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| EMBO J, 1990 Nov, 9(11), 3767 - 75 Control of replication of plasmid R1: structures and sequences of the antisense RNA, CopA, required for its binding to the target RNA, CopT; Persson C et al.; The replication frequency of plasmid R1 is determined by the availability of the RepA protein, which acts at the origin of replication to promote initiation . Synthesis of RepA is negatively regulated both at the transcriptional and post-transcriptional levels . Post-transcriptional control is exerted through the action of an antisense RNA, CopA RNA . The target of CopA RNA, CopT RNA, is located in the leader region of the RepA mRNA . Binding between CopA and CopT inhibits repA expression . We have previously presented an in vitro analysis of the binding reaction between CopA and CopT RNAs . In this communication, we extend the in vitro analysis by determining the regions of CopA required for binding, and also demonstrate that binding occurs in at least two steps . The first step is the formation of an initial, transient complex; stem-loop II is the structure in CopA necessary and sufficient for this step . The subsequent step(s), resulting in the formation of a complete duplex, requires a stretch of single-stranded nucleotides located 5' to stem-loop II in CopA, and its counterpart in CopT . We show that the single-stranded region can be positioned on either side of stem-loop II provided that there is a complementary stretch of nucleotides in CopT, indicating that the second step(s) is not sequence-specific . Furthermore, the effects of salt concentration and temperature on the binding reaction indicate that duplex formation occurs through a mechanism of gradual intra-strand breaking and inter-strand formation of hydrogen bonds. Biotechnology (N Y), 1990 Nov, 8(11), 1036 - 40 High-level direct expression of semi-synthetic human interleukin-6 in Escherichia coli and production of N-terminus met-free product; Yasueda H et al.; We have developed a direct expression system for high-level production of recombinant human interleukin-6 (rhIL-6) in Escherichia coli . In this system, (i) the natural N-terminal coding region of the hIL-6 gene was replaced by a synthetic sequence containing A-T rich codons, (ii) dual Shine-Dalgarno (SD) sequences were employed, (iii) an A-T rich segment was inserted in front of the initiation codon to avoid putative mRNA secondary structure in the region and (iv) the natural amber termination codon of the hIL-6 gene was changed to an ocher stop codon . The hIL-6 polypeptide, synthesized at a high level, formed cytoplasmic inclusion bodies . After refolding, the N-terminal methionine was removed by aminopeptidase-P in vitro . The purified recombinant hIL-6 had B-cell differentiation activity equivalent to natural IL-6 from a human T-cell culture. Biotechnology (N Y), 1990 Nov, 8(11), 1030 - 3 Protective surface antigen P69 of Bordetella pertussis: its characterization and very high level expression in Escherichia coli; Makoff AJ et al.; The surface antigen, P69 of Bordetella pertussis, an N-terminal fragment of the precursor protein, P93, is likely to be an important component of future subunit vaccines against whooping cough . We have expressed several defined N-terminal fragments of P93 in E . coli and compared their electrophoretic mobilities with that of purified P69 from B . pertussis . These experiments show that P69 is considerably smaller than the 69 kD originally estimated from its gel mobility and is probably 60.4 kD in size . Our initial plasmids expressed only very low levels of this antigen . We diagnosed the limiting factor to be a poor ribosome binding site (RBS) by demonstrating a large stimulation of expression on a two-cistron plasmid . The limitation of expression could be completely overcome by only two base changes close to the initiation codon, resulting in a further increase in expression of P69 at levels to 30-40% total cell protein . Although the protein accumulated as insoluble inclusion bodies, it could be solubilized by guanidinium chloride. Biotechnol Prog, 1990 Nov-Dec, 6(6), 494 - 7 Fractionation of recombinant tumor necrosis factor using hydrophobic and hydrophilic membranes; Dorin G et al.; Recombinant tumor necrosis factor (TNF) is expressed in Escherichia coli as a soluble intracellular protein . A purification process is described that incorporates a hydrophilic membrane (cellulosic) separation followed by a hydrophobic one (PTFE) . The hydrophilic step is a traditional one in that species are separated primarily on the basis of size . The hydrophobic step separates species on the basis of parameters apparently not related to size . By combining these two steps, an increase in TNF purity of 7-10-fold can be achieved with a yield of 50% . The effects of cellular debris and pH on selectivity and recovery of the hydrophobic filtration step are discussed. Gene, 1990 Oct 30, 95(1), 1 - 7 Cloning and sequencing the gene encoding Escherichia coli ribonuclease I: exact physical mapping using the genome library; Meador J 3rd et al.; The amino acid (aa) sequence of the N terminus of Escherichia coli RNase I was determined . A mixed oligodeoxynucleotide coding for that sequence was used to probe the 476 lambda clones of Kohara et al . {Cell 50 (1987) 495-508} . DNA from these clones carry almost the entire E . coli chromosome in overlapping segments . Two overlapping clones hybridized to the probe sequence . From one of them DNA containing the rna gene was subcloned and sequenced . The inferred protein contains 245 aa residues and has an Mr of 27,156, which agrees with earlier estimates from sodium dodecyl sulfate-polyacrylamide-gel electrophoresis . RNase I is close to twice the size of pancreatic RNase A, but both enzymes contain eight Cys and four His; those aa are important for structure and function of RNase A . Proximal to the rna gene is a sequence that would code for a 23-aa peptide which conforms to consensus rules for signal peptides, and thus should transport this periplasmic enzyme . Sites for eight restriction enzymes had been mapped on each lambda clone . By relating to the map for that specific region, it was possible to position the rna gene exactly at 659 kb from the thr locus (time zero on a time scale of 100 min) . This physical mapping gave a more precise (exact) map position based on distance than was possible using genetic mapping based on a time scale derived from conjugation, and should be applicable for mapping many other E . coli genes. Biochem Biophys Res Commun, 1990 Oct 30, 172(2), 669 - 75 Glutathione S-transferase in yeast: induction of mRNA, cDNA cloning and expression in Escherichia coli; Tamaki H et al.; Glutathione S-transferase Y-2 mRNA synthesis was induced in yeast Issatchenkia orientalis approximately 37-fold by cultivation with o-dinitrobenzene . A cDNA library complementary to poly (A)+RNA of I . orientalis grown with o-dinitrobenzene was screened by colony hybridization . Twenty positive clones were obtained from 6,000 clones and seven of twenty positive clones expressed glutathione S-transferase activity in E . coli . One of the expressing clones harboring plasmid pHT108 had 28 times more glutathione S-transferase activity induced by Isopropyl-beta-D-thio-galactopyranoside than a strain harboring plasmid pUC118 . Expressed glutathione S-transferase Y-2 protein comigrated with yeast glutathione S-transferase Y-2 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis as detected by immunoblot analysis. Biochem Biophys Res Commun, 1990 Oct 30, 172(2), 432 - 8 The binding of ATP and AMP to Escherichia coli adenylate kinase investigated by 1H and 15N NMR spectroscopy; Glushka J et al.; {N6 15N}ATP and {N6 15N}AMP, complexed with E.coli adenylate kinase (AKe), were observed with 15N isotope-filtered NMR pulse sequences and 1H{15N} heterocorrelated experiments to determine differences between binding sites based on chemical shifts and competition by substrate analogs . The chemical shifts of the N6 amino proton and nitrogen signals changed significantly after mixing with adenylate kinase . Differences in chemical shifts between the bound ATP and AMP signals are slight . The response of these shifts to further addition of other substrates or Mg2+ supports the view that the unchelated nucleotides can bind to both the sites, whereas the metal complexed species are restricted to the MgATP/MgADP binding site. Biochemistry, 1990 Oct 30, 29(43), 10120 - 5 Subunit structure and activity of the mannitol-specific enzyme II of the Escherichia coli phosphoenolpyruvate-dependent phosphotransferase system solubilized in detergent; Lolkema JS et al.; The original proposal of Saier stating that P-enolpyruvate-dependent mannitol phosphorylation is catalyzed by the monomeric form of the bacterial phosphotransferase enzyme IImtl, which would be the form predominantly existing in the phospholipid bilayer, whereas mannitol/mannitol-P exchange would depend on the transient formation of functional dimers, is refuted {Saier, M.H . (1980) J . Supramol . Struct . 14, 281-294} . The correct interpretation of the proportional relation between the rate of mannitol phosphorylation in the overall reaction and the enzyme concentration is that enzyme IImtl is dimeric under the conditions employed . Differences measured in the enzyme concentration dependency of the overall and exchange reactions were caused by different assay conditions . The dimer is favored over the monomer at high ionic strength and basic pH . Mg2+ ions bind specifically to enzyme IImtl, inducing dimerization . A complex formed by mixing inorganic phosphate, F-, and Mg2+ at sufficiently high concentrations inhibits enzyme IImtl, in part, by dissociation of the dimer . Enzyme IImtl was dimeric in 25 mM Tris, pH 7.6, and 5 mM Mg2+ over a large enzyme concentration range and under many different turnover conditions . The association/dissociation equilibrium was demonstrated in phosphate bufers, pH 6.3 . The dimer was the most active form both in the overall and in the exchange reaction under the conditions assayed . The monomer was virtually inactive in mannitol/mannitol-P exchange but retained 25% of the activity in the overall reaction. FEBS Lett, 1990 Oct 29, 273(1-2), 46 - 50 Time-resolved fluorescence studies on mutants of the dihydrolipoyl transacetylase (E2) component of the pyruvate dehydrogenase complex from Azotobacter vinelandii; Schulze E et al.; Fluorescence anisotropy decays were measured for the wild-type dihydrolipoyl transacetylase (E2) component of pyruvate dehydrogenase complex from Azotobacter vinelandii and E . coli and for E2-mutants from A . vinelandii in which the alanine-proline-rich sequence between the binding domain and the catalytic domain is partially or completely deleted . In both E2-mutants the rotational mobility of the lipoyl domain and the overall activity after reconstitution of the complex are significantly decreased indicating the important role of the deleted sequence for the movement of the lipoyl domain and the transfer of substrates between the different active sites within the complex. FEBS Lett, 1990 Oct 29, 273(1-2), 23 - 6 Enhanced expression of group II phospholipase A2 gene in the tissues of endotoxin shock rats and its suppression by glucocorticoid; Nakano T et al.; We studied the regulation of group II phospholipase A2 (PLA2-II) gene in vivo, using endotoxin shock rat as a model for systemic inflammation . Administration of endotoxin into rats increased PLA2 activity in the plasma, as described by Vadas and Hay, using endotoxin-challenged rabbit . Specific absorption of this activity by anti-PLA2-II antibody indicated that the released PLA2 was PLA2-II . The levels of PLA2-II mRNA were elevated in the aorta, spleen, lung, and thymus but not in the liver and kidney . The tissues with high PLA2-II mRNA contents released a greater amount of PLA2-II than the tissues of control rats . These results suggest that in endotoxin shock rats, PLA2-II is synthesized de novo in the above tissues and released into circulation . Furthermore, our present study demonstrates that glucocorticoid suppresses the enhanced expression of the PLA2-II gene in the tissues of endotoxin shock rats. FEBS Lett, 1990 Oct 29, 273(1-2), 208 - 10 Influence of the aminoacyl-tRNA synthetase inhibitors and the diadenosine-5'-tetraphosphate phosphonate analogues on the catalysis of diadenosyl oligophosphates formation; Biryukov AI et al.; Well-known aminoacyl-tRNA synthetase (ARSase) inhibitors, namely the analogues of amino acids and aminoacyl adenylates (aminoalkyl- and aminophosphonyl adenylates with Ki congruent to 0.1 microM) as well as the diadenosine 5',5'''-p1,p4-tetraphosphate (Ap4A) phosphonoanalogues, were for the first time used for the Ap4A biosynthesis regulation . Effects of a set of such compounds on lysyl-, phenylalanyl- and alanyl-tRNA synthetases from E . coli, capable of synthesizing Ap4A in the presence of Zn2+ ions and pyrophosphatase, have been studied . The adenylate analogues were found to inhibit the Ap4A and Ap3A formation (I50 congruent to 6 mM) . Aminophosphonic and aminophosphonous acids are not involved in Ap3A and Ap4A biosynthesis and inhibited it at high concentrations . The Ap4A phosphoanalogues slightly inhibited the major reactions of ARSases, as well as the biosynthesis of Ap3A and Ap4A, at a concentration of 5 mM. FEBS Lett, 1990 Oct 29, 273(1-2), 147 - 9 Tight ATP and ADP binding in the noncatalytic sites of Escherichia coli F1-ATPase is not affected by mutation of bulky residues in the 'glycine-rich loop'; Pagan J et al.; It is shown that ATP dissociates very slowly (koff less than 6.4 x 10(5) s-1, t1/2 greater than 3 h) from the three noncatalytic sites of E . coli F1-ATPase and that ADP dissociates from these three sites in a homogeneous fashion with koff = 1.5 x 10(-4) s-1 (t1/2 = 1.35 h) . Mutagenesis of alpha-subunit residues R171 and Q172 in the 'glycine-rich loop' (Homology A) consensus region of the noncatalytic sites was carried out to test the hypothesis that unusually bulky residues at these positions are responsible wholly or partly for the observed tight binding of adenine nucleotides . The mutations alpha Q172G or alpha R171S,Q172G had no effects on ATP or ADP binding to or rates of dissociation from F1 noncatalytic sites . KdATP and KdADP of isolated alpha-subunit were weakened by approximately 1 order of magnitude in both mutants . The results suggest that neither residue alpha R171 nor alpha Q172 interacts directly with bound nucleotide, and show that the presence of bulky residues per se in the glycine-rich loop region of F1-alpha-subunit is not responsible for tight binding in the noncatalytic sites. Science, 1990 Oct 26, 250(4980), 553 - 6 Protection of mice against the Lyme disease agent by immunizing with recombinant OspA; Fikrig E et al.; Lyme borreliosis is a tick-borne illness caused by Borrelia burgdorferi . The gene for outer surface protein A (OspA) from B . burgdorferi strain N40 was cloned into an expression vector and expressed in Escherichia coli . C3H/HeJ mice actively immunized with live transformed E . coli or purified recombinant OspA protein produced antibodies to OspA and were protected from challenge with several strains of B . burgdorferi . Recombinant OspA is a candidate for a vaccine for Lyme borreliosis. Science, 1990 Oct 26, 250(4980), 528 - 32 DNA looping and unlooping by AraC protein; Lobell RB et al.; Expression of the L-arabinose BAD operon in Escherichia coli is regulated by AraC protein which acts both positively in the presence of arabinose to induce transcription and negatively in the absence of arabinose to repress transcription . The repression of the araBAD promoter is mediated by DNA looping between AraC protein bound at two sites near the promoter separated by 210 base pairs, araI and araO2 . In vivo and in vitro experiments presented here show that an AraC dimer, with binding to half of araI and to araO2, maintains the repressed state of the operon . The addition of arabinose, which induces the operon, breaks the loop, and shifts the interactions from the distal araO2 site to the previously unoccupied half of the araI site . The conversion between the two states does not require additional binding of AraC protein and appears to be driven largely by properties of the protein rather than being specified by the slightly different DNA sequences of the binding sites . Slight reorientation of the subunits of AraC could specify looping or unlooping by the protein . Such a mechanism could account for regulation of DNA looping in other systems. Nucleic Acids Res, 1990 Oct 25, 18(20), 6025 - 9 Thiophosphate interference experiments locate phosphates important for the hammerhead RNA self-cleavage reaction; Ruffner DE et al.; A hammerhead domain of less than 50 nucleotides is responsible for a self-cleavage reaction in the replication of plant RNA pathogens . The hammerhead is composed of three helices joining at a central conserved core of 11 single stranded nucleotides . The core is believed to fold into a tertiary structure that provides functional groups for catalysis and to coordinate one or more divalent metal ions . In this study we use a phosphorothioate substitution interference assay to identify four phosphates in the conserved core which also play a role in the self-cleavage reaction. Nucleic Acids Res, 1990 Oct 25, 18(20), 5945 - 8 Sequence analysis of two temperature-sensitive mutations in the alpha subunit gene (rpoA) of Escherichia coli RNA polymerase; Igarashi K et al.; The rpoA gene of Escherichia coli encodes the alpha subunit of the DNA-dependent RNA polymerase . Two mutant alleles, rpoA101 and rpoA112, both of which produce RNA polymerase with altered thermostability and reduced fidelity of transcription in vitro (Ishihama et al . (1980) J . Mol . Biol . 137, 137-150), have been analyzed in details . The mutations were found to be responsible for the temperature-sensitive growth by complementation test using a rpoA-expression plasmid . Each mutant allele was amplified from total cell DNA by PCR (polymerase chain reaction) and directly sequenced . Both the mutant rpoA genes were found to carry a single base transition which leads to a substitution of Cys for Arg at the position 191 (rpoA101) or 45 (rpoA112), respectively . Since the rpoA112 mutation causes the defect in RNA polymerase assembly (Kawakami & Ishihama (1980) Biochemistry 19, 3491-3495), the amino-terminal region of alpha including the position 45 was considered to play an important role in subunit assembly. J Biol Chem, 1990 Oct 25, 265(30), 18656 - 62 The folate cofactor of Escherichia coli DNA photolyase acts catalytically; Hamm-Alvarez S et al.; Escherichia coli DNA photolyase catalyzes the light-driven (300-500 nm) repair of pyrimidine dimers formed between adjacent pyrimidine bases in DNA exposed to UV light (200-300 nm) . The light-driven repair process is facilitated by two enzyme-bound cofactors, FADH2 and 5,10-methenyltetrahydrofolate . The function of the folate has been characterized in greater detail in this series of experiments . Investigations of the relative binding affinities of photolyase for the monoglutamate and polyglutamate forms of 5,10-methenyltetrahydrofolate show that the enzyme has a greater affinity for the naturally occurring polyglutamate forms of the folate and that the exogenously added monoglutamate derivative is less tightly associated with the protein . Multiple turnover experiments reveal that the folate remains bound to photolyase even after 10 turnovers of the enzyme . Examination of the rates of repair by photolyase containing stoichiometric folate in the presence or absence of free folate under multiple turnover conditions and at micromolar concentrations of enzyme also demonstrates that the folate acts catalytically . The stimulation of turnover by exogenous folate seen at low concentrations of photolyase is shown to be due to the lower affinity of photolyase for the monoglutamate derivative used in reconstitution procedures . These results demonstrate that the folate of E . coli DNA photolyase is a bona fide cofactor and does not decompose or dissociate during multiple turnovers of the enzyme. J Biol Chem, 1990 Oct 25, 265(30), 18213 - 8 Escherichia coli formate-hydrogen lyase . Purification and properties of the selenium-dependent formate dehydrogenase component; Axley MJ et al.; The formate-hydrogen lyase complex of Escherichia coli decomposes formic acid to hydrogen and carbon dioxide under anaerobic conditions in the absence of exogenous electron acceptors . The complex consists of two separable enzymatic activities: a formate dehydrogenase and a hydrogenase . The formate dehydrogenase component (FDHH) of the formate-hydrogen lyase complex was purified to near homogeneity in two column chromatographic steps . The purified enzyme was composed of a single polypeptide of molecular weight 80,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis . Metal analysis showed each mole of enzyme contained 3.3 g atoms of iron . Denaturation of FDHH released a compound which, when oxidized, displayed a fluorescence spectrum similar to that of the molybdopterin cofactor found in certain other enzymes . The enzyme contained selenium in the form of selenocysteine as determined by radioactive labeling of the enzyme with 75Se and amino acid analysis . FDHH activity was maximal between pH 7.5 and 8.5; however, the enzyme was maximally stable at pH 5.3-6.4 and highly unstable above pH 7.5 . Nitrate and nitrite salts caused a drastic reduction in activity . Although azide inhibited FDHH activity, it also protected the enzyme from inactivation by oxygen. J Biol Chem, 1990 Oct 25, 265(30), 18185 - 91 Identification of a region of Escherichia coli ribosomal protein L2 required for the assembly of L16 into the 50 S ribosomal subunit; Romero DP et al.; In vitro mutagenesis of rplB was used to generate changes in a conserved region of Escherichia coli ribosomal protein L2 between Gly221 and His231 . Mutants were selected by temperature sensitivity using an inducible expression system . A mutant L2 protein with the deletion of Thr222 to Asp228 was readily distinguishable from wild-type L2 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and ribosomes from the strain overexpressing this mutant protein were characterized by sucrose density gradient centrifugation and protein composition . In addition to 30 S and 50 S ribosomal subunits, cell lysates contained a new component that sedimented at 40 S in 1 mM Mg2+ and at 48 S in 10 mM Mg2+ . These particles contained mutant L2 protein exclusively, completely lacked L16, and had reduced amounts of L28, L33, and L34 . They did not reassociate with 30 S ribosomal subunits and were inactive in polyphenylalanine synthesis . Other mutants in the same conserved region, including the substitution of His229 by Gln229, produced similar aberrant 50 S particles that sedimented at 40 S and failed to associate with 30 S subunits. J Biol Chem, 1990 Oct 25, 265(30), 18154 - 60 The presence of both the signal sequence and a region of mature LamB protein is required for the interaction of LamB with the export factor SecB; Altman E et al.; In the accompanying paper (Altman, E., Bankaitis, V.A., and Emr, S.D . (1990) J . Biol . Chem . 