibited the sialyltransferase activity . In vitro synthesis of colominic acid is strongly inhibited by CMP by blocking the incorporation of {14C}Neu5Ac into a protein-complex intermediate and therefore into free polymer . CDP and CTP also inhibited (91% and 84%) this enzyme activity whereas cytosine and cytidine had no effect . CMP inhibition corresponded to a competitive model the calculated Ki was 30 microM . Incubations of protein{14C}Neu5Ac with CMP, CDP and CTP led to de novo synthesis of CMP-{14C}Neu5Ac . The presence of colominic acid, which usually displaces the reaction equilibrium towards polymer synthesis, did not affect this de novo CMP-{14C}Neu5Ac formation . CMP also inhibited in vivo colominic acid biosynthesis. Eur J Biochem, 1989 Jan 2, 178(3), 627 - 34 Purification of GTP cyclohydrolase I from human liver and production of specific monoclonal antibodies; Schoedon G et al.; GTP cyclohydrolase I, the first enzyme in the de novo biosynthesis of tetrahydrobiopterin, was enriched more than 13,000-fold from human liver by preparative isoelectric focusing using Sephadex G-200 SF gels . The pI of the active enzyme was determined as 5.6 by analytical isoelectric focusing in the same matrix . The native enzyme has an apparent molecular mass of 440 kDa and appears to be composed of eight 50-kDa subunits as estimated from SDS/PAGE . The enriched enzyme preparation was used to produce specific monoclonal antibodies . From 11 monoclonal antibodies obtained, one was extensively characterized for further applications . This monoclonal antibody belongs to the IgM class and shows immunoreactivity with GTP cyclohydrolase I both from man and from Escherichia coli . It is capable of highly sensitive detection of GTP cyclohydrolase I by ELISA and by Western blot analysis . The monoclonal antibody was used for the immunoenzymatic localisation of GTP cyclohydrolase I in human peripheral blood mononuclear cells . Furthermore, it was possible to demonstrate the absence of immunoreactivity in cells with GTP cyclohydrolase I deficiency . The antibody's use as a tool either for differential diagnosis of atypical phenylketonuria due to GTP cyclohydrolase I deficiency or prenatal diagnosis of this severe inherited metabolic disease is now under investigation. Zentralbl Bakteriol Mikrobiol Hyg {A}, 1989 Jan, 270(3), 424 - 33 Duck hepatitis B virus: cloning and subcloning of the viral genome; Oexle K et al.; In the course of studies on the biology of hepadnavirus infections, duck hepatitis B virus (DHBV) DNA was isolated from the serum of a German Pekin duck . Viral DNA was cloned in E . coli using pBR 322 DNA as a vector . The cloned DHBV DNA F 1-6 was characterised by restriction enzyme analyses . DHBV DNA F 1-6 was subcloned in both orientations in plasmid pSP 65 to produce strand-specific RNA probes . These probes specifically identified asymmetrically replicating nascent minus-strand DHBV DNA species or plus-strand viral RNA transcripts. Protein Eng, 1989 Jan, 2(5), 387 - 91 Chemical synthesis, expression and product assessment of a gene coding for biologically active human tumour necrosis factor alpha; Ashman K et al.; A gene encoding human tumour necrosis factor alpha (TNF-alpha) has been chemically synthesized, cloned and expressed to yield a biologically active protein in Escherichia coli . The 480-bp gene was assembled by enzymic ligation of 32 oligonucleotides, cloned directly into M13mp18 for sequence verification and expressed in the broad host range high-level expression vector pMMB66EHST . Expressed recombinant TNF-alpha was shown to have the correct molecular weight, processed N-terminal sequence, antibody cross-reactivity and tumour cell killing activity . The expression product of the synthetic gene has been purified to homogeneity by a two-step ion-exchange procedure and the purified material shown to be active. Protein Eng, 1989 Jan, 2(5), 353 - 7 Selective proton labelling of amino acids in deuterated bovine calbindin D9K . A way to simplify 1H-NMR spectra; Brodin P et al.; A technique for proton labelling of selected amino acids in deuterated calbindin D9K, heterologously expressed in E . coli, was developed in order to simplify and obtain higher resolution in 1H-NMR spectra . The spect