265, 18148-18153) a putative SecB binding site was identified in the mature LamB protein . The export of wild-type LamB was unperturbed when this region was removed, however, suggesting the presence of a second site of interaction between SecB and LamB . In this paper we show that the interference caused by export-defective LamB proteins is influenced by the amount of signal sequence that is present . If a large portion of the signal sequence is deleted then the interference levels are significantly reduced . This result suggests that a region of the signal sequence contributes to the interaction of SecB with the LamB protein . Using anti-SecB affinity chromatography, we demonstrated directly that the association of SecB protein with precursor LamB is dependent on the presence of both the LamB signal sequence and the interfering region which maps to amino acids 320-380 of mature LamB . Although the interfering region is not necessary for the export of wild-type LamB under normal conditions, when the signal sequence is mutationally altered the interfering region is required to promote the efficient export of LamB protein . Also, deletion of the interfering region eliminates the ability of wild-type LamB precursor to be maintained in an export competent conformation in vivo . Collectively, our results indicate that efficient export of the LamB protein is achieved by an interaction with SecB that involves both the LamB signal sequence and the interfering region in mature LamB. J Biol Chem, 1990 Oct 25, 265(30), 18148 - 53 Characterization of a region in mature LamB protein that interacts with a component of the export machinery of Escherichia coli; Altman E et al.; It has been shown that the synthesis of an export-defective protein can interfere with the normal export process in Escherichia coli by limiting the availability of SecB protein, a component of the export apparatus (Collier, D.N., Bankaitis, V.A., Weiss, J.B., and Bassford, P.J . (1988) Cell 53, 273-283) . Consistent with this observation, we find that the interference elicited by an export-defective LamB protein is a titratable response resulting from the limitation of a single ligand . We have mapped the interfering region in LamB to between amino acids 320 and 380 of the mature protein . Expression of this sequence in the form of a LacZ-LamB-LacZ fusion protein elicits the export interference phenotype . Deletion of the sequence from an export-defective LamB protein eliminates the ability of this protein to interfere with the export of other secreted proteins . Together, these findings show that this sequence is both necessary and sufficient to cause export interference . Surprisingly, deletion of this sequence from an otherwise wild-type LamB protein does not cause the mutant LamB product to exhibit any obvious export defect . Based on our results, we propose that SecB interacts with both amino acids 320-380 of mature LamB and the LamB signal sequence during initiation of the export process. Nucleic Acids Res, 1990 Oct 25, 18(20), 6097 - 100 Sequence logos: a new way to display consensus sequences; Schneider TD et al.; A graphical method is presented for displaying the patterns in a set of aligned sequences . The characters representing the sequence are stacked on top of each other for each position in the aligned sequences . The height of each letter is made proportional to its frequency, and the letters are sorted so the most common one is on top . The height of the entire stack is then adjusted to signify the information content of the sequences at that position . From these 'sequence logos', one can determine not only the consensus sequence but also the relative frequency of bases and the information content (measured in bits) at every position in a site or sequence . The logo displays both significant residues and subtle sequence patterns. J Biol Chem, 1990 Oct 25, 265(30), 18366 - 71 Pyrophosphate-dependent phosphofructokinase . Conservation of protein sequence between the alpha- and beta-subunits and with the ATP-dependent phosphofructokinase; Carlisle SM et al.; Full-length cDNA clones for the alpha- and beta-subunits of pyrophosphate-fructose 6-phosphate 1-phosphotransferase have been isolated from a cDNA expression library derived from potato tuber poly(A)+ RNA . The nucleotide sequences indicate that the alpha- and beta-subunits are related with about 40% of amino acid residues being identical . A comparison of the deduced amino acid sequences of both subunits of this enzyme with that of the major ATP-dependent fructose 6-phosphate 1-phosphotransferase from Escherichia coli (Shirakihara, Y., and Evans, P . R . (1988) J . Mol . Biol . 204, 973-994) showed little homology between the proteins except for regions involved in the binding of fructose 6-phosphate/fructose, 1,6-bisphosphate and possibly between regions binding pyrophosphate and the beta- and gamma-phosphates of ADP/ATP . A comparison of the derived secondary structures of the two subunits of the PPi-dependent enzyme with the known secondary structure of the E . coli ATP-dependent enzyme indicated that the overall structure of these enzymes is similar . These data suggest that catalytic activity resides on the beta-subunit of the pyrophosphate-dependent enzyme. J Biol Chem, 1990 Oct 25, 265(30), 18095 - 7 Oxygen regulation of nitrate transport by diversion of electron flow in Escherichia coli; Denis KS et al.; Anaerobic nitrate respiration is regulated by oxygen at the level of nitrate transport; however, the mechanism of O2 inhibition is unknown . Potentially, oxygen could inhibit directly by causing conformational changes in the porter system or indirectly through diversion of electron flow from the nitrate reductase complex to oxygen reduction . Inhibition due to electron diversion implies that nitrate reduction is required for nitrate transport . In this regard, nitrate uptake and its regulation by oxygen were studied in mutants of Escherichia coli (strain AN386) deficient in cytochrome d (RG98), cytochrome o (RG101), and a mutant deficient in both cytochrome d and cytochrome o (RG99) . Respiratory nitrate uptake in RG99 was highly resistant to the effects of oxygen supporting the indirect mechanism of electron diversion in oxygen regulation . Nitrate transport in RG98 and RG101 is highly sensitive to oxygen; these mutants exhibited 81 and 85% inhibition, respectively, which is similar to inhibition in the wild type . These results indicate that during nitrate respiration, O2 inhibits transport by limiting the supply of electrons to the nitrate reductase complex. Nature, 1990 Oct 25, 347(6295), 780 - 2 The Drosophila claret segregation protein is a minus-end directed motor molecule; Walker RA et al.; A product encoded at the claret locus in Drosophila is needed for normal chromosome segregation in meiosis in females and in early mitotic divisions of the embryo . The predicted amino-acid sequence of the segregation protein was shown recently to be strikingly similar to Drosophila kinesin heavy chain . We have expressed the claret segregation protein in bacteria and have found that the bacterially expressed protein has motor activity in vitro with several novel features . The claret motor is slow (4 microns min-1), unlike either kinesin or dyneins . It has the directionality, the ability to generate torque and the sensitivity to inhibitors reported previously for dyneins . The finding of minus-end directed motor activity for a protein with sequence similarity to kinesin suggests that the dynein and kinesin motor domains are ancestrally related . The minus-end directed motor activity of the claret motor is consistent with a role for this protein in producing chromosome movement along spindle microtubules during prometaphase and/or anaphase. J Biol Chem, 1990 Oct 25, 265(30), 18408 - 13 A short intervening structure can block rho factor helicase action at a distance; Steinmetz EJ et al.; We have characterized the helicase activity of transcription termination factor rho on a variety of substrates . Helicase activity requires specific recognition of a single-stranded region of RNA upstream (5') of the nucleic acid duplex on which rho acts . Spacer sequences of at least 450 nucleotides can be inserted between the rho-binding signals and the duplex region with little effect on activity . RNA-DNA helices of up to 120 base pairs, but not as long as 210 base pairs, can be disrupted efficiently by rho . The stoichiometry of release of substrates with long spacer sequences, as with the standard substrate, approaches a value of one RNA released per rho hexamer; thus cooperative binding by rho does not account for action at a distance . Instead, these results are consistent with a model in which a single rho hexamer binds initially to terminator sequences and then either loops out or tracks along the intervening RNA to reach the duplex region . Results with complex substrates are inconsistent with looping and support the tracking model: under conditions that allow disruption of RNA-DNA, but not RNA-RNA helices (0.4 mM Mg2+), the presence of a short RNA-RNA helix acts as a block to the disruption of an RNA-DNA helix downstream . These findings are discussed in relation to the mechanism of the helicase activity as well as its role in rho-dependent transcription termination. J Biol Chem, 1990 Oct 25, 265(30), 18379 - 85 Mutational and immunochemical analysis of plasminogen activator inhibitor 1; Shubeita HE et al.; We have undertaken a structural and functional analysis of recombinant plasminogen activator inhibitor type 1 (PAI-1) produced in Escherichia coli using site-directed mutagenesis and immunochemistry . Expression of recombinant PAI-1 yielded an inhibitor that was functionally indistinguishable from PAI-1 made in human endothelial cells . Mutations in both the reactive center P1 and P1' residues (Arg-Met) and a putative secondary binding site for plasminogen activators on PAI-1 have been engineered to assess their functional effects . The inhibition of a panel of serine proteases, including plasminogen activators, trypsin, elastase, and thrombin, has been studied . Substitution of the P1 arginine residue with lysine or the P1' residue with either valine or serine had no detectable effect on the rate of inhibition of plasminogen activators . However, replacement of both P1 and P1' by Met-Ser produced a variant with no detectable plasminogen activator inhibitor activity . Mutations introduced into either Asp102 or Lys104 in the second site did not affect the rate of inhibition of plasminogen activators . Complementary immunochemical experiments using antibodies directed against the same two regions of the PAI-1 protein confirm that the reactive center is the primary determinant of inhibitory activity and that the putative second site is not a necessary functional region. Nucleic Acids Res, 1990 Oct 25, 18(20), 5969 - 73 Enzymatic recognition of DNA modifications induced by singlet oxygen and photosensitizers; Muller E et al.; DNA modifications induced either by photosensitization (illumination in the presence of methylene blue) or by chemically generated singlet oxygen (thermal decomposition of an 1,4-etheno-2,3-benzodioxin) are recognized and incised by repair endonucleases present in crude bacterial cell extracts . Only a small fraction of the incised modifications are sites of base loss (AP-sites) sensitive to exonuclease III, endonuclease IV from E . coli or to the UV-endonuclease from M . luteus . Cell extracts from E . coli strains overproducing or defective in endonuclease III recognize the modifications induced by illumination in the presence of methylene blue just as well as do those from wild-type E . coli strains . This indicates that dihydropyrimidine derivatives, which are characteristic of hydroxyl radical-induced DNA modifications, are absent . In contrast, most of the modifications induced are not recognized by a cell extract from a fpg strain defective in formamidopyrimidine-DNA glycosylase FPG protein) . Furthermore, incision by a cell extract from an E . coli strain overproducing FPG protein takes place at much lower protein concentration than with the wild-type strain . Experiments with purified FPG protein confirm that this enzyme is responsible for the recognition of singlet oxygen-induced DNA base modifications. Eur J Biochem, 1990 Oct 24, 193(2), 361 - 6 Distribution of the early light-inducible protein in the thylakoids of developing pea chloroplasts; Cronshagen U et al.; The distribution of the early light-inducible protein (ELIP) of pea (Pisum sativum) between grana and stroma thylakoids was studied . An antibody raised against a bacterial-expressed fusion protein containing ELIP sequences was used . Illumination of dark-grown pea seedlings causes an accumulation of the ELIP in the thylakoid membranes with a maximum level at 16 h . During continuous illumination exceeding 16 h the level decreases again . The fractionation of thylakoid membranes of 48-h-illuminated pea seedlings in grana and stroma thylakoids reveals that there is no uniform distribution of ELIP in the thylakoids . Rather 60-70% of ELIP was found in the stroma thylakoids and 30-40% in the grana thylakoids . This distribution is in accordance with that of photosystem I but not with that of photosystem II . After Triton-X-100 solubilization almost all ELIP is found in the photosystem-I-containing fraction . This also supports an association of ELIP with photosystem I. Eur J Biochem, 1990 Oct 24, 193(2), 325 - 30 Export and purification of a cytoplasmic dimeric protein by fusion to the maltose-binding protein of Escherichia coli; Blondel A et al.; A hybrid between the maltose-binding protein (MalE) of Escherichia coli and the gene 5 protein (G5P) of phage M13 was constructed at the genetic level . MalE is a monomeric and periplasmic protein while G5P is dimeric and cytoplasmic . The hybrid (MalE-G5P) was synthesized in large amounts from a multicopy plasmid and efficiently exported into the periplasmic space of E . coli . The export was dependent on the integrity of the signal peptide . MalE-G5P was purified from a periplasmic extract by affinity chromatography on cross-linked amylose, with a yield larger than 50,000 molecules/E . coli cell . The hybrid specifically bound denatured but not double-stranded DNA cellulose, as native G5P . Sedimentation velocity and gel-filtration experiments showed that MalE-G5P exists as a dimer . Thus, it was possible to efficiently translocate through the membrane a normally cytoplasmic and dimeric protein, by fusion to MalE . Moreover, the passenger protein kept its activity, specificity and quaternary structure in the purified hybrid . MalE-G5P will enable the study of mutant G5P that no longer binds single-stranded DNA and therefore cannot be purified by DNA-cellulose chromatography. Eur J Biochem, 1990 Oct 24, 193(2), 387 - 94 Overexpression in Escherichia coli of a methionine-free designed interleukin-2 receptor (Tac protein) based on a chemically cleavable fusion protein; Husken D et al.; Several fusion proteins of our previously chemically synthesized gene encoding the interleukin-2-receptor alpha subunit (IL-2R alpha or Tac protein) were constructed . They were designed in order to be cleavable by cyanogen bromide . Thus, the original internal methionines of the IL-2R alpha were replaced by either alanine, valine, leucine or isoleucine, based on secondary structure predictions . Additionally, aspartate at position 6 was substituted for glutamate in order to stabilize the acid-labile Asp-Pro bond . Direct C-terminal fusion of total beta-galactosidase and portions thereof did not result in substantial amounts of the expected construct . Ternary fusions consisting of beta-galactosidase domains N- and C-terminally fused to the mutant synthetic methionine-free interleukin-2 receptor alpha subunit (synIL-2R alpha) yielded inclusion bodies amounting to 4-7% of the total protein . This first overexpression of a type I membrane receptor can be rationalized by the known beta-galactosidase structure models . The fusion protein can be cleaved with cyanogen bromide, isolated and the resulting synIL-2R alpha detected by Western blot analysis. Eur J Biochem, 1990 Oct 24, 193(2), 319 - 24 Expression of three plant glutamine synthetase cDNA in Escherichia coli . Formation of catalytically active isoenzymes, and complementation of a glnA mutant; Bennett M et al.; Three cDNA clones encoding the closely related glutamine synthetase (GS) alpha, beta and gamma polypeptides of Phaseolus vulgaris (French bean) were recombinantly expressed in Escherichia coli . The GS expression plasmids correctly synthesised the recombinant alpha, beta and gamma polypeptides which then assembled into catalytically active homo-octameric isoenzymes . These isoenzymes behaved similarly to their native homologues on ion-exchange and gel-filtration chromatography . Furthermore, the alpha and gamma isoenzymes complemented a GS(glnA)-deficient mutant, thus demonstrating their physiological activity in E . coli . Differences were observed between the three recombinant GS plasmids in their quantitative expression of the GS polypeptides and their ability to complement the E . coli mutant . These differences were correlated to the degree of solubility of the polypeptide, which was observed to be dependent on the temperature of expression . The production of active GS isoenzymes in E . coli facilitates the isolation and characterisation of the individual P . vulgaris homo-octameric GS isoenzymes. Biochemistry, 1990 Oct 23, 29(42), 9863 - 71 Engineering enzyme subsite specificity: preparation, kinetic characterization, and X-ray analysis at 2.0-A resolution of Val111Phe site-mutated calf chymosin; Strop P et al.; Comparison of the three-dimensional structure of bovine chymosin with the structures of homologous aspartic proteinases complexed with peptide inhibitors shows that Val111 in chymosin occupies a position between the specificity subsites S1 and S3 . A mutation corresponding to Val111 to Phe has been introduced in an intermediary plasmid construct of prochymosin by bridging its unique restriction sites by a synthetic mutant oligonucleotide duplex . A prochymosin fusion product was expressed in Escherichia coli in such a way that the extension and substitution of the propart does not interfere with the activation of the zymogen . After activation of the crude prochymosin, the enzyme was purified by affinity chromatography on Sepharose with V-dL-P-F-F-V-dL as ligand . This procedure provided large amounts of pure protein as judged by FPLC, the activity/protein ratio, and SDS-PAGE . The enzymatic properties were determined by using a variety of peptide substrates and inhibitors; KM values for the mutant enzyme were approximately twice those of the wild type, but the kcat values were little changed . The mutant enzyme was crystallized, X-ray data were collected to 2.0-A resolution by using a FAST area detector, and the structure was solved by using difference Fourier methods and refined to an R factor of 19.5% . The mutation leads to only local changes in conformation, with the phenylalanine side chain occupying part of the S1 and S3 pockets . This accounts for the increased KM of this mutant for a substrate with a large phenylalanine side chain at P1 . It is also consistent with the higher affinity of the mutant for an inhibitor with small side chains at P1 and P3 when compared with the wild-type enzyme. Biochim Biophys Acta, 1990 Oct 23, 1087(2), 219 - 25 Partial apolipoprotein E-beta-galactosidase fusion protein expressed in Escherichia coli retains binding activity to the LDL(B/E) receptor; Ribeiro A et al.; A partial rat apo E-beta-galactosidase fusion protein was produced in Escherichia coli Y1089 infected with recombinant lambda GT11 obtained by immunoscreening of a rat liver cDNA library with an anti-rat LDL antiserum . Partial cDNA overlapped the apo E mRNA sequence coding for apo E binding domain towards the LDL(B/E) receptor up to codon for Arg-139 . Fusion protein specifically bound to human fibroblasts . The high-affinity component exhibited a Kd of 5 x 10(-8) M and 4.1 x 10(5) sites per cell . Fusion protein binding to fibroblasts was mediated by their apo E moiety and not by beta-galactosidase since: (1) specific binding of fusion protein was competed out by human LDL; (2) beta-galactosidase did not compete with fusion protein binding; and (3) human fibroblasts from a patient with familial hypercholesterolemia, deficient in LDL(B/E) receptor, bound fusion protein 10-times lower than control fibroblasts . It was demonstrated that partial fusion protein retained the functional activity of the native apo E . However, compared to full-length native or engineered apo E, fusion protein was able to bind fibroblasts without being complexed with phospholipids . Fusion proteins might be a useful tool for studying the functional efficiency of the LDL(B/E) receptor and for mapping residues and domains involved in the binding process. Biochim Biophys Acta, 1990 Oct 23, 1087(2), 205 - 11 Expression of active human hypoxanthine-guanine phosphoribosyltransferase in Escherichia coli and characterisation of the recombinant enzyme; Free ML et al.; A plasmid, pRG1, has been constructed by incorporating the coding sequence of human hypoxanthine-guanine phosphoribosyltransferase (HPRT) into the expression vector pT7-7 . Expression of human HPRT has been achieved in HPRT- Escherichia coli cells transformed with pRG1 and pGP1-2, as shown by: (1) exclusive labelling with {35S}methionine of a polypeptide with the same mobility as purified human HPRT on SDS-PAGE; and (2) measurement of HPRT activity after cell lysis . Although the majority of the recombinant HPRT was present in the particulate fraction after cell lysis and centrifugation, sufficient HPRT activity was present in the supernatant fraction to allow comparison with the HPRT purified from human erythrocytes and the activity in human haemolysates and lymphoblast lysates . Small differences in electrophoretic mobility on native gels were found between HPRT activity from these sources . The Km values of recombinant HPRT for the substrates 5-phospho-alpha-D-ribosyl-1-pyrophosphate and guanine were compared with those of lymphoblast and erythrocyte HPRT. Biochim Biophys Acta, 1990 Oct 23, 1087(2), 212 - 8 Nuclease S1 mapping of 16S ribosomal RNA in ribosomes; Rahman MA et al.; Escherichia coli 16S rRNA and 16S-like rRNAs from other species have several universally conserved sequences which are believed to be single-stranded in ribosomes . The quantitative disposition of these sequences within ribosomes is not known . Here we describe experiments designed to explore the availability of universal 16S rRNA sequences for hybridization with DNA probes in 30S particles and 70S ribosomes . Unlike previous investigations, quantitative data on the accessibility of DNA probes to the conserved portions of 16S rRNA within ribosomes was acquired . Uniquely, the experimental design also permitted investigation of cooperative interactions involving portions of conserved 16S rRNA . The basic strategy employed ribosomes, 30S subunits, and 16S rRNAs, which were quantitatively analyzed for hybridization efficiency with synthetic DNA in combination with nuclease S1 . In deproteinated E . coli 16S rRNA and 30S subunits, the regions 520-530, 1396-1404, 1493-1504, and 1533-1542 are all single-stranded and unrestricted for hybridization to short synthetic DNAs . However, the quantitative disposition of the sequences in 70S ribosomes varies with each position . In 30S subunits there appear to be no cooperative interactions between the 16S rRNA universal sequences investigated. Biochemistry, 1990 Oct 23, 29(42), 9956 - 62 Escherichia coli maltodextrin phosphorylase: contribution of active site residues glutamate-637 and tyrosine-538 to the phosphorolytic cleavage of alpha-glucans; Schinzel R et al.; The role of Escherichia coli maltodextrin phosphorylase (EC 2.4.1.1) active site residues Glu637 and Tyr538 which line the sugar-phosphate contact region of the enzyme was investigated by site-directed mutagenesis . Substitution of Glu637 by an Asp or Gln residue reduced kcat to approximately 0.2% of wild-type activity, while the Km values were affected to a minor extent . This indicated participation of Glu637 in transition-state binding rather than in ground-state binding . 31P NMR analysis of the ionization state of enzyme-bound pyridoxal phosphate suggested that Glu637 is also involved in modulation of the protonation state of the coenzyme phosphate observed during catalysis . Despite loss of proposed hydrogen-bonded substrate contacts, the Tyr538Phe mutant enzyme retained more than 10% activity; the apparent affinity of all substrates was slightly decreased . Mutations at either site affected the error rate of the enzyme (ratio of hydrolysis/phosphorolysis) . Besides a role in substrate binding, the hydrogen-bond network of Tyr538 supports the exclusion of water from the active site. Mol Cell Endocrinol, 1990 Oct 22, 73(2-3), 83 - 91 Production of rat renin fusion protein in Escherichia coli and the preparation of renin-specific antisera; Campbell DJ et al.; Rat renin fused at the N-terminus with Sj26, a 26,000 Da glutathione S-transferase of Schistosoma japonicum, was expressed in Escherichia coli . The fusion protein was soluble and easily purified from crude bacterial lysates by affinity chromatography on immobilised glutathione . The fusion protein possessed no detectable renin activity . Antisera raised in rabbits against the fusion protein were specific for renin . These antisera did not bind soluble renin but bound immobilized renin . By immunoblotting, these antisera demonstrated rat renin to migrate on sodium dodecyl sulfate-polyacrylamide gel electrophoresis as two broad bands of 33,000-34,000 and 35,000-37,000 Da . By immunocytochemistry of rat tissues, these antisera stained renin containing cells in the afferent arteriole of the glomerulus of the kidney, the zona glomerulosa of the adrenal and the corpus luteum of the ovary . However, apart from the afferent arteriole of the kidney, no immunoreactive renin was identified in blood vessels of the kidney, adrenal or ovary . These studies demonstrate that a recombinant renin fusion protein is a valuable alternative approach for the preparation of renin-specific antisera. J Mol Biol, 1990 Oct 20, 215(4), 549 - 57 Novel mutants of elongation factor G; Richter Dahlfors AA et al.; A novel mutant form of elongation factor G (EF-G) in Escherichia coli is described . This variant EF-G restricts reading frame errors by a factor of 2 to 3 in vivo at two different positions in a lacIZ fusion . In addition, a conventional fusidic acid resistant (fusR) mutant of EF-G was compared with the restrictive mutant . Both mutants were characterized in vitro in a steady-state poly(U) translating system . The data indicate that the restrictive EF-G variant has an altered interaction with the ribosome both in vivo and in vitro . In contrast, the conventional fusR variant is altered in its interaction with GTP, which is evident in vitro. J Mol Biol, 1990 Oct 20, 215(4), 497 - 510 Characterization of the Escherichia coli araFGH and araJ promoters; Hendrickson W et al.; The identities of two cloned, arabinose-inducible promoters were tested by hybridizing promoter DNA fragments with restriction digests of chromosomal DNA containing Mudlac phage inserted in either araFGH or in araE transport operons . One promoter, thought to be araE, is within 10(3) base-pairs of a Mudlac insertion in the araE gene . The second promoter was not found within several thousand base-pairs of either of the known transport genes . This promoter is now named araPJ (araJ) . The DNA sequence of the fragment containing the araFGH promoter was determined . The start site of transcription in vivo was located to within +/- 1 base-pair (bp) by S1 nuclease mapping . DNase 1 footprinting revealed that, in comparison with the araBAD and araE promoters, the locations of the AraC and cyclic AMP receptor protein (CRP) binding sites are reversed with CRP lying between AraC and RNA polymerase . The central location of the CRP binding site may explain why the araFGH promoter is more catabolite sensitive than the other ara promoters . AraC and CRP were both required for maximal transcription in vitro, although a low level of transcription was detected with CRP alone . S1 nuclease mapping of mRNA-DNA hybrids from the araJ promoter located the transcription start point to within #/- 3 bp, and demonstrates that the promoter is dependent upon AraC protein and CRP in vivo . DNase footprinting showed that the location of the AraC protein binding site on araJ is adjacent to the RNA polymerase site, as seen at the araBAD and araE promoters . Two CRP sites were observed; one is upstream from the AraC site and one is downstream from the transcription start site. J Mol Biol, 1990 Oct 20, 215(4), 493 - 5 Preliminary crystallographic analysis of the breakage-reunion domain of the Escherichia coli DNA gyrase A protein; Reece RJ et al.; The 64 x 10(3) Mr N-terminal breakage-reunion domain of the Escherichia coli DNA gyrase A protein was purified from an over-expressing strain . When complexed with the gyrase B protein, this truncated A protein has all of the enzymic properties of the full-length counterpart, although with reduced efficiency in some cases . The 64 x 10(3) Mr protein has been crystallized in several forms, a number of which were too small for crystallographic analysis . However, two forms grew to sufficient size for preliminary X-ray analysis . Both forms were tetragonal with a primitive lattice . One form (type I) had cell dimensions of a = b = 170 A, c = 145 A a space group of either P41212 (P43212) or P42212, and diffracted to 6 A resolution . The type II crystals had cell dimensions of a = b = 177 A, c = 175 A, a space group of P41212 (P43212) or P42212, and diffracted to at least 4.5 A resolution . Both crystal forms apparently contained four subunits (possibly a tetramer) in the asymmetric unit . We are attempting to increase the size and quality of these crystals. J Immunol Methods, 1990 Oct 19, 133(2), 227 - 33 A comparison of immunoblotting, flow cytometry and ELISA to monitor the binding of anti-lipopolysaccharide monoclonal antibodies; Nelson D et al.; This study was designed to assess the use of flow cytometry to observe the binding, under physiological conditions, of anti-lipopolysaccharide (LPS) monoclonal antibodies (mAbs) to whole bacteria, and to compare this with the more conventional whole cell ELISA and immunoblotting techniques . The bacteria consisted of two clinical isolates of E . coli 018:K1 and 06:K5 and two isogenic mutants of the 018 parent: a non-capsulate (018:K-) and a rough mutant (018rf) . Two cross-reactive anti-core mAbs and one 018 0-antigen-specific mAb were used . ELISA and flow cytometry showed that capsule and O-polysaccharide influenced the binding of mAbs to the bacteria, whilst the latter technique demonstrated that sub-populations existed . Immunoblotting showed the two anti-core mAbs to be different, one bound only to core which was not substituted with O-antigen, whilst the other bound both to substituted and unsubstituted core . This comparison for monitoring the binding of anti-LPS mAbs demonstrates the potential use of flow cytometry in bacterial cell surface research, and complements results obtained by ELISA and immunoblotting. Biochemistry, 1990 Oct 16, 29(41), 9667 - 77 Partial 1H NMR assignments of the Escherichia coli dihydrofolate reductase complex with folate: evidence for a unique conformation of bound folate; Falzone CJ et al.; Sequence-specific 1H assignments have been made for over 25% of the amino acid side chains of Escherichia coli dihydrofolate reductase complexed with folate by using a variety of two-dimensional techniques . Proton resonances were assigned by using a combination of site-directed mutagenesis and a knowledge of the X-ray crystal structure . Unique sets of NOE connectivities present in hydrophobic pockets were matched with the X-ray structure and used to assign many of the residues . Other residues, particularly those near or in the active site, were assigned by site-directed mutagenesis . The ability to assign unambiguously the proton resonances of these catalytically important residues allowed for extensive networks of NOE connectivities to follow from these assignments . As a consequence of these assignments, the orientation of the pterin ring of folate could be determined, and its conformation is similar to that of the productive dihydrofolate complex . Under these experimental conditions, only one bound form of the pterin ring could be detected. Biochemistry, 1990 Oct 16, 29(41), 9591 - 9 Folding of a predominantly beta-structure protein: rat intestinal fatty acid binding protein; Ropson IJ et al.; The equilibrium and kinetic properties of the unfolding-refolding transitions of Escherichia coli derived rat intestinal fatty acid binding protein have been examined using several different denaturants . This protein, which contains 2 tryptophans but no prolines or cysteines, has a predominantly beta-structure: its 10 antiparallel beta-strands are organized into 2 orthogonal sheets surrounding a large solvent-filled internal cavity . For urea and guanidine hydrochloride, the completely reversible transition was monitored by circular dichroism, absorbance, and fluorescence spectroscopy . Each of these data sets was best fit by a simple, two-state model involving only native and unfolded forms . However, linear extrapolation to determine the free energy of folding in the absence of denaturant resulted in different values for the free energy of folding depending upon which denaturant was used . When fluorescence was used to monitor the transition, the extrapolated free energy estimates for the two denaturants were markedly different: 10.03 +/- 0.24 kcal mol-1 for urea versus 5.22 +/- 0.33 kcal mol-1 for guanidine hydrochloride . The midpoints of these transitions were 5.51 and 1.36 M, respectively . The transition caused by either denaturant as monitored by circular dichroism and absorbance spectroscopy was virtually coincident with that monitored by fluorescence, further supporting the assignment of a two-state model for the equilibrium results . The addition of a 2-fold molar excess of ligand (oleate) increased the extrapolated estimates approximately 2.5 kcal mol-1 for both denaturants.(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1990 Oct 16, 29(41), 9522 - 31 Comparison of chemical reactivity, cytotoxicity, interstrand cross-linking and DNA sequence specificity of bis(platinum) complexes containing monodentate or bidentate coordination spheres with their monomeric analogues; Farrell N et al.; The properties of a new bis(platinum) complex containing two monodentate coordination spheres, {(trans-PtCl(NH3)2)2H2N(CH2)4NH2}Cl2 (1,1/t,t), are reported . Comparison is made with respect to chemical reactivity, in vitro biological activity in murine and tumor cells, DNA conformational changes, cross-linking efficiency, and sequence specificity between this complex and the previously reported complex containing two bidentate platinum atoms, {(Pt(mal)(NH3))2H2N(CH2)4NH2} (2,2/c,c), as well as with their respective monomeric analogues, {PtCl(dien)}Cl and cis-{PtCl2(NH3)2}(cis-DDP) . While both bis(platinum) complexes are active against cis-DDP-resistant cells, the monodentate bis(platinum) complex (1,1/t,t) has a lower resistance factor than the complex with bidentate coordination spheres (2,2/c,c) . More importantly, this property is repeated in a human ovarian carcinoma cell line . DNA-binding studies show that DNA interstrand cross-linking is more efficient for the 1,1/t,t complex . DNA sequencing studies employing the exonuclease activity of T4-polymerase demonstrate that there are a variety of binding sites; some are common to all complexes and some common to both bis(platinum) complexes, while the monodentate 1,1/t,t species also reacts at unique sites, not attacked by any of the other complexes studied . The circular dichroism of CT DNA modified by the 1,1/t,t complex is also unique and is not seen for any of the other agents. Biochemistry, 1990 Oct 16, 29(41), 9572 - 84 Activator-dependent preinduction binding of sigma-70 RNA polymerase at the metal-regulated mer promoter; Heltzel A et al.; Expression of the Tn21 mercury-resistance (mer) locus is controlled by the merR gene product, which represses mer structural gene (merTPCAD) transcription in the absence of mercuric ion {Hg(II)} and activates it in the presence of Hg(II) . In vivo DNA methylation of the mer regulatory region (merOP) shows that, with or without the inducer Hg(II), MerR strongly protects four guanine residues in a dyadic region located between the -10 and -35 hexamers of the structural gene promoter (PTPCAD) . Prior to induction by Hg(II), RNA polymerase is also bound at PTPCAD; occupancy of the uninduced promoter by RNA polymerase is dependent on MerR . Methylation and permanganate footprinting demonstrate that induction by Hg(II) results in MerR/Hg(II)-dependent promoter DNA melting in the -10 region of PTPCAD and in additional DNA structural distortions within the region of dyad symmetry . Thus, MerR fosters the binding of RNA polymerase to an inactive promoter, and upon induction, MerR/Hg(II) facilitates DNA distortions suitable for efficient formation of the active transcription complex. Biochemistry, 1990 Oct 16, 29(41), 9728 - 33 Purified internal G-domain of translational initiation factor IF-2 displays guanine nucleotide binding properties; Vachon G et al.; Translational initiation factor IF-2 is involved in a multistep pathway leading to the synthesis of the first peptide bond . IF-2 is a guanine nucleotide binding protein (G-protein) and catalyzes GTP hydrolysis in the presence of ribosomes . According to sequence homologies with other G-proteins, particularly EF-Tu, a theoretical model for the tertiary structure of the putative G-domain of IF-2 has been previously proposed {Cenatiempo, Y., Deville, F., Dondon, J., Grunberg-Manago, M., Hershey, J . W . B., Hansen, H . F., Petersen, H . U., Clark, B . F . C., Kjeldgaard, M., La Cour, T . F . M., Mortensen, K . K., & Nyborg, J . (1987) Biochemistry 26, 5070-5076} . A short fragment of IF-2 encompassing the putative G-domain was purified by limited proteolysis of a chimeric protein, synthesized from a gene fusion, between a segment of the IF-2 gene and lacZ . The N- and C-terminal sequences of this IF-2 peptide were characterized . Its calculated length is 181 amino acids and its molecular mass 19.4 kDa, whereas it migrates at 14 kDa in SDS-polyacrylamide gels . This segment of IF-2 can form binary complexes with GDP and can be cross-linked to GTP, therefore indicating that it really corresponds to the G-domain . However, in contrast to the situation described for the purified G-domain of EF-Tu, the IF-2 fragment did not hydrolyze GTP even in the presence of ribosomes . It is assumed that active centers of IF-2 located outside the G-domain are needed for the latter reaction.(ABSTRACT TRUNCATED AT 250 WORDS) FEBS Lett, 1990 Oct 15, 272(1-2), 55 - 7 Importance of the G27-A43 mismatch at the anticodon stem of Escherichia coli tRNA(Thr2); Komine Y et al.; The tRNA(Thr2) isoacceptor of E . coli has a G-A mismatch at positions 27-43 . When the anticodon of this tRNA was converted to an amber anticodon (CUA), this tRNA showed suppressor activity in E . coli . Moreover, introduction of the base pair (G-C or U-A) at positions 27-43 of this suppressor tRNA reduced its suppressor activity . These results indicate that the G27-A43 mismatch is necessary for full function of tRNA(Thr2). Biochem Biophys Res Commun, 1990 Oct 15, 172(1), 357 - 63 An active site of growth hormone for eliciting the differentiation of preadipose 3T3-F442A cells to adipose cells; Uchida E et al.; In order to elucidate the active sites of growth hormone for eliciting the differentiation of preadipose 3T3-F442A cells to adipocytes, four artificial mutant variants of human growth hormone (hGH) modified in the loop region of amino acid residues 54-74 were prepared in Escherichia coli by site-directed mutagenesis . Although the P59A (replacement of Pro59 with Ala) variant retained almost the same biological- and receptor binding-activity as hGH, the P61A (replacement of Pro61 with Ala) and the P59A-P61A (replacement of both Pro59 and Pro61 with Ala) both exhibited about half the activity, and the delta (62-67) variant (deletion of the residues 62-67) exhibited only about 0.1% the activity of those of intact hGH . The results suggest that Pro61 may be involved in formation of the active conformation of hGH, but Pro59 may not, and that the amino acid residues around 62-67 may be critical for the specific biological features of hGH. J Immunol, 1990 Oct 15, 145(8), 2440 - 7 Germinal center cells are a major IL-5-responsive B cell population in peripheral lymph nodes engaged in the immune response; Rabinowitz JL et al.; Germinal center formation and the development of B cell memory in lymphoid tissue is a T cell-dependent process . The specific B cell-T cell interactions, and/or cytokines, resulting in germinal center cell growth have not yet been identified . Germinal center B cells were separated from other lymph node (LN) B cells by panning on peanut agglutinin (PNA)-coated dishes . Resulting fractions enriched for PNA+ (germinal center) B cells, and the PNA- (other) LN B cells from immune SJL mice were assayed for proliferation in the presence of cytokines . PNA+ and PNA- B cells responded equally to IL-4 in the anti-mu co-stimulator assay . In contrast, PNA+ B cells responded to murine (r)IL-5 or human B cell growth factor in the dextran sulfate (DxS) co-stimulator assay, to a much greater degree than did PNA- B cells . The same results were obtained with PNA+ and PNA- cells from LAF1 mice . Unfractionated LN B cells from nonimmunized SJL or BALB/c mice did not respond to IL-5 with or without DxS . B cell populations from BALB/c mice such as from spleen and peritoneal cavity, which are known to be high in Ly-1+B cells, responded to IL-5 alone, and more dramatically, to IL-5 as a co-stimulator with DxS . Such populations of cells from SJL mice, which are known to contain low numbers of Ly-1+B cells, responded markedly less . These results are consistent with those of others which show that in nonimmunized mice, Ly-1+B cells are a major IL-5 responsive subpopulation . IL-1 enhanced the proliferation of PNA+ cells in response to rIL-5 and had no effect on PNA- cells . IL-4 and IL-5 did not enhance each other's effects as co-stimulators of proliferation . In contrast to PNA+ B cells from immune LN, B cells activated by Escherichia coli endotoxin exhibited no responses to rIL-5 . The present results indicate that in immune LN, PNA+, germinal center B cells constitute a prominent IL-5-responsive population. J Biol Chem, 1990 Oct 15, 265(29), 17980 - 7 Expression of actin in Escherichia coli . Aggregation, solubilization, and functional analysis; Frankel S et al.; Wild type Dictyostelium discoideum actin (42 kDa) and a truncated form of actin were expressed in Escherichia coli . Amino-terminal sequencing indicated that the truncated species was composed of two peptides, which were the result of internal translation initiation at Met-119 and Met-123 . After sonication or French press lysis, all of the actin was present in highly insoluble aggregates . When bacteria were lysed directly into Sarkosyl detergent, most of the actin was soluble, and greater than 50% remained soluble after Sarkosyl was removed . Full-length wild type actin was purified using DNase I affinity chromatography and gel filtration . This species was able both to polymerize and to bind myosin in an ATP-sensitive manner, indicating it was native . Affinity chromatography demonstrated that the truncated form of actin bound DNase I to the same extent as actin synthesized in eukaryotes, indicating the applicability of this approach to mutant forms of actin . Thus, lysis procedures utilizing Sarkosyl may prove useful in isolating some of the other proteins which are normally soluble but become insoluble after bacterial expression. J Biol Chem, 1990 Oct 15, 265(29), 17665 - 72 Variation of cofactor levels in Escherichia coli . Sequence analysis and expression of the pncB gene encoding nicotinic acid phosphoribosyltransferase; Wubbolts MG et al.; The pncB gene from Escherichia coli, which encodes nicotinic acid phosphoribosyltransferase (EC 2.4.2.11), was cloned on a 1.5-kilobase TaqI-EcoRI fragment . Its position on the E . coli chromosome was determined at 20.8 min between the asnS and pepN loci . The nucleotide sequence of the gene and the transcription and translation initiation sites were determined . Expression of pncB on a multicopy plasmid leads to a 25-fold increase in nicotinic acid phosphoribosyltransferase activity . Growth of E . coli in the presence of nicotinic acid leads to strong repression of nicotinic acid phosphoribosyltransferase activity, indicating that the cloned pncB sequence contains its own control sequences . It is shown that increased nicotinic acid phosphoribosyltransferase activity effects a 5-fold increase in the intracellular concentration of NAD . The cloned pncB gene can therefore be used as a tool to raise intracellular cofactor levels. J Biol Chem, 1990 Oct 15, 265(29), 17627 - 36 RNA primary sequence or secondary structure in the translational initiation region controls expression of two variant interferon-beta genes in Escherichia coli; Gross G et al.; Efficient expression in Escherichia coli (E . coli) of the human interferon-beta gene (IFN-beta) gene and of a chemically synthesized IFN-beta gene variant (506 base pairs; synIFN-beta) adapted to the E . coli codon usage, both fused to the E . coli atpE ribosome-binding site, is controlled either by primary sequence or by mRNA secondary-structure in the translational initiation region . High level expression of the natural human atpE/IFN-beta gene fusion is governed by the nucleotide composition preceding the initiator codon AUG . A single U----C exchange in the -2 or -1 position preceding the initiator codon AUG reduces the translational efficiency from 18% of total cellular protein to only 8% or 4%, respectively, while both U----C substitutions reduce IFN-beta expression below 1% . These sequence alterations interfere with efficient ribosome binding as revealed by toeprinting . They provide further evidence for the influence of the anticodon-flanking regions of tRNA(fMet) upon the initiation rate of translation . In contrast, translation of the synthetic variant atpE/synIFN-beta gene fusion is controlled by a moderately stable stem-loop structure (delta G = -4 kcal/mol; 37 degrees C) located within the coding region and overlapping the 30 S ribosomal subunit attachment site . That the stability of the hairpin interferes with the initiation of translation is inferred from site-directed mutagenesis and toeprint analyses . mRNA half-life in these variants is positively correlated with the rate of translation and involves two major endonucleolytic cleavage site 5'-upstream of the Shine-Dalgarno region. J Biol Chem, 1990 Oct 15, 265(29), 17493 - 8 Construction and properties of active chimeric enzymes between human aldolases A and B . Analysis of molecular regions which determine isozyme-specific functions; Kitajima Y et al.; To study the structure/function relationships of human aldolase isozymes, particularly isozyme-specific functions, we constructed Escherichia coli expression plasmids for six BA chimeric enzymes (BA34, BA108, BA137, BA212, BA306, and BA306*), each composed of the N-terminal side of isozyme B and the C-terminal side of isozyme A, and one BAB chimeric enzyme which contains a fragment of isozyme A (residues 213-306) inserted in between the N-terminal and the C-terminal fragments of isozyme B . They were transfected into E . coli, and the generated enzymes were characterized . This study reveals the following . (i) For isozyme A, the C-terminal Tyr-363 and the N-terminal region bearing isozyme group-specific sequences 1-3 and Lys-107 (the C-6 phosphate-binding site) are responsible for the higher catalytic activity toward fructose 1,6-bisphosphate, which is 7 times higher than that of aldolase B . Conversely, an internal region spanning positions 108-212 is required for the lower activity toward fructose 1-phosphate . (ii) For isozyme B, an internal sequence spanning positions 108-212 which includes some isozyme B-specific residues and a postulated C-1 phosphate-binding site (Lys-146 or Arg-148) is responsible for a higher catalytic activity toward fructose 1-phosphate, which is 8-10 times that of isozyme A . The more upstream sequence containing positions 1-107 is responsible for the lower catalytic activity toward fructose 1,6-bisphosphate . (iii) At least residues 212-306, composing a long stretch near the active-site Lys-229 and highly conserved among isozymes A, B, and C, may be required for the basal framework of the aldolase molecule to exhibit the activity common to the three isozymic forms. J Biol Chem, 1990 Oct 15, 265(29), 17451 - 5 Molecular cloning of wheat dihydrodipicolinate synthase; Kaneko T et al.; Four forms of dihydrodipicolinate synthase (DHDPS), which catalyzes the first reaction in the lysine-specific biosynthetic pathway in higher plants, were purified to homogeneity from a suspension culture of wheat (Triticum aestivum) . These polypeptides have similar N-terminal amino acid sequences . Two different cDNA clones were isolated by screening a wheat cDNA library with oligonucleotide probes based on these amino acid sequences . The predicted amino acid sequences indicate that both clones encode putative chloroplast transit peptides that have little homology to each other as well as the conserved mature protein portions of Mr 35,737 and 35,776 (94% identity) . Mature wheat DHDPS has 30% amino acid identity to Escherichia coli DHDPS . One of the cloned cDNAs, which had been fused to bacterial transcription/translation signals, genetically complemented a strain of E . coli that lacks endogenous DHDPS activity . Moreover, the expression of wheat DHDPS cDNA in wild-type E . coli increased the enzymatic activity 10-fold in the transformed bacteria. Gene, 1990 Oct 15, 94(2), 237 - 41 Expression in Escherichia coli of an immunoreactive visna virus transactivating protein; Watson G et al.; We have cloned the gene encoding the visna 1514 transactivating protein, Tat, into the Escherichia coli lambda pR expression plasmid, pRIT2T . Efficient synthesis of the protein A::Tat fusion protein was obtained in host strains which carried either wild-type or temperature-sensitive (ts) lambda repressors . However, constitutive synthesis of the fusion protein in these host strains resulted in selection against plasmids which synthesized the fusion protein . Efficient repression of the lambda pR promoter was obtained using a ts repressor gene carried on a multicopy plasmid . Synthesis of the fusion protein in this strain was efficient on induction, and reproducible after subculture . Antisera generated against the termini of visna Tat were used to demonstrate that the fusion protein retained the antigenicity of both the N and C termini of the transactivating protein. J Biol Chem, 1990 Oct 15, 265(29), 17826 - 36 Specific DNA cleavage and binding by vaccinia virus DNA topoisomerase I; Shuman S et al.; Cleavage of a defined linear duplex DNA by vaccinia virus DNA topoisomerase I was found to occur nonrandomly and infrequently . Approximately 12 sites of strand scission were detected within the 5372 nucleotides of pUC19 DNA . These sites could be classified as having higher or lower affinity for topoisomerase based on the following criteria . Higher affinity sites were cleaved at low enzyme concentration, were less sensitive to competition, and were most refractory to religation promoted by salt, divalent cations, and elevated temperature . Cleavage at lower affinity sites required higher enzyme concentration and was more sensitive to competition and induced religation . Cleavage site selection correlated with a pentameric sequence motif (C/T)CCTT immediately preceding the site of strand scission . Noncovalent DNA binding by topoisomerase predominated over covalent adduct formation, as revealed by nitrocellulose filter-binding studies . The noncovalent binding affinity of vaccinia topoisomerase for particular subsegments of pUC19 DNA correlated with the strength and/or the number of DNA cleavage sites contained therein . Thus, cleavage site selection is likely to be dictated by specific noncovalent DNA-protein interactions . This was supported by the demonstration that a mutant vaccinia topoisomerase (containing a Tyr----Phe substitution at the active site) that was catalytically inert and did not form the covalent intermediate, nevertheless bound DNA with similar affinity and site selectivity as the wild-type enzyme . Noncovalent binding is therefore independent of competence in transesterification . It is construed that the vaccinia topoisomerase is considerably more stringent in its cleavage and binding specificity for duplex DNA than are the cellular type I enzymes. J Biol Chem, 1990 Oct 15, 265(29), 17713 - 9 Identification of a critical cysteine in EcoRI DNA methyltransferase by mass spectrometry; Everett EA et al.; EcoRI DNA methyltransferase (MTase) is rapidly inactivated by N-ethylmaleimide with concomitant incorporation of 2 mol of N-ethyl{2-3H}maleimide/mol of functional monomer . Preincubation of the enzyme with either S-adenosylmethionine or DNA reduces the rate of activity loss, whereas preincubation with DNA and the S-adenosylmethionine analog sinefungin completely protects the enzyme from inactivation . An endo proteinase Glu-C digest of N-ethyl{2-3H}maleimide-modified enzyme was prepared and separated by high pressure liquid chromatography . Modified and unmodified cysteine-containing peptides were located and identified by radioactivity, mass spectrometry, and tandem mass spectrometry . In the absence of any ligands, cysteines 25, 116, and 223 are modified by N-ethylmaleimide; in the presence of DNA and sinefungin, Cys-223 is essentially unmodified . Thus, N-ethylmaleimide modification of Cys-223 in EcoRI DNA MTase is responsible for the loss of enzyme activity . Cys-223 is preceded by Asn, and this (or Cys-Asn) occurs with high frequency in adenine and cytosine (N-4) DNA MTases . Direct involvement of cysteine in methyl transfer reactions to adenine N-6 and cytosine N-4 is supported by the similarity of the reactions catalyzed by adenine N-6 and cytosine N-4 DNA MTases, the frequent presence of Asn-flanking Cys, and the importance of Cys-223 to EcoRI MTase function. J Biol Chem, 1990 Oct 15, 265(29), 17706 - 12 Carbohydrate-binding protein 35 . Isoelectric points of the polypeptide and a phosphorylated derivative; Cowles EA et al.; Purified carbohydrate-binding protein 35 (CBP35) and extracts of mouse cells containing CBP35 were analyzed by two-dimensional gel electrophoresis . Such an analysis on recombinant CBP35, obtained by expression of a cDNA clone in Escherichia coli, yielded a pI value of 8.7 . When extracts of mouse 3T3 cells were subjected to two-dimensional gel electrophoresis and immunoblotting, two spots were observed, corresponding to pI values of 8.7 and 8.2 . The pI 8.2 species represents post-translational modification of the CBP35 polypeptide (pI 8.7) by the addition of a single phosphate group . This conclusion is based on the finding that purified CBP35 contained a pI 8.2 species that was labeled with 32PO4 and could be converted to the unlabeled pI 8.7 species by alkaline phosphatase treatment . The phosphorylated (pI 8.2) form of CBP35 is found in both the cytosolic and nuclear fractions, whereas the unphosphorylated (pI 8.7) species is found exclusively in the nuclei . Quiescent populations of 3T3 fibroblasts (confluent monolayers or serum-starved sparse cultures) are characterized by the predominance of phosphorylated CBP35 . Stimulation of the same cells into the proliferative state resulted in an increase in the amount of the phosphorylated species; more dramatic, however, is the elevation of the level of the unphosphorylated form, which is barely detectable in quiescent cells. J Biol Chem, 1990 Oct 15, 265(29), 17680 - 7 A tetrameric iron superoxide dismutase from the eucaryote Tetrahymena pyriformis; Barra D et al.; An iron-containing superoxide dismutase has been purified from the protozoan Tetrahymena pyriformis . It has a molecular weight of 85,000 and is composed of four subunits of equal size . The tetramer contains 2.5 g atoms of ferric iron . Visible absorption and electron spin resonance spectra closely resemble those of other iron-containing superoxide dismutases . The amino acid sequence of the iron superoxide dismutase was determined . Each subunit is made up of 196 residues, corresponding to a molecular weight of 22,711 . Comparison of the primary structure with the known sequences of other iron-containing superoxide dismutases reveals a relatively low degree of identity (33-34%) . However, a higher percentage identity is found with mammalian manganese-containing superoxide dismutases (41-42%) . The amino acid sequence is discussed in consideration of residues that may distinguish iron from manganese or dimeric from tetrameric superoxide dismutases. Biochem J, 1990 Oct 15, 271(2), 415 - 20 cDNA structure and expression of calpactin, a peptide involved in Ca2(+)-dependent cell aggregation in sponges; Robitzki A et al.; Aggregation of cells of the marine sponge Geodia cydonium is mediated by an aggregation factor (AF) particle of Mr 1.3 X 10(8) . It is now reported that the AF particle is associated with calpactin, which was ascribed a role in the cell-adhesion process . In order to identify the sequence similarity to other members of the lipocortin family, the cDNA of sponge calpactin was cloned and found to display an 80% sequence similarity to vertebrate calpactin II but only a 47% similarity to calpactin I . The calpactin gene, which contains the consensus sequence coding for the amino acids G-T-D-E, was expressed in Escherichia coli and subsequently purified to a 37000-Mr polypeptide . Both the p32 and the p37 are provided with approximately two Ca2+ ions/molecule and the property to bind to phospholipids . The dissociation constant (calpactin-Ca2+) was in the absence of phospholipids in the range 500-700 microM-Ca2+ but in their presence about 20-30 microM-Ca2+ . On the basis of (i) inhibition studies with antibodies to calelectrin and (ii) competition experiments with soluble phospholipids (both chemically defined as well as total homologous membrane lipids) we conclude that the AF-associated calpactin and plasma-membrane-bound phospholipid(s) are involved in cell-cell aggregation in sponges. Biochem J, 1990 Oct 15, 271(2), 487 - 91 Investigation of putative active-site lysine residues in hydroxymethylbilane synthase . Preparation and characterization of mutants in which (a) Lys-55, (b) Lys-59 and (c) both Lys-55 and Lys-59 have been replaced by glutamine; Hadener A et al.; A new construct carrying the hemC gene was transformed into Escherichia coli, resulting in approx . 1000-fold over-expression of hydroxymethylbilane synthase (HMBS) . This construct was used to generate HMBS in which (a) Lys-55, (b) Lys-59 and (c) both Lys-55 and Lys-59 were replaced by glutamine (K55Q, K59Q and K55Q-K59Q respectively) . All three modified enzymes are chromatographically separable from wild-type enzyme . Kinetic studies showed that the substitution K55Q has little effect whereas K59Q causes a 25-fold decrease in Kapp . cat./Kapp . m . Treatment of K55Q, K59Q and K55Q-K59Q separately with pyridoxal 5'-phosphate and NaBH4 resulted in incomplete and non-specific reaction with the remaining lysine residues . Pyridoxal modification of Lys-59 in the K55Q mutant caused greater enzymic inactivation than similar modification of Lys-55 in K59Q . The results in sum show that, though Lys-55 and Lys-59 may be at or near the active site, neither is indispensable for the catalytic activity of HMBS. Biochem Biophys Res Commun, 1990 Oct 15, 172(1), 211 - 6 Cloning and sequencing of the structural gene for the small subunit of methylamine dehydrogenase from Methylobacterium extorquens AM1: evidence for two tryptophan residues involved in the active center; Chistoserdov AY et al.; In two independent clone libraries, clones were identified that hybridized with oligonucleotide probes based on N- or C-terminal polypeptide sequence of the small subunit of methylamine dehydrogenase from Methylobacterium extorquens AM1 . Plasmids from all clones had in common a 5.2 kb Bam HI-HindIII DNA fragment . A 0.57 kb SacII-BclI subfragment that hybridized to the oligonucleotide probes was sequenced . Nucleotide sequence analysis coincided with polypeptide sequence data in the structural part of the small subunit with a single contradiction: amino acid 17 is Asp rather than Asn . The two amino acids that are involved in the active center which had not been determined from previous polypeptide sequencing proved to be tryptophans. Gene, 1990 Oct 15, 94(2), 195 - 9 Stem-loop structures at the 3' end of tobacco Rubisco large subunit mRNA; Akada S et al.; There are two inverted repeat nucleotide (nt) sequences, each capable of forming a stem-loop structure (sls) at the 3' end of the tobacco Rubisco large subunit mRNA (rbcL) . The smaller sls is followed by a larger sls . The in vivo functions of the 3' sls of the rbcL mRNA were characterized using the Escherichia coli system . S 1 mapping of the rbcL transcripts synthesized in E . coli revealed that the 3' end of a major transcript in the bacterial cell is almost identical to the 3' end of authentic chloroplast (cp) rbcL mRNA . This native 3' end is located 4 nt downstream from the larger sls for the cp mRNA and 6 nt for the bacterial transcript, respectively . Deletion experiments show that the larger sls is essential for producing the native 3' end of rbcL mRNA in E . coli . The sls do not function as an efficient transcription terminator but can stabilize upstream mRNA segments in vivo. J Biol Chem, 1990 Oct 15, 265(29), 17673 - 9 Osmotic control of proU transcription is mediated through direct action of potassium glutamate on the transcription complex; Prince WS et al.; Osmoregulated transcription from the proU promoter of Escherichia coli has been successfully reconstituted from purified components in a simple in vitro system consisting of plasmid DNA template, RNA polymerase, and nucleotides in the absence of any other protein factor . proU transcription is stimulated by addition of high concentrations of potassium glutamate, the ionic compound accumulated in vivo during hyperosmotic stress . Transcription from the nonosmoregulated promoters beta la, lac, and pepN is inhibited under the same conditions, demonstrating the specificity of potassium glutamate as an inducer of proU transcription . proU transcription requires a circular DNA template, but stable alterations in the degree of supercoiling are unnecessary for this potassium glutamate-dependent signaling . These results agree well with previous data obtained in an S-30 coupled transcription/translation system and suggest that physiological changes in the ionic composition of the intracellular millieu can regulate gene expression. Nucleic Acids Res, 1990 Oct 11, 18(19), 5787 - 92 Transcription of single-copy hybrid lacZ genes by T7 RNA polymerase in Escherichia coli: mRNA synthesis and degradation can be uncoupled from translation; Chevrier-Miller M et al.; In Escherichia coli transcription of individual genes generally requires concomitant translation, and thus the decay of mRNAs cannot be studied without the complication of translation . Here we have used T7 RNA polymerase to transcribe in vivo lacZ genes carrying ribosome binding sites of variable efficiency . We show that neither cell viability nor growth rate is affected by the T7-driven transcription of these genes, provided that they are present as single chromosomal copy . Furthermore, transcription is now completely uncoupled from translation, allowing large amounts of even completely untranslated mRNAs to be synthesized . Taking advantage of these features, we discuss the influence of the frequency of translation upon the processing and degradation of the lac message. Nucleic Acids Res, 1990 Oct 11, 18(19), 5713 - 6 A single-stranded DNA-binding protein promotes the binding of the purified oestrogen receptor to its responsive element; Mukherjee R et al.; The purified human oestrogen receptor (hER) does not form a detectable complex with an oestrogen responsive element (ERE) under conditions where hER-ERE complexes are readily formed with crude extracts from Hela or yeast cells expressing the hER . This indicates that other factor(s) are necessary for ER-ERE binding . Such a ER DNA binding stimulatory factor (DBSF) has been purified from the yeast Saccharomyces cerevisiae . It is a 45 kDa single-stranded DNA-binding protein (SSB) which cannot be substituted for by the purified E . coli SSB. Nucleic Acids Res, 1990 Oct 11, 18(19), 5673 - 6 Rat DNA polymerase beta gene can join in excision repair of Escherichia coli; Ohnishi T et al.; Though DNA polymerase I (poll) of Escherichia (E.) coli is understood to play a role in repair synthesis of excision repair, it is still obscure whether DNA polymerase beta (pol beta) plays a similar role in eukaryotic cells . To estimate the role of pol beta in excision repair processes, we inserted the rat pol beta gene into several mutant E . coli defective in a diverse set of enzymatic activities of poll . UV resistance was seen only when the 5'----3' exonuclease (exo) activity of poll molecules remained . Therefore it is suggested that 5'----3' exo activity as well as pol beta activity are essential for repair synthesis of excision repair in eukaryotic cells. Nucleic Acids Res, 1990 Oct 11, 18(19), 5659 - 65 In vivo and in vitro analyses of an intron-encoded DNA endonuclease from yeast mitochondria . Recognition site by site-directed mutagenesis; Sargueil B et al.; The pal 4 nuclease (termed I-Sce II) is encoded in the group I al 4 intron of the COX I gene of Saccharomyces cerevisiae . It introduces a specific double-strand break at the junction of the two exons A4-A5 and thus mediates the insertion of the intron into an intronless strain . To define the sequence recognized by pal 4 we introduced 35 single mutations in its target sequence and examined their cleavage properties either in vivo in E . coli (when different forms of the pal 4 proteins were artificially produced) or in vitro with mitochondrial extracts of a mutant yeast strain blocked in the splicing of the al 4 intron . We also detected the pal 4 DNA endonuclease activity in extracts of the wild type strain . The results suggest that 6 to 9 noncontiguous bases in the 17 base-pair region examined are necessary for pal 4 nuclease to bind and cleave its recognition site . We observed that the pal 4 nuclease specificity can be significantly different with the different forms of the protein thus explaining why only some forms are highly toxic in E . coli . This study shows that pal 4 recognition site is a complex phenomenon and this might have evolutionary implications on the transfer properties of the intron. Nucleic Acids Res, 1990 Oct 11, 18(19), 5633 - 6 T7 endonuclease I resolves Holliday junctions formed in vitro by RecA protein; Muller B et al.; T7 endonuclease I is known to bind and cleave four-way junctions in DNA . Since these junctions serve as analogues of Holliday junctions that arise during genetic recombination, we have investigated the action of T7 endonuclease I on recombination intermediates containing Holliday junctions . We find that addition of T7 endonuclease I to strand exchange reactions catalysed by RecA protein of Escherichia coli leads to the formation of duplex products that correspond to 'patch' and 'splice' type recombinants . Resolution of the recombination intermediates occurs by the introduction of nicks at the site of the Holliday junction . The recombinant molecules contain 5'-phosphate and 3'-hydroxyl termini which may be ligated to restore the integrity of the DNA. Nucleic Acids Res, 1990 Oct 11, 18(19), 5625 - 32 The involvement of base 1054 in 16S rRNA for UGA stop codon dependent translational termination; Hanfler A et al.; The deletion of the highly conserved cytidine nucleotide at position 1054 in E . coli 16S rRNA has been characterized to confer an UGA stop codon specific suppression activity which suggested a functional participation of small subunit rRNA in translational termination . Based on this structure-function correlation we constructed the three point mutations at site 1054, changing the wild-type C residue to an A, G or U base . The mutations were expressed from a complete plasmid encoded rRNA operon (rrnB) using a conditional expression system with the lambda PL-promoter . All three altered 16S rRNA molecules were expressed and incorporated into 70S ribosomal particles . Structural analysis of the protein and 16S rRNA moieties of the mutant ribosomes showed no differences when compared to wild-type particles . The phenotypic analysis revealed that only the 1054G base change led to a significantly reduced generation time of transformed cells, which could be correlated with the inability of the mutant ribosomes to specifically stop at UGA stop codons in vivo . The response towards UAA and UAG termination codons was not altered . Furthermore, in vitro RF-2 termination factor binding experiments indicated that the association behaviour of mutant ribosomes was not changed, enforcing the view that the UGA stop codon suppression is a direct consequence of the rRNA mutation . Taken together, these results argue for a direct participation of that 16S rRNA motif in UGA dependent translational termination and furthermore, suggest that termination factor binding and stop codon recognition are two separate steps of the termination event. Nature, 1990 Oct 11, 347(6293), 572 - 5 Altered protein conformation on DNA binding by Fos and Jun; Patel L et al.; The protein products of the c-fos and c-jun proto-oncogenes (Fos and Jun, respectively) form a heterodimeric protein complex that interacts with the activator protein-1 (AP-1) binding site and regulates gene transcription in response to extracellular stimuli . Protein dimerization is mediated primarily by a coiled-coil-like structure termed the leucine-zipper and DNA binding occurs primarily through regions of each protein rich in basic amino acids that contact both strands of the AP-1 site . The precise nature of the protein-DNA interaction is unknown as studies concerned with dimerization and DNA binding by Fos and Jun have relied on indirect methods to investigate protein-protein-DNA interactions . Here we have developed assay systems using fluorescence spectroscopy and circular dichroism to monitor dimerization and DNA binding directly . The results indicate that the interaction of Fos and Jun with DNA results in an altered conformation of the protein dimers and an increased alpha-helical content . These techniques may have general application in studies concerning the interaction of transcriptional regulatory proteins with specific DNA target sequences. Biochemistry, 1990 Oct 9, 29(40), 9395 - 402 Glycine to alanine substitutions in helices of glyceraldehyde-3-phosphate dehydrogenase: effects on stability; Ganter C et al.; Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from chicken was expressed in and purified from Escherichia coli . To investigate the physical basis of possible protein stabilization strategies, the effect of substitutions of glycine residues by alanine in helical regions was determined . One Gly to Ala substitution (G316A) located in the central core of the subunit was found to strongly stabilize the protein, while the other mutations are neutral or destabilize the protein . The effect seen for the stabilizing mutant in irreversible heat denaturation correlates with the first transition in folding equilibrium experiments that is observable by fluorescence, but not with the one detected by circular dichroism measurements or in dilution-induced dissociation experiments . The stabilizing effect of a Gly to Ala substitution therefore does not seem to be caused by an entropic effect on the unfolded state . Rather, an internal cavity is filled by the substitution G316A, probably stabilizing the native state . In large oligomeric proteins, imperfect packing may be a frequent cause of limited stability. Biochemistry, 1990 Oct 9, 29(40), 9352 - 7 Purification of recombinant ribulose-1,5-bisphosphate carboxylase/oxygenase large subunits suitable for reconstitution and assembly of active L8S8 enzyme; Lee B et al.; Ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) from Anacystis nidulans was reconstituted in vitro from extracts of Escherichia coli strains that separately express large and small subunits . This reconstitution system was shown to be useful for monitoring the appearance of dissociated or fractionated subunit preparations . Recombinant large subunits were purified to a state of homogeneity and retained reconstitution capacity in the presence of added small subunits . The purified large subunits appeared to be in the form of an octamer, probably an L8 structure, and showed 0.15% of the carboxylase activity of the purified L8S8 enzyme . Purified large subunit octamers are disrupted by nondenaturing PAGE; however, the octamer is stable to electrophoresis in the presence of exogenous protein. Mol Cell Biochem, 1990 Oct 15-Nov 8, 98(1-2), 95 - 9 Crystal structure of chicken liver basic fatty acid-binding protein at 2.7 A resolution; Scapin G et al.; The three-dimensional structure of chicken liver basic fatty acid-binding protein has been determined at 2.7 A resolution by X-ray crystallography . Phases were calculated using the multiple isomorphous replacement procedure and a preliminary model was built . This model, with an initial R-factor of 0.57, was then improved by a cycle of refinement by simulated annealing which brought the R factor down to 0.32 . The protein is structured as a compact 10-stranded-beta-barrel which encapsulates a residual electron density that can be interpreted as a fatty acid molecule . The NH2-terminus portion of the molecule contains two short alpha-helices . The structure of this liver protein appears very similar to that of the Escherichia coli derived rat intestinal FABP recently determined by X-ray diffraction methods. Mol Cell Biochem, 1990 Oct 15-Nov 8, 98(1-2), 75 - 9 Expression of fatty acid-binding protein from bovine heart in Escherichia coli; Oudenampsen E et al.; The coding part of the cDNA of cardiac fatty acid-binding protein (cFABP) from bovine heart was cloned into the vector pKK233-2 . After induction with isopropyl-beta-D-thiogalactopyranoside cFABP was found in a soluble form in the cytosol of plasmid transformed E . coli amounting up to 5.7% of the soluble protein . cFABP was detected after SDS-polyacrylamide gelelectrophoresis and/or isoelectric focusing and Western blot by immuno-staining and was determined quantitatively by a solid phase enzyme-linked immuno sorbent assay . The cFABP produced by bacteria binds oleic acid with high affinity as shown by comigration of protein and ligand in both gelfiltration and isoelectric focusing . cFABP was purified from bacterial lysates to near homogeneity and resolved into four isoproteins. J Theor Biol, 1990 Oct 7, 146(3), 395 - 406 Analysis of a model for minichromosome segregation in Escherichia coli; Schurr T et al.; The present article contains a theoretical, quantitative analysis of the implications of the Helmstetter-Leonard model (1987, J . molec . Biol . 197, 195-204.) for the segregation of chromosomal DNA in Escherichia coli, on the expected copy-number distribution of minichromosomes in a culture in steady-state exponential growth . According to the model, two determinants are involved in the mechanism of chromosome segregation: a partition system that assures the equal allotment of chromosomes between daughter cells at cell division, and a locus within the minimal oriC region that specifies the attachment site of the chromosomes to the cell envelope at initiation of replication . There are many parameters that must be taken into account in such a study, and since some of them are probabilistic in nature, a strictly analytical approach is not feasible and we had to resort to computer simulation . A wide range of parameter values was tested, in all combinations . The minichromosome copy-number distributions obtained all had a prominent mode equal to the number of oriC binding sites and their main features were determined essentially by that and very little by any of the other parameters of the model . In order to avoid the unrealistic situation in which this one feature completely dominates the results, the original model was modified so that each individual minichromosome is no longer required to replicate during every cell generation, by introducing a limit to the number of unsuccessful attempts to locate a suitable binding site . The copy-number distributions predicted by this version of the model are quantitatively and qualitatively very different and depend on all the components of the model . The simulation results are sufficiently well-behaved to allow consideration as to whether a particular empirical minichromosome copy-number distribution--when such data become available--could in fact be governed by the proposed model; it may even be possible to get a rough estimate for the different parameters involved. J Biol Chem, 1990 Sep 25, 265(27), 16102 - 7 Site-directed mutagenesis of rat liver S-adenosylhomocysteinase . Effect of conversion of aspartic acid 244 to glutamic acid on coenzyme binding; Gomi T et al.; Aspartic acid 244 that occurs at the putative NAD(+)-binding site of rat liver S-adenosylhomocysteinase was replaced by glutamic acid by oligonucleotide-directed mutagenesis . The mutant enzyme was purified to homogeneity as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis . Gel permeation chromatography showed that the purified mutant enzyme was a tetramer as is the wild-type enzyme . In contrast to the wild-type enzyme, which possesses 1 mol of tightly bound NAD+ per mol of enzyme subunit, the mutant enzyme had only 0.05 mol of NAD+ but contained about 0.6 mol each of NADH and adenine per mol of subunit . The mutant enzyme, after removal of the bound compounds by acid-ammonium sulfate treatment, exhibited S-adenosylhomocysteinase activity when assayed in the presence of NAD+ . From the appearance of activity as a function of NAD+ concentration, the enzyme was shown to bind NAD+ with a Kd of 23.0 microM at 25 degrees C, a value greater than 280-fold greater than that of the wild-type enzyme . In the presence of a saturating concentration of NAD+, the mutant enzyme showed apparent Km values for substrates similar to those of the wild-type enzyme . Moderate decreases of 8- and 15-fold were observed in Vmax values for the synthetic and hydrolytic directions, respectively . These results indicate the importance of Asp-244 in binding NAD+, and are consistent with the idea that the region of S-adenosylhomocysteinase from residues 213 to 244 is part of the NAD+ binding site . This region has structural features characteristic of the dinucleotide-binding domains of NAD(+)- and FAD-binding proteins (Ogawa, H., Gomi, T., Mueckler, M . M., Fujioka, M., Backlund, P.S., Jr., Aksamit, R.R., Unson, C.G., and Cantoni, G.L . (1987) Proc . Natl . Acad . Sci . U.S.A . 84, 719-723). Eur J Biochem, 1990 Oct 5, 193(1), 91 - 5 Subcellular localisation of dihydrolipoamide dehydrogenase and detection of lipoic acid in bloodstream forms of Trypanosoma brucei; Jackman SA et al.; In the long-slender bloodstream form of Trypanosoma brucei, the enzyme dihydrolipoamide dehydrogenase exists in the absence of the 2-oxo-acid dehydrogenase complexes of which it is normally a component, and appears to be associated with the plasma membrane of the organism {Danson, M . J., Conroy, K., McQuattie, A . & Stevenson, K . J . (1987) Biochem . J . 243, 661-665} . In the present paper, a complete subcellular fractionation of T . brucei has been carried out and, by comparison with marker enzymes, it is confirmed that the dihydrolipoamide dehydrogenase is indeed associated with the plasma membrane . In addition, we now provide evidence that the distribution of the enzyme is over the whole surface of the membrane, including the flagellar pocket region, and that the enzyme is not found in any other cellular fraction . A study of the latency of the enzyme suggests that it is located on the cytoplasmic surface of the plasma membrane . The discovery of the presumed substrate of dihydrolipoamide dehydrogenase, lipoic acid, is reported for T . brucei . Using a biological assay involving a strain of Escherichia coli that requires lipoic acid for growth, we have found that acid-hydrolysed extracts of T . brucei contain 1.7 (+/- 0.2) ng of the cofactor/mg protein . The chemical nature of the lipoic acid was confirmed by gas chromatography/mass spectrometry. J Biol Chem, 1990 Oct 5, 265(28), 17348 - 54 Characterization of an active single polypeptide form of the human immunodeficiency virus type 1 protease; DiIanni CL et al.; The pepsin-like aspartyl proteases consist of a single polypeptide chain with topologically similar amino- and carboxyl-terminal domains, each of which contributes 1 aspartic acid residue to the active site . This structure has been proposed to have evolved by gene duplication and fusion from a dimeric enzyme composed of two identical polypeptide chains, such as the aspartyl protease (PRT) of human immunodeficiency virus type 1 (HIV-1) . To determine if a single polypeptide form of the HIV-1 protease would be enzymatically active, two protease coding regions were linked to form a dimeric gene (pFGGP) . Expression of this gene in Escherichia coli yielded a protein with the expected molecular mass of 22 kDa . The in vitro kinetic parameters of PRT and FGGP (where FGGP is the single polypeptide form of the HIV-1 protease with 2 glycine residues connecting the two subunits) for three peptide substrates are similar . Construction and analysis of a CheY-GAG-FGGP fusion protein demonstrated that FGGP is capable of precursor processing in vivo . Mutation of one or both of the active site aspartates to either asparagine or glutamate rendered the enzyme inactive, demonstrating that both active site aspartate residues are required for enzymatic activity. J Biol Chem, 1990 Oct 5, 265(28), 17341 - 7 Translocation of ProOmpA possessing an intramolecular disulfide bridge into membrane vesicles of Escherichia coli . Effect of membrane energization; Tani K et al.; In the absence of delta mu H+, the in vitro translocation of proOmpA resulted in the stable accumulation of a possible translocation intermediate in addition to a transiently accumulating one . The stable intermediate was detected on a polyacrylamide gel as two proteinase K-resistant bands corresponding to a molecular weight of about 28,000 . The appearance of the bands was appreciably enhanced when proOmpA was oxidized with ferricyanide . No mature OmpA appeared . When proOmpA reduced with dithiothreitol was used, on the other hand, the bands did not appear at all . Upon the replacement of Cys302 of OmpA with Gly, the intermediate accumulation was abolished . The proOmpA treated with dithiothreitol was labeled with N-{3H}-ethylmaleimide, whereas that treated with ferricyanide was not . The ferricyanide-treated proOmpA was translocated into membrane vesicles in the presence of delta mu H+ . The mature OmpA thus translocated and processed was not labeled with N-{3H}ethylmaleimide . It is concluded that proOmpA possessing the Cys290-Cys302 disulfide bridge can be translocated without cleavage of the bridge, when delta mu H+ is imposed . The accumulation of the disulfide bridge-containing intermediate was ATP-dependent, whereas its conversion to the translocated mature form was not blocked in the presence of adenosine 5'-(beta, gamma-imino)triphosphate . It is concluded that the early and late stages of the translocation reaction require ATP and delta mu H+ differently. J Biol Chem, 1990 Oct 5, 265(28), 17215 - 21 Characterization of Escherichia coli cells deficient in 1-acyl-sn-glycerol-3- phosphate acyltransferase activity; Coleman J; A mutant of Escherichia coli K-12 defective in 1-acyl-sn-glycerol-3-phosphate acyltransferase has been isolated . At the permissive temperature for growth, 30 degrees C, 20% of the total cellular glycerophospholipids is 1-acyl-sn-glycerol-3-phosphate, as identified by mass spectral analysis and proton NMR . This percentage of 1-acyl-sn-glycerol-3-phosphate rises to about 30% when the temperature of the culture is shifted to 42 degrees C . This increase is primarily at the expense of phosphatidylethanolamine . Extracts from cells harboring the plsC mutation have no detectable 1-acyl-sn-glycerol-3-phosphate acyltransferase activity . The fatty acid composition of the accumulated 1-acyl-sn-glycerol-3-phosphate is about 60% cis-vaccenate and 40% palmitate, with no detectable amounts of palmitoleate or other fatty acids, consistent with the known fatty acid composition of the sn-1 position of glycerophospholipids . The isolation of this gene, plsC, completes the list of genes known to be required for the synthesis of the major glycerophospholipids in E . coli. J Biol Chem, 1990 Oct 5, 265(28), 17050 - 4 recA protein filaments bind two molecules of single-stranded DNA with off rates regulated by nucleotide cofactor; Zlotnick A et al.; To probe the role of nucleotide cofactor in the binding of single-stranded DNA to recA protein, we have developed a sedimentation assay using 5'-labeled 32P-poly(dT).recA.poly(dT) complexes sediment quantitatively when centrifuged at 100,000 x g for 45 min, whereas free poly(dT) remains in the supernatant . In the presence of ATP, between 6 and 7 bases cosediment per recA monomer; but when ADP is present or in the absence of added nucleotide cofactor, only 3-3.5 bases/recA monomer cosediment . In competition experiments in which recA.32P-poly(dT) complexes are incubated with unlabeled poly(dT), we again find 3-3.5 bases of labeled poly(dT) cosedimenting per recA monomer when no nucleotide cofactor is present . However, when the same experiment is performed with ATP, only half of the expected 6-7 bases of labeled poly(dT) remain bound to the DNA, demonstrating that half of the poly(dT) in the complex exchanges rapidly with free poly(dT), whereas the other half equilibrates slowly, like poly(dT) in the absence of nucleotide . The rate of exchange of the second more tightly bound poly(dT) is accelerated when ADP is present . Our observations are rationalized by a model in which each recA protein helical filament binds two strands of poly(dT) with a stoichiometry of 3-3.5 bases/recA monomer/strand. Cell, 1990 Oct 5, 63(1), 203 - 11 Primary structure and functional expression of rat and human stem cell factor DNAs; Martin FH et al.; Partial cDNA and genomic clones of rat stem cell factor (SCF) have been isolated . Using probes based on the rat sequence, partial and full-length cDNA and genomic clones of human SCF have been isolated . Based on the primary structure of the 164 amino acid protein purified from BRL-3A cells, truncated forms of the rat and human proteins have been expressed in E . coli and mammalian cells and have been shown to possess biological activity . SCF is able to augment the proliferation of both myeloid and lymphoid hematopoietic progenitors in bone marrow cultures . SCF exhibits potent synergistic activities in conjunction with colony-stimulating factors, resulting in increased colony numbers and colony size. J Biol Chem, 1990 Oct 5, 265(28), 17167 - 73 The origin binding protein of herpes simplex virus 1 binds cooperatively to the viral origin of replication oris; Elias P et al.; The origin binding protein (OBP) or herpes simplex virus 1 has been expressed in Escherichia coli and used to study the role of multiple OBP binding sites in the herpes simplex virus #1 origin of replication, oris . Our results showed that the sequence CGTTCGCACTT was required for the binding of OBP to duplex DNA with high affinity . The minimal oris contains three repeats of this sequence or close derivatives thereof . Filter binding experiments have demonstrated that specific binding occurs to two of these repeats, box I and box II . An investigation using the DNase I footprinting technique revealed that the binding of OBP to box I and box II was cooperative and led to the formation of a highly organized complex in which the entire oris sequence was induced . We observed furthermore that the AT-rich sequence of the oris dyad was readily accessible to macromolecules even in the OBP.oris complex . The DNase I cleavage pattern of this sequence was, however, altered radically, indicating that a significant conformational change had occurred . A tentative structural model for the OBP-oris interaction is discussed on the basis of these observations. J Biol Chem, 1990 Oct 5, 265(28), 17078 - 83 Differential ATP requirements distinguish the DNA translocation and DNA unwinding activities of the Escherichia coli PRI A protein; Lee MS et al.; The Escherichia coli primosome is a mobile multiprotein DNA replication-priming apparatus that assembles at a specific site (termed a primosome assembly site (PAS} on single-stranded DNA-binding protein-coated single-stranded DNA . The PRI A protein (factor Y, protein n') is a PAS sequence-specific (d)ATPase as well as a DNA helicase and is believed to direct the assembly of the primosome at a PAS . In this report, the PRI A DNA helicase reaction is dissected in vitro, by use of a strand displacement assay, into three steps with distinct ATP requirements . First, the PRI A protein gains entry to the DNA via an ATP-independent, PAS sequence-specific binding event . Second, the PRI A protein translocates along the single-stranded DNA in the 3'----5' direction at a maximal rate of 90 nucleotides/s . DNA translocation requires ATP hydrolysis . The ATP concentration required to support half of the maximal translocation rate is 100 microM, which is identical to the Km for ATP of the PRI A protein DNA-dependent ATPase activity . Finally, the PRI A protein unwinds duplex DNA . The ATP concentration required for duplex DNA unwinding depends upon the length of the duplex region to be unwound . Displacement of a 24-nucleotide long oligomer required no more ATP than that required for the translocation of PRI A protein along single-stranded DNA, whereas displacement of a 390-nucleotide long DNA fragment required a 10-fold higher concentration of ATP than that required for oligomer displacement. J Biol Chem, 1990 Oct 5, 265(28), 16898 - 912 Triple-helical DNA pairing intermediates formed by recA protein; Umlauf SW et al.; RecA protein aligns homologous single- and double-stranded DNA molecules in three-stranded joints that can extend over thousands of base pairs . When cross-linked by 4'-amino-4,5',8-trimethyl-psoralen the joint structure observed in nonuniform and divided into multiple substructures each a few hundred base pairs long . Two paired substructures are observed; at least one, and possibly both, are right-handed triple helices . Sites of homologous contact are interspersed with regions where the DNA molecules are arranged side-by-side without contact . These substructures alternate in all combinations . The length and frequency of joints is much greater when one of the DNA substrates is linear, and interwinding is unrestricted, than when there are topological restrictions between the pairing partners . The results are consistent with the idea that recA protein facilitates the formation of a right-handed triple-helical DNA pairing intermediate during strand exchange . The results further suggest that recA filaments do not promote the formation of structures that provide efficient topological compensation for right-handed interwinding of two paired DNA molecules. Cell, 1990 Oct 5, 63(1), 87 - 95 The avian retroviral IN protein is both necessary and sufficient for integrative recombination in vitro; Katz RA et al.; The integration of viral DNA into the host cell chromosome is an essential feature of the retroviral life cycle . The integration reaction requires cis-acting sequences at the ends of linear viral DNA and a trans-acting product of the pol gene, the integration protein (IN) . Previously, we demonstrated that avian sarcoma-leukosis virus (ASLV) IN is able to carry out the first step in the integration process in vitro: nicking of the ends of linear viral DNA . In this paper, using two independent assays, we demonstrate that IN, alone, is sufficient to carry out the second step: cleavage and joining to the target DNA . These results demonstrate that the retroviral IN protein is an integrase. J Biol Chem, 1990 Oct 5, 265(28), 17094 - 100 Mammalian heterogeneous nuclear ribonucleoprotein A1 . Nucleic acid binding properties of the COOH-terminal domain; Kumar A et al.; A1 is a core protein of the eukaryotic heterogeneous nuclear ribonucleoprotein complex and is under study here as a prototype single-stranded nucleic acid-binding protein . A1 is a two-domain protein, NH2-terminal and COOH-terminal, with highly conserved primary structure among vertebrate homologues sequenced to date . It is well documented that the NH2-terminal domain has single-stranded DNA and RNA binding activity . We prepared a proteolytic fragment of rat A1 representing the COOH-terminal one-third of the intact protein, the region previously termed COOH-terminal domain . This purified fragment of 133 amino acids binds to DNA and also binds tightly to the fluorescent reporter poly(ethenoadenylate), which is used to access binding parameters . In solution with 0.41 M NaCl, the equilibrium constant is similar to that observed with A1 itself, and binding is cooperative . The purified COOH-terminal fragment can be photochemically cross-linked to bound nucleic acid, confirming that COOH-terminal fragment residues are in close contact with the polynucleotide lattice . These binding results with isolated COOH-terminal fragment indicate that the COOH-terminal domain in intact A1 can contribute directly to binding properties . Contact between both COOH-terminal domain and NH2-terminal domain residues in an intact A1:poly(8-azidoadenylate) complex was confirmed by photochemical cross-linking. Eur J Biochem, 1990 Oct 5, 193(1), 243 - 7 Linear dichroism studies of tryptophanase and its quasisubstrate complexes oriented in polyacrylamide gel; Zakomirdina LN et al.; Tryptophanase from Escherichia coli was oriented in a compressed slab of polyacrylamide gel and its linear dichroism (LD) and absorption spectra have been measured . The free enzyme displays four LD bands at 305, 340, 425 and 490 nm . Two bands at 340 and 425 nm belong to the internal coenzyme-lysine aldimine . The 305-nm band apparently belongs to an aromatic amino acid residue . The 490-nm band disappears after treatment with NaBH4 or after incubation with L-alanine and subsequent dialysis . It is suggested that the 490-nm band belongs to a quinonoid enzyme subform . The reaction of tryptophanase with threo-3-phenyl-DL-serine, L-threonine and D-alanine leads to formation of an external aldimine with an intense absorption band at 420-425 nm . The values of reduced LD (delta A/A) in this band strongly differ from that in the 420-nm band of the free enzyme . The LD value of the complex with D-alanine is intermediate between those of the free enzyme and the complex with 3-phenylserine . In the presence of indole the complex with D-alanine displays the same LD as that observed with 3-phenylserine . The reaction of tryptophanase with L-alanine or oxindolyl-L-alanine leads to formation of a quinonoid intermediate with an absorption band near 500 nm . The LD value in this band is close to that of an external aldimine with L-threonine . It is concluded that reorientations of the coenzyme occur in the course of the tryptophanase reaction. J Biol Chem, 1990 Oct 5, 265(28), 17044 - 9 Ribosomal protein S1 of Escherichia coli is the effector for the regulation of its own synthesis; Skouv J et al.; To facilitate the study of the regulation of the rpsA gene, a translational fusion between the rpsA gene and the lacZ gene was constructed . Synthesis of the fusion protein was repressed about 10-fold when rpsA was supplied in trans on a multicopy plasmid . This repression is similar to the post-transcriptional regulation previously found for the wild type rpsA gene . Addition of purified protein S1 to a coupled in vitro transcription-translation system caused a specific reduction in the synthesis of the rpsA-lacZ fusion protein . Addition of various subdomain fragments of protein S1 to the coupled in vitro system showed that the N-terminal fragment, possessing the ribosome binding domain of protein S1, was able to repress the synthesis of the rpsA-lacZ fusion protein . In contrast, fragments from the C-terminal region, containing the nucleic acid binding domain of protein S1, were inactive in this repression . Induction of truncated rpsA genes, coding for either the N-terminal 101 or 329 amino acids caused a reduction in the synthesis of the chromosomally encoded protein S1, thus confirming in vivo that the N-terminal part of protein S1 represses rpsA expression. J Biol Chem, 1990 Oct 5, 265(28), 17162 - 6 Site-directed mutagenesis of Moloney murine leukemia virus reverse transcriptase . Demonstration of lysine 103 in the nucleotide binding site; Basu A et al.; Lys103 and Lys421 of Moloney murine leukemia virus reverse transcriptase have been implicated in the dNTP binding function as judged by their reactivity to a substrate binding site-directed reagent, pyridoxal 5'-phosphate (Basu, A., Nanduri, V . B., Gerard, G . F., and Modak, M . J . (1988) J . Biol . Chem . 263, 1648-1653) . To assess the true catalytic importance of the individual lysine residues in Moloney murine leukemia virus reverse transcriptase, we mutated Lys103 and Lys421 to leucine and alanine, respectively . Analysis of the mutant enzymes revealed that mutation at the 103 position had a drastic effect on the DNA polymerase activity whereas the 421 mutation had no effect . Both mutants exhibited normal RNase H activity as well as the ability to bind to RNA or DNA templates as judged by UV-mediated cross-linking of the enzyme to the template primers . The enzyme with mutation at codon 421 (Lys----Ala) exhibited properties that were indistinguishable from the wild type with respect to its mode of catalysis, i.e . preference of template primer and divalent metal ion, RNA- or DNA-dependent DNA polymerase activity, RNase H activity, and the processive mode of DNA synthesis . These observations suggest that only Lys103 and not Lys421 is the catalytically important residue that is involved in the binding of substrate dNTP in Moloney murine leukemia virus reverse transcriptase. Nature, 1990 Oct 4, 347(6292), 497 - 8 Antibodies from Escherichia coli; Pluckthun A; Use of Escherichia coli as an expression host has opened up new possibilities in antibody research and its applications . It greatly facilitates rational engineering and random mutagenesis. Biochemistry, 1990 Oct 2, 29(39), 9274 - 81 Construction and expression of a novel recombinant anaphylatoxin, C5a-N19, as a probe for the human C5a receptor; Gerard NP et al.; We have constructed a novel recombinant C5a anaphylatoxin (C5a-N19) containing a 19-residue amino-terminal extension peptide, using a plasmid vector which secretes the nascent polypeptide to the Escherichia coli periplasmic space . C5a-N19 was purified from cell lysates by immunoaffinity chromatography using a monoclonal antibody which recognizes a portion of the amino-terminal extension peptide . C5a-N19 was characterized as biologically indistinguishable from the unmodified recombinant anaphylatoxin for release of lysosomal enzymes from dibutyryl-cAMP-differentiated U937 cells . In contrast to unmodified C5a, which is not recognized by anti-C5a antibodies following binding to its cellular receptor, receptor-bound C5a-N19 is recognized by the monoclonal antibody directed against the amino-terminal extension sequence . Because the monoclonal antibody recognizes the C5a-receptor complex on cells, this methodology is useful in fluorescence sorting of C5a receptor-positive cells . A C5a receptor affinity column was constructed by saturating monoclonal antibody bound to agarose with C5a-N19 . Digitonin-solubilized C5a receptor from dibutyryl-cAMP-induced U937 cells was adsorbed to the matrix and eluted by dissociation of the ligand-receptor complex from the antibody . Analysis by SDS-polyacrylamide gel electrophoresis revealed a unique protein band at 41K, consistent with the molecular weight predicted from cross-linking experiments when the contribution of C5a is subtracted . Development of this recombinant C5a derivative provides a useful probe previously unavailable for the C5a receptor molecule. Biochemistry, 1990 Oct 2, 29(39), 9218 - 25 Cadmium-113 NMR studies of the DNA binding domain of the mammalian glucocorticoid receptor; Pan T et al.; The DNA binding domain of the mammalian glucocorticoid hormone receptor (GR) contains nine highly conserved cysteine residues, a conservation shared by the superfamily of steroid and thyroid hormone receptors . A fragment {150 amino acids (AA) in length} consisting of GR residues 407-556, containing within it the entire DNA binding domain (residues 440-525), has been overexpressed and purified from Escherichia coli previously . This fragment has been shown to contain 2.3 +/- 0.2 mol of Zn(II) per mole of protein {Freedman, L . P., Luisi, B . F., Korszun, Z . R., Basavappa, R., Sigler, P . B., & Yamamoto, K . R . (1988) Nature 334, 543} . Zn(II) {or Cd(II) substitution} has been shown to be essential for specific DNA binding . 113Cd NMR of a cloned construct containing the minimal DNA binding domain of 86 AA residues {denoted GR(440-525)} with 113Cd(II) substituted for Zn(II) identifies 2 Cd(II) binding sites by the presence of 2 113Cd NMR signals each of which integrates to 1 113Cd nucleus . The chemical shifts of these two sites, 704 and 710 ppm, suggest that each 113Cd(II) is coordinated to four isolated -S- ligands . Shared -S- ligands connecting the two 113Cd(II) ions do not appear to be present, since their T1s differ by 10-fold, 0.2 and 2.0 s, respectively . Addition of a third 113Cd(II) or Zn(II) to 113Cd2GR(440-525) results in occupancy of a third site, which introduces exchange modulation of the two original 113Cd NMR signals causing them to disappear . Addition of EDTA to the protein restores the original two signals.(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1990 Oct 2, 29(39), 9212 - 8 Synthetic peptide model of an essential region of an aminoacyl-tRNA synthetase; Park SJ et al.; A 40 amino acid sequence of the unsolved structure of Escherichia coli alanine-tRNA synthetase is essential for tRNA binding and encodes an immunological determinant that cross-reacts with antibodies raised against a eukaryote (insect Bombyx mori) alanine enzyme . The secondary structure of this sequence is predicted to be an amphiphilic alpha-helix that includes one aspartyl and eight glutamyl side chain carboxyl groups . The antibody reactivity and the conformation of a synthetic peptide model of this region (Glu346 to Ser385) were investigated . In addition, double Arg----Gln and Leu----Ala substitutions were separately placed in the enzyme on the hydrophilic and hydrophobic face, respectively, of the predicted helix . These mutations conserve the polar/nonpolar character of each face and retain the potential for helix formation . Circular dichroism spectra of the synthetic peptide model demonstrate the potential for amphiphilic helix formation for the segment from Glu346 to Ser385 . The behavior of the mutations in the enzyme, together with earlier data and immunological assays presented here, suggests that one face of the putative helix is an antigenic region of the surface of the enzyme where it contributes to the interaction with alanine tRNA and that the specific sequence of the helix is an important determinant of enzyme stability. Biochemistry, 1990 Oct 2, 29(39), 9249 - 56 micF RNA binds to the 5' end of ompF mRNA and to a protein from Escherichia coli; Andersen J et al.; micF RNA regulates the levels of outer membrane protein F (OmpF) in Escherichia coli in response to temperature increase and other stress conditions by decreasing the levels of ompF mRNA (Andersen et al., 1989) . A 93-nucleotide micF RNA was synthesized in vitro directly from polymerase chain reaction generated DNA which was designed to contain a functional T7 RNA polymerase promoter upstream of the micF RNA gene and an appropriate restriction site for transcription termination . A transcript (150 nucleotides) containing the ribosomal binding domain of ompF mRNA messenger was synthesized in vitro from the ompF gene cloned into a T7 expression vector . A stable duplex was formed between micF RNA and the 150-nucleotide 5' transcript of ompF mRNA after incubation at 37 degrees C in a physiological buffer . The melting curve of the duplex formed by micF RNA and 150-nucleotide transcript revealed a Tm of 56 degrees C and a delta Tm that spans about 20 degrees C; both are consistent with the proposed structure for the micF/ompF duplex . In addition, as determined by competition studies and UV cross-linking/label-transfer analyses, an E . coli protein was found to bind specifically to micF RNA . The protein also bound weakly to the 150-nucleotide ompF transcript . The data are the first to demonstrate the complex between micF RNA and the 5' end of ompF mRNA and suggest that in vivo a micF ribonucleoprotein (RNP) particle may participate in the destabilization ompF mRNA during thermoregulation of OmpF porin. J Biochem (Tokyo), 1990 Oct, 108(4), 609 - 13 Mechanisms of inhibition of various cellular DNA and RNA polymerases by several flavonoids; Ono K et al.; Four flavonoids (i.e., baicalein, quercetin, quercetagetin, and myricetin), known to be inhibitors of HIV-reverse transcriptase, have been shown to be more or less inhibitory to the activities of various cellular DNA and RNA polymerases . The degree of the inhibition varied depending on the combination of the flavonoid and the enzyme species: baicalein was moderately inhibitory to DNA polymerase gamma and E . coli DNA polymerase I; quercetin was strongly inhibitory to DNA polymerase beta and E . coli RNA polymerase and moderately inhibitory to DNA polymerase I; quercetagetin was a potent inhibitor for all of DNA polymerases alpha, beta, gamma, and I and RNA polymerase; myricetin was a strong inhibitor of DNA polymerases alpha and I and RNA polymerase . However, terminal deoxynucleotidyltransferase was virtually insensitive to inhibition by these flavonoids . The inhibition by the flavonoids was due to competition with the template.primer in the case of the DNA polymerases, whereas the inhibition was due to competition with the triphosphate substrate (GTP) in the case of RNA polymerase . The Ki values of these flavonoid inhibitors for DNA and RNA polymerases was determined. Protein Eng, 1990 Oct, 4(1), 69 - 71 Site-directed mutagenesis reveals functional contribution of Thr218, Lys220 and Asp304 in chymosin; Suzuki J et al.; The functional contributions of amino acid residues Thr218 and Asp304 of chymosin, both of which are highly conserved in the aspartic proteinases, are analysed by means of site-directed mutagenesis . The optimum pH values, milk-clotting (C) and proteolytic (P) activities and kinetic parameters for synthetic oligopeptides as substrates were examined for the mutant enzymes . The mutation Thr218Ser caused a marked increase in the C/P ratio, which seemed to be due to a change in substrate recognition . Although the negative charge of Asp304 had been expected to play a role in lowering the optimum pH values in the aspartic proteinases, this turned out not to be the case in chymosin because both the mutations Asp304Ala and Asp304Glu caused a similar shift of the optimum pH towards the acidic side . In addition, the mutation Lys220Leu, which we generated previously, was found to cause a decrease in the C/P ratio, mainly due to the increase in the proteolytic activity. Anal Biochem, 1990 Oct, 190(1), 120 - 8 Isolation and separation of the glycan strands from murein of Escherichia coli by reversed-phase high-performance liquid chromatography; Harz H et al.; The length distribution of the glycan strands in the murein (peptidoglycan) sacculus of Escherichia coli has been analyzed after solubilization of the murein by complete digestion with human serum amidase . The glycan strands released were separated according to length by reversed-phase HPLC on wide-pore Nucleosil 300 C18 material at 50 degrees C, employing a convex gradient from 5 to 11% acetonitrile . The length of the fractionated glycan strands, which carry a nonreducing 1,6-anhydromuramic acid as a natural end group, was calculated from the ratio of total to nonreducing terminal muramic acid residues . This was possible after complete hydrolysis of the isolated glycan strands by muramidase followed by separation of the released nonreducing and reducing di- and tetrasaccharides by reversed-phase HPLC on Hypersil C18 . The method established allows the separation of the glycan strands of murein, a poly-GlcNAc(beta 1-4)MurNAc-polysaccharide, up to a degree of polymerization of approximately 60 . The predominant lengths of the glycan strands were 5 to 10 GlcNAc(beta 1-4)MurNAc disaccharide units. FEMS Microbiol Lett, 1990 Oct, 60(1-2), 103 - 7 Cloning, mapping and gene product identification of rhaT from Escherichia coli K12; Baldoma L et al.; Rhamnose utilization requires the function of a specific rhamnose transport system . Rhamnose transport mutants have been isolated and characterized . The structural gene, rhaT, encoding the rhamnose permease has been cloned from Escherichia coli . rhaT has been mapped in the rha locus (87.7 min) by analysis of cotransduction with glpK and other rha markers . The precise location of the gene has been determined by complementation analysis of rhamnose transport mutants transformed with recombinant plasmids containing different fragments of the cloned region . Gene order (counterclockwise) is established as glpK . . . rhaT-rhaR-rhaS-rhaB-rhaA-rhaD . The gene product has been identified by expression of rhaT in a T7 RNA polymerase/promoter system . This 23 kDa protein has been assigned to the rhaT product and has been shown to be located in the cell membrane. Biofactors, 1990 Oct, 2(4), 207 - 11 DNA photorepair: chromophore composition and function in two classes of DNA photolyases; Jorns MS; DNA photolyase catalyzes the repair of pyrimidine dimers in UV-damaged DNA in a reaction which requires visible light . Class I photolyases (Escherichia coli, yeast) contain 1,5-dihydroFAD (FADH2) plus a pterin derivative (5,10-methenyltetrahydropteroylpolyglutamate) . In class II photolyases (Streptomyces griseus, Scenedesmus acutus, Anacystis nidulans, Methanobacterium thermoautotrophicum) the pterin chromophore is replaced by an 8-hydroxy-5-deazaflavin derivative . The two classes of enzymes exhibit a high degree of amino acid sequence homology, suggesting similarities in protein structure . Action spectra studies show that both chromophores in each enzyme tested act as sensitizers in catalysis . Studies with E . coli photolyase show that the pterin chromophore is not required when FADH2 acts as the sensitizer but that FADH2 is required when the pterin chromophore acts as sensitizer . FADH2 is probably the chromophore that directly interacts with substrate in a reaction which may be initiated by electron transfer from the excited singlet state (1FADH2*) to form a flavin radical plus an unstable pyrimidine dimer radical . Pterin, the major chromophore in E . coli photolyase, may act as an antenna to harvest light energy which is then transferred to FADH2. Avian Dis, 1990 Oct-Dec, 34(4), 848 - 54 Effect of beta carotene on disease protection and humoral immunity in chickens; Tengerdy RP et al.; The effect of beta carotene on disease protection and humoral immunity in chickens was investigated in comparison with the effect of other lipid-soluble antioxidant vitamins, vitamin E and A, which are both proven immunoenhancers and contributors to disease protection . Beta carotene alone was not as effective as either vitamin in protecting chickens from Escherichia coli infection, nor did it significantly enhance humoral immunity . In combination with vitamin E, however, beta carotene significantly increased disease protection and reduced hepatomegaly caused by E . coli infection. Nippon Juigaku Zasshi, 1990 Oct, 52(5), 923 - 30 Biological effects of leptospiral lipopolysaccharide to mouse B, T and NK cells; Isogai E et al.; Leptospiral lipopolysaccharides (LPSs) extracted from Leptospira interrogans serovars copenhageni and hebdomadis were tested for the biological effect to mouse B, T and NK cells . Each leptospiral LPS was a potent mitogen for spleen B cells . Activation of the cells was also expressed by polyclonal B cell activation . In contrast, mitogenicity for T cells, induction of interleukin-2 (IL-2) secretion in T cells and increase of tumor-killing activity and chemiluminescence in NK cells were not observed after stimulation with leptospiral LPS . After intravenous injection of leptospiral LPS in mice, the spleen and lymphnodes were examined by histocytochemical technique . Increase of Ig-bearing lymphocytes was recognized while decrease of T cells was observed in the lymphoid organs . Mitogenic response to PHA, Con A and PWM decreased with relation to the T cell depletion . In conclusion, it is apparent that leptospiral LPS possess marked immunological potencies on B cells but not T and NK cells . The biological effects of leptospiral LPS were common ones as LPS but the level was considered to be different from classical LPS such as Escherichia coli LPS. Antibiot Khimioter, 1990 Oct, 35(10), 46 - 50 {Nucleotide sequence of aacC2 gene from a clinical strain of Escherichia coli}; Vakulenko SB et al.; The nucleotide sequence of the aacC2 gene encoding ++aminoglycoside acetyltransferase AAC(3)-II was determined . The structural part of the 852-bp aacC2 gene encoded the protein with the molecular weight of 30.6 consisting of 286 amino acids . The promotor region and the structural part of the gene were compared with the known sequences of the aacC2 gene . Evolutionary changes in the regulatory region as well as a number of nucleotide substitutes in the structural part of the gene and amino acid substitutes in the enzyme molecule were detected. Antibiot Khimioter, 1990 Oct, 35(10), 43 - 5 {Cloning of aacC2 gene from a clinical strain of Escherichia coli}; Entina EG et al.; The 2.3-kb Hind-III fragment containing the gentamicin resistance aacC2 gene was cloned from a clinical strain of E . coli with the aminoglycoside resistance pattern indicative of ++aminoglycoside acetyltransferase AAC (3)-II . An approach based on the phenomenon of the replicon dissociation of high molecular weight plasmids was applied . The restriction map of the cloned fragment was constructed . It was shown by subcloning the fragment that the aacC2 gene was localized within the 1.1-kb Alu I-Sal I fragment. Mol Gen Genet, 1990 Oct, 224(1), 24 - 32 N-terminal truncated forms of the bifunctional pi initiation protein express negative activity on plasmid R6K replication; Greener A et al.; The replication initiation protein pi of the Escherichia coli plasmid R6K is a dual regulator in the control of plasmid copy number, functioning both as a specific initiator and inhibitor of replication . While the biochemical basis of these activities is not known, initiator activity requires binding of the protein to the seven 22 bp direct repeats within the gamma-origin region . By deleting C-terminal segments of the pi coding region, we have found that the N-terminal polypeptides of pi that are produced, corresponding to the first 117 and 164 amino acids, respectively, retain the negative activity of the bifunctional protein, i.e . these truncated pi proteins specifically inhibit R6K replication in vivo . These negatively acting polypeptides, however, are incapable of initiating replication in vivo and fail to bind to the gamma-origin of the R6K DNA in vitro . A correspondence between the observed negative activity of the N-terminal peptide and the negative regulatory activity of the intact pi protein is supported by the finding that point mutations introduced into the 164 amino acid N-terminal peptide that result in a decrease in its inhibitory activity also produce a plasmid high-copy phenotype when these mutations are incorporated into the full-length pi protein . These findings demonstrate that the negative domain of pi resides in the N-terminal segment of the protein . Furthermore, the data obtained suggest that inhibition of R6K replication by pi does not require direct binding to DNA. J Gen Microbiol, 1990 Oct, 136 ( Pt 10), 2113 - 8 Requirement of the Escherichia coli dnaK gene for thermotolerance and protection against H2O2; Delaney JM; Thermotolerance in Escherichia coli is induced by exposing cells to a brief heat shock (42 degrees C for 15 min) . This results in resistance to the lethal effect of exposure to a higher temperature (50 degrees C) . Mutants defective in the recA, uvrA and xthA genes are more sensitive to heat than the wild-type . However, after development of thermotolerance these mutants are like the wild-type in their heat sensitivity . This suggests that thermotolerance is an inducible response capable of protecting cells from the lethal effects of heat, independently of recA, uvrA and xthA . Thermotolerance does not develop in a dnaK mutant . In addition, the dnaK mutant is sensitive to heat and H2O2, but is resistant to UV irradiation . This implies that the E . coli heat-shock response includes a mechanism that protects cells from heat and H2O2, but not from UV. J Muscle Res Cell Motil, 1990 Oct, 11(5), 378 - 91 Expression of human beta-myosin heavy chain fragments in Escherichia coli; localization of actin interfaces on cardiac myosin; Eldin P et al.; A cDNA clone coding for an internal fragment of slow-cardiac beta-myosin heavy chain was isolated from a lambda gt10 human skeletal muscle library . Six overlapping cDNA subclones, which span myosin heavy chain subregions and presumably interact with actin, were derived from this clone, fused to a beta-galactosidase vector and expressed in Escherichia coli . Three of the subclones were obtained by PCR (polymerase chain reaction) which enables gene or cDNA fragments to be amplified independently of preexisting restriction sites . Initially, various experiments were carried out using a long MHC (myosin heavy chain) fusion protein containing the 50 kDa-20 kDa connecting region, the whole 20 kDa region and the short subfragment 2 region . This MHC fusion protein was chemically or proteolytically cleaved in the same conditions as the native myosin molecule . Whole and truncated forms of the MHC fusion protein were separated on polyacrylamide gels, electroblotted on nitrocellulose sheets and renatured . They were then assayed in overlay experiments with F-actin and/or myosin light chains in solution . Specific antibodies were used to detect interactions between heavy chain fragments and F-actin or light chains . We thus observed that one long heavy chain fragment synthesized by E . coli behaved like proteolytic or chemical MHC preparations made from native myosin molecules . Two chymotryptic fragments of the MHC fusion protein, which are soluble at low ionic strength, cosedimented with F-actin in solution . Our results demonstrate that, in actin overlay experiments with whole fusion proteins, interactions seem to be due to the heavy chain fragment, not to the bacterial component . All interactions were non ATP-sensitive . We further investigated the possible participation of the six recombinant MHC fragments in contributing to the actomyosin interfaces on the 50 kDa-20 kDa regions of the human cardiac beta-MHC . The present procedure, which enables the synthesis of any MHC fragment independent of any protease site, is a powerful new tool for studying structure-function relationships within the myosin molecule family. Res Commun Chem Pathol Pharmacol, 1990 Oct, 70(1), 125 - 7 Beneficial effect of cod liver oil in murine endotoxemia; Rosa DM et al.; Cod liver oil (CLO), a marine fish oil, contains approximately 20% omega-3 fatty acids (OFA) . When CLO is administered to humans, inhibition of platelet aggregation, decreased platelet arachidonic acid levels, and reduced levels of endotoxin-induced thromboplastin synthesis by monocytes are observed . Since endotoxin causes both increased platelet aggregation and monocyte generation of thromboplastin with resultant microvascular compromise, the purpose of this study was to determine whether CLO is protective in murine endotoxemia . Swiss Webster mice were given CLO (1.0mg, 10.0mg, or 100mg), or 100mg triolein (an unsaturated fat containing no OFA) or saline (control) intraperitoneally (IP) three hours prior to IP administration of 0.3mg E.coli endotoxin . Survivals at 48 hours post-endotoxin administration were as follows: (A) 1.0mg CLO (4/20, 20%); (B) 10mg CLO (5/20, 25%); (C) 100.0mg CLO (11/20, 55%); (D) 100mg triolein (1/20, 5%); (E) 0.13cc saline (1/20, 5%) . The significance of groups A,B,C,D verses the control group E is as follows: A vs E, p = 0.15; B vs E, p = 0.08; C vs E, p = 0.0006; D vs E, p = 0.76 . This study demonstrates the beneficial effect of 100mg parenteral CLO in murine endotoxemia . This effect is probably mediated through antiplatelet and/or antimonocyte activating mechanisms. Genome, 1990 Oct, 33(5), 696 - 706 Use of interplasmid recombination to generate stable selectable markers for yeast transformation: application to studies of actin gene control; Hubberstey AV et al.; A plasmid recombination system has been developed that relies upon interplasmid exchanges for yeast cell viability . Two types of plasmids, one carrying the LEU2 allele inserted within yeast actin gene sequences and the other carrying 2-microns plasmid DNA and an intact actin gene, were constructed . Neither plasmid alone yielded transformants in the haploid Leu- strain AH22, but when cotransformed, a number of colonies were obtained . Southern blot analysis revealed that transformants arose because of recombination events within the homologous actin sequences that transferred the LEU2 gene to the actin gene on the 2-microns plasmid . The recombinant plasmids could be recovered, and sequence analysis of one recombination site revealed that the exchange event was faithful at the nucleotide level . The resulting recombinant plasmids carried a defective actin gene and presumably arose because of a double-crossover event . Deletion mutations that prevented actin gene expression on one donor plasmid enabled the recovery at a high frequency of transformants resulting primarily from single-crossover events between the two plasmids . This was presumably because such events no longer generated an intact actin gene on a multicopy plasmid . Infrequently a transformant from a plasmid with an intact gene was recovered, but in these cases the plasmid was not present in multiple copies . These cells exhibited a slower growth rate, and Northern blot analysis revealed an elevated level of actin mRNA. J Comp Pathol, 1990 Oct, 103(3), 289 - 300 Fever and changes in plasma zinc and iron concentrations in the goat . The effects of interferon inducers and recombinant IFN-alpha 2a; Van Miert AS et al.; Inflammation or invasion by pathogenic micro-organisms induces systemic changes, collectively known as the acute phase response . Among the varied alterations that together produce this response are fever, hypoferraemia and hypozincaemia . It is likely that these responses are mediated, in part, by production and release of cytokines such as interleukin-1 (Il-1), interferons (IFN-alpha) and tumour necrosis factor (TNF) . The present report describes a comparative study in dwarf goats on recombinant human IFN-alpha 2a (0.5 x 10(5) IU per kg intravenously (i.v.) and 0.5 x 10(6) IU per kg intramuscularly (i.m.}, Poly I:Poly C (an interferon inducer; 30 micrograms per kg i.v.), Newcastle disease virus La Sota strain (an interferon inducer; 0.5 ml per kg i.v.) and Escherichia coli endotoxin (an Il-1 and TNF inducer; 0.1 microgram per kg i.v.) . The i.v . injection of recombinant IFN-alpha 2a caused characteristic monophasic febrile reactions, but no significant changes in plasma zinc and iron concentrations . The temperature responses were not due to endotoxin contamination because polymyxin B, which blocks endotoxin, had no inhibitory effect on the pyrogenicity of IFN-alpha 2a . In contrast, the IFN-alpha 2a-induced fever was completely prevented by flurbiprofen pretreatment (1 mg per kg i.v.) . In contrast to the i.v . administration, i.m . injection of IFN-alpha 2a caused fever, hypoferraemia and hypozincaemia . Similar results were obtained after E . coli endotoxin, NCD La Sota strain and Poly I:Poly C injection . However, the shapes of the temperature curves and the changes in trace metal concentrations were markedly different . These data support the theory that fever and the changes in plasma zinc and iron concentrations are regulated by different mechanisms. Liver, 1990 Oct, 10(5), 302 - 12 Inhibitory effects of hepatitis B virus antigen on induction of lymphokine-activated killer cell activity; Shirai M et al.; We investigated the inhibitory effects of purified recombinant hepatitis B virus (HBV) surface antigen (rHBsAg) and core antigen (rHBcAg) on lymphokine-activated killer cell (LAK) activity . Either peripheral blood mononuclear cells (PBMCs) or CD16+ CD3- LAK precursors, both of which were pre-incubated with interleukin-2 (IL-2) and rHBsAg or rHBcAg for 72 h, showed a significant decrease in LAK cytotoxicity against Daudi cells, in comparison to the results recorded in the presence of IL-2 alone, or IL-2 and E . coli extracts . This inhibitory effect was dose-dependent and was observed to be time-dependent from 24 to 72-h-cultures with these HBV antigens . This influence was not mediated with either adherent cells or other accessory cells . The proliferative reaction of either PBMCs or the LAK precursors after being cultured with IL-2 and rHBsAg or rHBcAg for 72 h was significantly diminished compared with the levels of reaction of those cells after a 72-h culture with IL-2 alone or with IL-2 and E . coli extracts . The levels of IL-2-driven IL-2 receptor (p55) expression of either PBMCs or the LAK precursors in the presence of rHBsAg or rHBcAg were higher than the levels seen in the absence of these HBV antigens . These results suggest that HBsAg and HBcAg may inhibit the induction of LAK activity by interfering with the proliferative reaction of the LAK precursors to IL-2 without inhibiting the IL-2 receptor expression of the cells . Cytofluorographic analysis of PBMCs, cultured with rIL-2, showed lower percentages of CD3+ and CD16+ cells in the presence of these HBV antigens than those in the absence of antigens. J Biol Response Mod, 1990 Oct, 9(5), 475 - 9 Stimulation of granulopoiesis in patients with malignancy by recombinant human granulocyte-macrophage colony-stimulating factor: assessment of two routes of administration; Herrmann F et al.; We administered Escherichia coli-derived recombinant human granulocyte-macrophage colony-stimulating factor to 61 patients with malignancy, 36 of whom had normal peripheral blood counts and 25 of whom had peripheral cytopenia due to underlying bone marrow disease, to compare the efficacy of two different routes of administration to stimulate the in vivo granulopoiesis: i.e., continuous i.v . infusion and s.c . injection . Three well-tolerated dose levels were investigated . Application of granulocyte-macrophage colony-stimulating factor resulted in dose-dependent increases in circulating neutrophils, eosinophils, and monocytes and an increase in bone marrow cellularity, irrespective of route of administration . In some patients, mild side effects, including bone pain, dyspnea, flu-like symptoms, and a decrease of platelet counts, were recorded, but they were less pronounced when the hormone was administered subcutaneously. Isr J Med Sci, 1990 Oct, 26(10), 564 - 7 Pyogenic liver abscess; Shpitz B et al.; A retrospective study of 18 adults admitted to Meir General Hospital with pyogenic liver abscess during the years 1982-88 was conducted . Our most useful diagnostic tool was ultrasound, which was accurate in all 15 patients in whom it was performed . Only two patients presented with multiple abscesses . Increased alkaline phosphatase was the most predictive laboratory finding, seen in all but one patient . One person was treated conservatively by antibiotics only . In one patient the abscess drained spontaneously into the right thoracic cavity . Initial surgical drainage was done in nine patients and percutaneous catheter drainage in seven . Only one patient died, of septic shock, yielding a mortality rate of 5.5% . The treatment-related complication rate, observed in almost half of the patients, was high . All patients with such complications required repeated drainage, which was achieved surgically in all cases. Inflammation, 1990 Oct, 14(5), 499 - 508 A monoclonal-antibody-defined adhesion-related antigen on bovine neutrophils is required for neutrophil aggregation; Bochsler PN et al.; Surface adhesion molecules present on human leukocytes are known to regulate certain adhesion-related events, such as adhesion to endothelium, extravasation, and aggregation . We have used a mouse anti-human monoclonal antibody designated 60.3 (MAb 60.3) and indirect immunofluorescence technique to identify an antigen on bovine neutrophils (PMNs) . MAb 60.3 bound to resting and stimulated bovine PMN in a surface-oriented pattern . Immunofluorescence flow cytometric analysis indicated that warming the PMNs from 4 degrees C to 37 degrees C slightly increased (13.9%) expression of the antigen recognized by MAb 60.3 . Zymosan-activated serum (ZAS, 10%) increased antigen expression by 12.4% over those PMNs in buffer alone, and phorbol 12-myristate 13-acetate (PMA; 100 ng/ml) by 65.6% . Bacterial lipopolysaccharide (LPS; 1 micrograms/ml) from E . coli 0111:B4 did not enhance antigen expression . The functional nature of this antigen was demonstrated by use of MAb 60.3 and PMN aggregation . Preincubation of bovine PMN with MAb 60.3 for 10 min resulted in nearly complete inhibition of PMN-PMN aggregation upon subsequent stimulation with PMA (100 ng/ml); preincubation with a control antibody did not inhibit aggregation . These results indicate that bovine PMNs possess surface molecule(s) that may function in adhesion-related events, and surface expression may be enhanced by PMN stimulation. Biophys J, 1990 Oct, 58(4), 897 - 903 Study of mechanisms of electric field-induced DNA transfection . II . Transfection by low-amplitude, low-frequency alternating electric fields; Xie TD et al.; Electroporation for DNA transfection generally uses short intense electric pulses (direct current of kilovolts per centimeter, microseconds to milliseconds), or intense dc shifted radio-frequency oscillating fields . These methods, while remarkably effective, often cause death of certain cell populations . Previously it was shown that a completely reversible, high ionic permeation state of membranes could be induced by a low-frequency alternating electric field (ac) with a strength one-tenth, or less, of the critical breakdown voltage of the cell membrane (Teissie, J., and T . Y . Tsong . 1981 . J . Physiol . (Paris) . 77:1043-1053) . We report the transfection of E . coli (JM105) by plasmid PUC18 DNA, which carries an ampicillin-resistance gene, using low-amplitude, low-frequency ac fields . E . coli transformants confer the ampicillin resistance and the efficiency of the transfection can be conveniently assayed by counting colonies in a selection medium containing ampicillin . For the range of ac fields employed (peak-to-peak amplitude 50-200 V/cm, frequency 0.1 Hz-1 MHz, duration 1-100 s), 100% of the E . coli survived the electric field treatment . Transfection efficiencies varied with field strength and frequency, and as high as 1 x 10(5)/micrograms DNA was obtained with a 200 V/cm square wave, 1 Hz ac field, 30 s exposure time, when the DNA/cell ratio was 50-75 . Control samples gave a background transfection of much less than 10/micrograms DNA . With a square wave ac field, the transfection efficiency showed a frequency window: the optimal frequency was 1 Hz with a 200 V/cm field, and was approximately 0.1 Hz with a 50 V/cm field.(ABSTRACT TRUNCATED AT 250 WORDS) Radiat Res, 1990 Oct, 124(1), 57 - 61 Homologous recombination and mutagenesis of gamma-irradiated plasmid DNA in Escherichia coli host cells; Mudgett JS et al.; Plasmid DNA was used to study gamma-radiation-induced recombination and mutagenesis in Escherichia coli host cells . Plasmid pBRP1, a derivative of pBR322 containing the lac operon of E . coli, was irradiated with 60Co gamma rays prior to transformation into E . coli strains of different recA and lac genotypes . Plasmid-chromosome recombination was assayed in lacY1 host cells, whereas plasmid mutagenesis was assayed in delta lac host cells lacking chromosomal sequences homologous to the plasmid . Both recombinant and mutant plasmids were identified by the phenotypic changes in lactose utilization, and confirmed by restriction analysis of isolated plasmids . Plasmid-chromosome recombination was induced to high levels (about 20% of survivors at 700 Gy) and was dependent on the host recA gene . Plasmid mutagenesis occurred at lower levels (about 1.5% of survivors at 600 Gy) and was relatively independent of the recA gene . Plasmid survival was unaffected by the presence or absence of host recA mutations or the potential for plasmid-chromosome recombination. Proc Natl Acad Sci U S A, 1990 Oct, 87(20), 7898 - 901 Induction of verotoxin sensitivity in receptor-deficient cell lines using the receptor glycolipid globotriosylceramide; Waddell T et al.; Verotoxin 1 is an Escherichia coli-derived subunit toxin that specifically binds to the glycolipid globotriosylceramide and is cytotoxic for cells that contain this plasma membrane glycolipid . Glycolipid incorporation experiments have now been performed using human lymphoid cells of the B lineage that lack this receptor, to conclusively demonstrate that globotriosylceramide alone is a functional receptor for this toxin . Globotriosylceramide incorporated into the membrane of toxin-resistant cells provides intracellular access to verotoxin by receptor-mediated endocytosis . Protein synthesis is then inhibited and globotriosylceramide-containing cells are killed. Lab Invest, 1990 Oct, 63(4), 551 - 6 Photoaffinity labeling of human c-myc protein with deoxythymidine triphosphate; Satav JG et al.; The recombinant human c-myc protein expressed in Escherichia coli can be efficiently labeled by ultraviolet-mediated cross-linking to dTTP and to a lesser extent to other nucleoside diphosphates and triphosphates, but not to nucleoside monophosphates . Specificity of nucleoside phosphate binding is suggested by (a) concentration-dependent competition by some nucleoside phosphates but not by others and (b) by the observation that the denatured myc protein does not bind the nucleotides . Competition experiments also indicate that the affinity of c-myc protein for nucleoside diphosphates and triphosphates is approximately the same irrespective of the nature of the base, or of the pentose sugar, but the thymine base permits the most efficient photoactivated cross-linking . The ultraviolet-mediated photoactivated cross-linking of deoxythymidine triphosphate has been used to identify the c-myc protein in extracts of cells which overexpress c-myc, and to identify the intermediates in myc oncoprotein degradation. J Gen Virol, 1990 Oct, 71 ( Pt 10), 2229 - 32 Antigenic analysis of the coat protein of beet necrotic yellow vein virus by means of monoclonal antibodies; Koenig R et al.; By means of monoclonal antibodies (MAbs), five (groups of) epitopes were identified on particles of beet necrotic yellow vein virus (BNYVV) . Epitopes 1 and 2, which were located on the opposite extremities of virus particles, are discontinuous (SDS-labile) epitopes which were destroyed when the particles were treated with trypsin . Epitope 3 is a continuous (SDS-stable) epitope located at the same extremity as epitope 2 . It was not destroyed when the particles were treated with trypsin and was present on an Escherichia coli-expressed fusion protein containing amino acids (aa) 1 to 103 of the BNYVV coat protein . The continuous epitope 4, which was located along the entire length of the particles, was found to be present on a fusion protein containing aa 104 to 188 of the BNYVV coat protein but not on trypsin-treated virus particles . In Western blots, these treated particles yielded two slightly smaller coat proteins which failed to react with MAbs specific for epitope 4 but did react with polyclonal antisera and MAbs specific for epitope 3 . BNYVV coat protein has a trypsin cleavage site on the carboxyl side of arginine in position 182, so it is therefore suggested that epitope 4 is located on the exposed C terminus, which is composed of aa 183 to 188 . Epitope 5 was also located along the entire length of the particles but in a more uneven distribution than epitope 4 . This may be because it is a discontinuous epitope that is very sensitive to subtle changes in protein conformation. J Gen Virol, 1990 Oct, 71 ( Pt 10), 2201 - 9 Expression of cauliflower mosaic virus gene I in insect cells using a novel polyhedrin-based baculovirus expression vector; Zuidema D et al.; An improved polyhedrin-based baculovirus expression vector was constructed to expedite distinguishing infections by putative baculovirus recombinants from infections by wild-type (wt) baculovirus . The vector utilizes the Escherichia coli beta-galactosidase gene (lacZ) as a genetic marker for positive recombination between wt Autographa californica nuclear polyhedrosis virus and the baculovirus transfer vector . The marker gene/expression cassette was constructed so that lacZ and the deleted polyhedrin gene were transcribed in opposite orientations, both terminating in a simian virus 40 DNA fragment which acts as a bidirectional terminator . In the constructed vector, lacZ is transcribed from the Drosophila melanogaster heat-shock promoter (hsp 70), which is constitutively expressed in baculovirus-infected Spodoptera frugiperda (Sf) cells, thereby making the site of the deleted polyhedrin gene available for the insertion and expression of foreign genes under the control of the polyhedrin promoter . Recombinant baculoviruses are readily selected in plaque assays by the development of a blue colour upon the addition of X-Gal . The colour selection renders the retrieval of recombinants less dependent on a high frequency of recombination between the transfer vector and wt baculovirus DNA . The usefulness of this new vector was illustrated by expressing gene I of cauliflower mosaic virus, which encodes a protein of Mr 46,000 . Expression of gene I was at the same level as in cells infected with a conventional polyhedrin-based expression vector . Gene I protein formed large hollow fibre-like structures in the cytoplasm of infected Sf cells . This is the first plant virus protein to be expressed in insect cells by a recombinant baculovirus. J Clin Microbiol, 1990 Oct, 28(10), 2264 - 8 Detection of colonization factor antigen I-positive enterotoxigenic Escherichia coli with a cloned polynucleotide probe; Grewal HM et al.; We compared a new colony hybridization assay with an established enzyme-linked immunosorbent assay for detection of enterotoxigenic Escherichia coli (ETEC) expressing colonization factor antigen I (CFA/I) . The tests were applied to 135 human ETEC strains . Of these isolates, 30 had previously been characterized for CFAs . A strain harboring the plasmid vector of the polynucleotide gene probe, nine non-ETEC strains from healthy infants, and eight ETEC strains of animal origin were included for further evaluation of probe specificity . The two assays showed a high level of concordance in the specific detection of ETEC strains expressing CFA/I . A total of 24 strains tested positive in the CFA/I hybridization assay, while 23 of those strains were positive in the CFA/I enzyme-linked immunosorbent assay . The single discrepant result could be explained by the loss of a regulatory gene . The strain harboring the plasmid vector of the probe, the non-ETEC E . coli strains, and the ETEC strains of animal origin were all negative in the CFA/I probe assay. FEBS Lett, 1990 Oct 1, 271(1-2), 203 - 6 Structural features of the McPC603 Fab fragment not defined in the X-ray structure; Skerra A et al.; The proteolytic Fab fragment of the well characterized antibody McPC603 was compared to the recombinant Fab fragment, which was obtained in functional form from an Escherichia coli expression system {(1989) Methods Enzymol . 178, 497-515} . We found evidence that the proteolytic fragment is glycosylated at Asn H160 in the CH1 domain, where additional electron density had been observed in the crystal structure {J . Mol . Biol . 190, 593-604} . In addition, its heavy chain is about 30 amino acids longer than visible in the electron density and thus contains the complete hinge region . These structural differences between the recombinant Fab fragment, which had been designed exactly according to the defined electron density, and the proteolytic Fab fragment of McPC603 had no effect on the hapten binding properties of these antigen binding fragments . Yet, it may be important to be aware of these structural features of McPC603 in folding studies and some comparative analyses of antibody structures. Arch Biochem Biophys, 1990 Oct, 282(1), 202 - 5 Base modifications in plasmid DNA caused by potassium permanganate; Akman SA et al.; KMnO4 is a powerful oxidizing agent which has been used to modify DNA bases . In previous studies, mild KMnO4 treatment has been shown to preferentially modify Thy; Cyt and Gua are modified only under harsher conditions to as yet unidentified products . In the present study, denatured plasmid pCMV beta gal DNA was exposed to 0.015-1.5 mM KMnO4, pH 8.6, at 4 degrees C for 5 min, after which the DNA was hydrolyzed in formic acid, trimethylsilylated, and analyzed for modified base content by gas chromatography-mass spectrometry/selected ion monitoring . KMnO4 treatment, even at concentrations as low as 0.015 mM, caused a concentration-dependent increase in the Thy products Thy glycol and 5-hydroxy-5-methylhydantoin, the Cyt products Cyt glycol, 5,6-dihydroxycytosine, and 5-hydroxyhydantoin, the Ade product 8-hydroxyadenine, and the Gua product 8-hydroxyguanine . The Ade product 4,6-diamino-5-formamidopyrimidine and the Gua product 2,6-diamino-4-hydroxy-5-formamidopyrimidine were minimally (less than or equal to 2-fold) increased by treatment with greater than or equal to 0.8 mM KMnO4 . These data demonstrate that, in addition to Thy, Cyt, Gua, and Ade bases in plasmid DNA may be modified by treatment with KMnO4, even under mild conditions . They represent the first identification of Cyt, Gua, and Ade products caused by KMnO4 treatment . Furthermore, these data suggest that previous studies which have used treatment with KMnO4 to study the mutagenicity of Thy glycol specifically or as a Thy-specific probe in DNA structure should be interpreted with caution. Arch Biochem Biophys, 1990 Oct, 282(1), 116 - 24 Structural and functional studies of the rat mitochondrial single strand DNA binding protein P16; Hoke GD et al.; The rat mitochondrial single strand DNA binding protein (SSB) P16 was purified to apparent homogeneity by elution from single strand DNA agarose with ethidium bromide . Each monomer of P16 contains two tryptophan residues, and the intrinsic fluorescence from these residues is quenched upon binding to single strand polynucleotides . From fluorescence quench titrations of ligand to fixed amounts of DNA lattice, a binding site size of 8 or 9 nucleotides per P16 monomer was found . Measurement of the affinity of P16 for isolated sites by titration with either oligo(dT)8 or 5'-dephosphorylated oligo(dT)8 indicated values on the order of 10(7) M-1 . P16 exhibited a binding preference for single strand DNA, poly(dT), and poly(dC) in comparison to double strand DNA, poly(U), or poly{d(A-T)} . Although it was not possible to show that P16 destabilizes double helical DNA or even poly{d(A-T)}, binding of P16 does inhibit the process of renaturation as shown by inhibition of duplex formation between poly(dA) and poly(dT) . The binding of saturating amounts of P16 to single strand poly(dT).oligo(dA)50 template-primers enhanced approximately 10-fold the activity of both the homologous mitochondrial DNA polymerase and the Escherichia coli DNA polymerase I Klenow fragment . However, the mitochondrial DNA primase was nearly completely inhibited by the saturation of the poly(dT) template with P16 . Amino-terminal sequence analysis of P16 and a protease-insensitive, DNA binding domain (Mr approximately 6000) revealed that the DNA binding domain residues, at least in part, in the amino-terminal third of the P16 molecule . Furthermore, the amino-terminal sequence was found to be strikingly similar to that of the Xenopus laevis mtSSB-1 and to a lesser extent similar to E . coli SSB and E . coli F sex factor SSB. Arch Biochem Biophys, 1990 Oct, 282(1), 110 - 5 Yeast thioltransferase--the active site cysteines display differential reactivity; Gan ZR et al.; Thioltransferase, catalyzing thiol-disulfide interchange between reduced glutathione and disulfides, was purified to homogeneity from Saccharomyces cerevisiae . The purification procedure included ammonium sulfate precipitation, Sephadex G-50 gel filtration, CM-Sepharose ion exchange chromatography, and C18 reverse phase high pressure liquid chromatography . Two thioltransferase activity peaks were resolved by CM-Sepharose chromatography . The protein from the major peak had a molecular weight of 12 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis while the minor peak protein migrated slightly faster in this gel system . Both proteins showed similar amino acid compositions and identical N-termini . The major peak of thioltransferase was extensively characterized . Plots of thioltransferase activity as a function of S-sulfocysteine or hydroxyethyl disulfide concentration did not show normal Michaelis-Menten kinetics . The enzyme activity had a pH optimum of 9.1 . The protein has 106 amino acid residues with two cysteines and no arginine . The active site amino acid sequence of the enzyme was identified as Cys26-Pro-Tyr-Cys29, which is similar to that of mammalian thioltransferase and Escherichia coli glutaredoxin . The two cysteines at the active site displayed different reactivities to iodoacetamide . Cys26 was alkylated by iodoacetamide at pH 3.5 while Cys29 was alkylated at pH 8.0 . The enzyme was completely inactivated when the Cys26 was carboxymethylated . A plot of incorporation of iodoacetamide into Cys29 at different pHs was similar to the pH dependence of the enzyme activity . The result suggested that Cys26 could readily initiate nucleophilic attack on disulfide substrates at physiological pH. Am Rev Respir Dis, 1990 Oct, 142(4), 775 - 81 Studies of an antiendotoxin antibody in preventing the physiologic changes of endotoxemia in awake sheep; Wheeler AP et al.; In sheep, endotoxin (LPS) causes pulmonary hypertension, hypoxemia, leukopenia, exudation of protein-rich lung lymph, reduced dynamic compliance (Cdyn), and increased resistance to airflow (RL), changes similar to those seen in human sepsis and sepsis-induced ARDS . We used well-described methods in the awake sheep-endotoxin model to evaluate the effectiveness of a commercially manufactured antibody to prevent the physiologic changes of endotoxemia . In awake sheep with chronic lung lymph fistulas, we used a whole-body plethysmograph to measure Cdyn, RL, and FRC . Pulmonary artery, left atrial, and systemic arterial pressures were recorded continuously . Arterial blood gases (for calculating AaPO2), leukocyte counts, and lymph samples were collected every 30 min . Animals received a 30-min (2 mg/kg) infusion of antiendotoxin antibody 4 h before LPS (0.75 micrograms/kg) challenge (n = 4), or were given a mixture of LPS (0.75 micrograms/kg) and antibody (2 mg/kg) that had been incubated in vitro at 37 degrees C for 30 min before infusion (n = 6) . A control group given only 2 mg/kg of antibody (n = 4) showed no change in any measured parameter, whereas control animals receiving LPS alone (n = 6) exhibited a typical endotoxin response . In all animals receiving endotoxin, Cdyn declined by approximately 50% within 30 to 60 min, and RL increased approximately sixfold over a similar time course . Accompanying the abnormalities in lung mechanics were pulmonary hypertension, leukopenia, and widening of the AaPO2.(ABSTRACT TRUNCATED AT 250 WORDS) Am J Physiol, 1990 Oct, 259(4 Pt 2), H1038 - 43 Loss of vascular responsiveness induced by endotoxin involves L-arginine pathway; Julou-Schaeffer G et al.; The involvement of L-arginine-dependent nitric oxide (NO) production in the vascular failure observed in endotoxemia was investigated in male Wistar rats treated with Escherichia coli lipopolysaccharide (LPS) . Contractile responses to norepinephrine (NE) were measured ex vivo in aortas isolated from rats treated with LPS (20 mg/kg ip, 4 h before experiments) and pressor responses to NE were recorded in vivo in rats infused with LPS (5 mg.kg-1.h-1 iv) . LPS pretreatment induced a rightward shift of the concentration-response curve to NE and a reduction of the maximal contraction by approximately 43% and 54% (P less than 0.05) in aortic rings with and without functional endothelium, respectively . This was not modified by the presence of indomethacin (10 microM) during the contractile experiments . In contrast, in the presence of NG-monomethyl-L-arginine (L-NMMA, 300 microM) or methylene blue (10 microM), maximal contractions to NE were restored to control values whether functional endothelium was present or not . The effects of L-NMMA were reversed by L- but not by D-arginine . Additionally, the effects of LPS pretreatment on vascular contractility were potentiated by L-arginine . In vivo, LPS infusion produced a reduction in pressor responsiveness to NE (0.1-10 mg/kg), which was also abolished by L-NMMA (30 mg/kg iv) . This effect of L-NMMA was reversed by L- but not by D-arginine (100 mg/kg iv) . These results demonstrate that activation of the L-arginine pathway has a major role in the production of vascular hyporeactivity in endotoxemia, ex vivo as well as in vivo . Additionally, they suggest that endothelium-independent vascular production of NO may be involved. Am J Physiol, 1990 Oct, 259(4 Pt 1), G519 - 23 Validation of tonometric measurement of gut intramural pH during endotoxemia and mesenteric occlusion in pigs; Antonsson JB et al.; Tonometry is a minimally invasive method for estimating gastrointestinal intramural pH (pHi) . Tissue pH is calculated by using the Henderson-Hasselbalch equation and measurements of arterial {HCO-3} and CO2 tension (PCO3) of saline contained in a Silastic balloon within the lumen of the gut . The validity of the method rests on two key assumptions: 1) PCO2 in saline in the tonometer balloon is similar to tissue PCO2 and 2) tissue and arterial {HCO-3} are similar . To validate this method, ileal pHi measured directly with a microelectrode was compared with pHi estimated tonometrically in four groups of anesthetized pigs . Group I (n = 4) were controls . In group II (n = 4), intestinal tissue acidosis was induced by total occlusion of the superior mesenteric artery (SMA) . In group III (n = 5), acidosis was induced by partial occlusion of the SMA . In group IV (n = 4), tissue acidosis was induced by endotoxemia . Agreement was excellent between direct and tonometric measurements in groups I and IV and less good in groups II and III . Weighted mean correlation coefficients (rw) for the two measurement methods were 0.743 and 0.9447 in groups II and IV, respectively . Correlation coefficients for the individual animals in group III were more variable than the other groups and ranged from 0.547 to 0.990 . The tonometric method for measuring GI pHi is invalid under conditions of zero flow and leads to error under conditions of low flow . However, the method is reliable in the setting of tissue acidosis induced by endotoxemia. Am J Physiol, 1990 Oct, 259(4 Pt 1), E498 - 505 Altered hepatic mitochondrial fatty acid oxidation and ketogenesis in endotoxic rats; Takeyama N et al.; Rat hepatic mitochondrial function, including oxidative phosphorylation, fatty acid oxidative capacity, kinetic parameters of carnitine palmitoyltransferase I (CPT I), and sensitivity of CPT I to malonyl-CoA inhibition were studied in vitro in isolated mitochondria following Escherichia coli lipopolysaccharide (LPS) . The hepatic mitochondrial CPT I in LPS-treated rats showed a lower apparent maximum velocity (Vmax) for palmitoyl-CoA and Ki for malonyl-CoA without changes in apparent Km for palmitoyl-CoA . The rate of oxygen consumption or end-product formation of palmitoyl-L-carnitine and octanoate was not altered, but the rate of CPT I-dependent palmitoyl-CoA (plus L-carnitine) oxidation was reduced by LPS, when acetyl-CoA produced via beta-oxidation was directed toward citrate . When acetyl-CoA was directed to acetoacetate, the oxygen consumption rates of palmitoyl-L-carnitine and palmitoyl-CoA (plus L-carnitine) were decreased by LPS, although mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase activity was not altered . These results indicate that hepatic mitochondria isolated from LPS-treated rats show lower ketogenic and long-chain acyl-CoA oxidative capacity than those of fasted controls, and inhibition of ketogenesis is elicited at a site distal to CPT I in addition to reduction in CPT I activity. Virology, 1990 Oct, 178(2), 626 - 30 Reverse guanine phosphoribosyltransferase selection of recombinant vaccinia viruses; Isaacs SN et al.; We have developed a procedure for the selection of recombinant vaccinia viruses with applicability to poxvirus mutagenesis studies and to the use of vaccinia virus as an expression vector . The method depends on the specific inability of a recombinant vaccinia virus expressing the Escherichia coli guanine phosphoriboxyltransferase gene (gpt) to form plaques on a hypoxanthine-guanine phosphoribosyltransferase-negative line of mouse fibroblasts in the presence of 6-thioguanine . Recombinant viruses that have the gpt removed can form plaques under selection conditions, thus providing a simple and efficient selection protocol . We have demonstrated the method by isolating a pseudo-wild type revertant virus and a simple deletion mutant virus from a recombinant vaccinia virus with gpt inserted into the vaccinia virus gene encoding the major 35,000-Da secretory protein. Proc Natl Acad Sci U S A, 1990 Oct, 87(19), 7668 - 72 Secondary structure of the ribosome binding site determines translational efficiency: a quantitative analysis; de Smit MH et al.; We have quantitatively analyzed the relationship between translational efficiency and the mRNA secondary structure in the initiation region . The stability of a defined hairpin structure containing a ribosome binding site was varied over 12 kcal/mol (1 cal = 4.184 J) by site-directed mutagenesis and the effects on protein yields were analyzed in vivo . The results reveal a strict correlation between translational efficiency and the stability of the helix . An increase in its delta G0 of -1.4 kcal/mol (i.e., less than the difference between an A.U and a G.C pair) corresponds to the reduction by a factor of 10 in initiation rate . Accordingly, a single nucleotide substitution led to the decrease by a factor of 500 in expression because it turned a mismatch in the helix into a match . We find no evidence that exposure of only the Shine-Dalgarno region or the start codon preferentially favors recognition . Translational efficiency is strictly correlated with the fraction of mRNA molecules in which the ribosome binding site is unfolded, indicating that initiation is completely dependent on spontaneous unfolding of the entire initiation region . Ribosomes appear not to recognize nucleotides outside the Shine-Dalgarno region and the initiation codon. Proc Natl Acad Sci U S A, 1990 Oct, 87(19), 7663 - 7 DNA polymerase II is encoded by the DNA damage-inducible dinA gene of Escherichia coli; Bonner CA et al.; The structural gene for DNA polymerase II was cloned by using a synthetic inosine-containing oligonucleotide probe corresponding to 11 amino acids, which were determined by sequencing the amino terminus of the purified protein . The labeled oligonucleotide hybridized specifically to the lambda clone 7H9 from the Kohara collection as well as to plasmid pGW511 containing the SOS-regulated dinA gene . Approximately 1400 base pairs of dinA sequence were determined . The predicted amino-terminal sequence of dinA demonstrated that this gene encoded DNA polymerase II . Sequence analysis of the upstream region localized a LexA binding site overlapping the -35 region of the dinA promoter, and this promoter element was found to be only two nucleotides downstream from the 3' end of the araD gene . These results demonstrate that the gene order is thr-dinA (pol II)-ara-leu on the Escherichia coli chromosome and that the DNA polymerase II structural gene is transcribed in the same direction as the araBAD operon . Based on the analysis of the predicted protein, we have identified a sequence motif Asp-Xaa-Xaa-Ser-Leu-Tyr-Pro-Ser in DNA polymerase II that is highly conserved among a diverse group of DNA polymerases, which include those from humans, yeast, Herpes and vaccinia viruses, and phages T4 and PRD1 . The demonstration that DNA polymerase II is a component of the SOS response in E . coli suggests that it plays an important role in DNA repair and/or mutagenesis. Proc Natl Acad Sci U S A, 1990 Oct, 87(19), 7593 - 7 Purification and characterization of recombinant Rev protein of human immunodeficiency virus type 1; Nalin CM et al.; Recombinant Rev protein of human immunodeficiency virus type 1 has been expressed in Escherichia coli and purified by ion-exchange and gel-filtration chromatography . Specific binding of the purified protein to the Rev-responsive element of the viral RNA is demonstrated . Physical characterization of the purified protein by circular dichroism and intrinsic fluorescence spectroscopy indicate that the protein preparation is suitable for structural analysis . Circular dichroism measurements show that the protein is approximately 40-45% alpha-helix . Tryptophan fluorescence measurements suggest that the single tryptophan residue is located near the surface of the protein . Gel-filtration chromatography of the protein indicates that it has an apparent molecular mass of 33,000 daltons . This suggests that the protein in solution forms a stable tetramer consisting of monomers having molecular mass of 13,000 daltons. Proc Natl Acad Sci U S A, 1990 Oct, 87(19), 7477 - 81 Cloning of the cDNA for human 12-lipoxygenase; Izumi T et al.; A full-length cDNA clone encoding 12-lipoxygenase (arachidonate:oxygen 12-oxidoreductase, EC 1.13.11.31) was isolated from a human platelet cDNA library by using a cDNA for human reticulocyte 15-lipoxygenase as probe for the initial screening . The cDNA had an open reading frame encoding 662 amino acid residues with a calculated molecular weight of 75,590 . Three independent clones revealed minor heterogeneities in their DNA sequences . Thus, in three positions of the deduced amino acid sequence, there is a choice between two different amino acids . The deduced sequence from the clone plT3 showed 65% identity with human reticulocyte 15-lipoxygenase and 42% identity with human leukocyte 5-lipoxygenase . The 12-lipoxygenase cDNA recognized a 3.0-kilobase mRNA species in platelets and human erythroleukemia cells (HEL cells) . Phorbol 12-tetradecanoyl 13-acetate induced megakaryocytic differentiation of HEL cells and 12-lipoxygenase activity and increased mRNA for 12-lipoxygenase . The identity of the cloned 12-lipoxygenase was assured by expression in a mammalian cell line (COS cells) . Human platelet 12-lipoxygenase has been difficult to purify to homogeneity . The cloning of this cDNA will increase the possibilities to elucidate the structure and function of this enzyme. Mutat Res, 1990 Oct, 232(2), 191 - 7 Studies on the effect of ascorbic acid and selenium on the genotoxicity of nitrofurans: nitrofurazone and furazolidone; Gajewska J et al.; The genotoxic properties of nitrofurazone and furazolidone were studied using the Ames test and SOS-chromotest . Both compounds were found to act as strong mutagens on the TA97 and TA102 strains of S . typhimurium and to induce the SOS-repair system in the PQ37 strain of E . coli . A good concordance was found between the mutagenic activity and the ability to induce the SOS system . Ascorbic acid and sodium selenite only very slightly lowered the genotoxic effect of the 2 nitrofurans studied both in the Ames test and in the SOS-chromotest. J Surg Res, 1990 Oct, 49(4), 328 - 32 Effect of prostaglandin E in multiple experimental models . IV . Effect on resistance to endotoxin and tumor necrosis factor shock; Waymack JP et al.; Administration of a long-acting prostaglandin E, 16,16-dimethyl-PGE (dPGE), to rats improves their survival of bacterial peritonitis . We examined the mechanism of this protective effect with reference to its interaction with the release of cachectin (TNF) . Sixty rats received saline, 20 micrograms/kg dPGE, or 80 micrograms/kg dPGE 12 hr prior to endotoxin and continuing for 48 hr . Survival rates for the saline, 20 micrograms/kg dPGE, and 80 micrograms/kg dPGE groups were 0, 40, and 85%, respectively . Forty rats received saline or 80 micrograms/kg dPGE, with the initial dose being 3 hr following endotoxin challenge and continuing for 48 hr . Survival rates for both groups were 0% . Sixty rats received saline or 80 micrograms/kg dPGE at 12 and 1 hr prior to endotoxin . Two hours after challenge, they were sacrificed and plasma TNF levels were assayed . The plasma TNF level in saline-treated rats was 22.72 +/- 0.83 ng/ml and in the dPGE-treated group, 16.03 +/- 1.13 ng/ml (P less than 0.001). J Cell Biol, 1990 Oct, 111(4), 1571 - 82 Cytoplasmic transport of ribosomal subunits microinjected into the Xenopus laevis oocyte nucleus: a generalized, facilitated process; Bataille N et al.; To study the biochemistry of ribonucleoprotein export from the nucleus, we characterized an in vivo assay in which the cytoplasmic appearance of radiolabeled ribosomal subunits was monitored after their microinjection into Xenopus oocyte nuclei . Denaturing gel electrophoresis and sucrose density gradient sedimentation demonstrated that injected subunits were transported intact . Consistent with the usual subcellular distribution of ribosomes, transport was unidirectional, as subunits injected into the cytoplasm did not enter the nucleus . Transport displayed properties characteristic of a facilitated, energy-dependent process; the rate of export was saturable and transport was completely inhibited either by lowering the temperature or by depleting nuclei of ATP; the effect of lowered temperature was completely reversible . Transport of injected subunits was likely a process associated with the nuclear pore complex, since export was also inhibited by prior or simultaneous injection of wheat germ agglutinin, a lectin known to inhibit active nuclear transport by binding to N-acetyl glucosamine-containing glycoproteins present in the NPC (Hart, G . W., R . S . Haltiwanger, G . D . Holt, and W . G . Kelly . 1989 . Annu . Rev . Biochem . 58:841-874) . Although GlcNAc modified proteins exist on both the nuclear and cytoplasmic sides of the nuclear pore complex, ribosomal subunit export was inhibited only when wheat germ agglutinin was injected into the nucleus . Finally, we found that ribosomal subunits from yeast and Escherichia coli were efficiently exported from Xenopus oocyte nuclei, suggesting that export of some RNP complexes may be directed by a collective biochemical property rather than by specific macromolecular primary sequences or structures. J Bacteriol, 1990 Oct, 172(10), 6090 - 7 Spiralins of Spiroplasma citri and Spiroplasma melliferum: amino acid sequences and putative organization in the cell membrane; Chevalier C et al.; Spiralin is the major membrane protein of the helical mollicute Spiroplasma citri . A similar protein occurs in the membrane of Spiroplasma melliferum, an organism related to S . citri . The gene encoding spiralin has been sequenced . A restriction fragment of the spiralin gene has been used as a probe to detect the gene encoding S . melliferum spiralin . A 4.6-kilobase-pair ClaI DNA fragment from S . melliferum strongly hybridized with the probe . This fragment was inserted in pBR322 and cloned in Escherichia coli . It was further subcloned in the replicative forms of M13mp18 and M13mp19, and its nucleotide sequence was determined (GenBank accession number M33991) . An open reading frame showing 88.6% base sequence homology with the S . citri spiralin gene could be identified and was assumed to be the gene encoding S . melliferum spiralin . The deduced amino acid sequence of the protein had 75% homology with the spiralin sequence . In particular, the two proteins possess a stretch of 20 amino acids which can form an alpha-helix, in which all polar amino acids occupy approximately one-third of the axial projection down the helix . On the basis of these data and published data, we propose a topological model for the structural organization of the spiralin in the cell membrane of spiroplasmas. J Bacteriol, 1990 Oct, 172(10), 6077 - 83 Evidence that there are only two tRNA(Phe) genes in Escherichia coli; Pittard J et al.; pheV, one of the genes that code for tRNA(Phe), was deleted from the chromosome of a strain of Escherichia coli K-12 . As a consequence of this mutation, expression of pheA, the gene for chorismate mutase P-prephenate dehydratase, the first enzyme in the terminal pathway of phenylalanine biosynthesis, was derepressed . Similar derepression of pheA has been reported in pheR mutants of E . coli K-12 (J . Gowrishankar and J . Pittard, J . Bacteriol . 150:1130-1137, 1982) . Attempts to introduce a pheR mutation into the delta pheV strain failed under circumstances suggesting that this combination of mutations is lethal . Southern blot analysis of pheV+ and delta pheV strains indicated that there are only two tRNA(Phe) genes in E . coli . It is recommended that the names pheU and pheV be retained for these genes. J Bacteriol, 1990 Oct, 172(10), 6026 - 34 The ClpP component of Clp protease is the sigma 32-dependent heat shock protein F21.5; Kroh HE et al.; The genes that encode the subunits of the Clp protease of Escherichia coli, clpA and clpP, appear to be regulated differently from each other . The clpA gene does not seem to be under heat shock control (Y . S . Katayama, S . Gottesman, J . Pumphrey, S . Rudikoff, W . P . Clark, and M . R . Maurizi, J . Biol . Chem . 263:15226-15236, 1988) . In contrast, the level of ClpP protein was increased in rpoH+ cells but not in null rpoH cells after an upshift in temperature from 17 to 43 degrees C . The level of ClpP protein in a null dnaK strain was also elevated relative to the level of ClpP protein in an otherwise isogenic dnaK+ strain . In two-dimensional gels, the ClpP protein was located in the position of the previously unidentified heat shock protein F21.5 . No protein spot corresponding to F21.5 was present in two-dimensional gels of a null clpP strain . The clpP gene, therefore, appears to be a heat shock gene, expressed in a sigma 32-dependent manner and negatively regulated by DnaK; the product of clpP is the previously unidentified heat shock protein F21.5. J Bacteriol, 1990 Oct, 172(10), 5852 - 5 Minicell-forming mutants of Escherichia coli: suppression of both DicB- and MinD-dependent division inhibition by inactivation of the minC gene product; Labie C et al.; We have determined the nucleotide sequence of the minB operon of 10 min mutants of Escherichia coli, characterized by impaired inhibition of polar divisions . These mutants were either sensitive or resistant to the division inhibitor DicB . All the mutations were found to lie in minC or minD, confirming the requirement of both gene products in the process of inhibition of polar sites . Mutations conferring resistance to inhibitor DicB were found exclusively in minC . In agreement with de Boer et al . (P . A . J . de Boer, R . E . Crossley, and L . I . Rothfield, Proc . Natl . Acad . Sci . USA 87:1129-1133, 1990), these results provide evidence that, in addition to promoting division inhibition with MinD, protein MinC acts in concert with DicB to inhibit division by a second, MinD-independent process. J Bacteriol, 1990 Oct, 172(10), 5767 - 73 Cloning, organization, and expression of the bioluminescence genes of Xenorhabdus luminescens; Frackman S et al.; The lux genes of Xenorhabdus luminescens, a symbiont of the nematode Heterorhabditis bacteriophora, were cloned and expressed in Escherichia coli . The expression of these genes in E . coli was qualitatively similar to their expression in X . luminescens . The organization of the genes is similar to that found in the marine luminous bacteria . Hybridization studies with the DNA that codes for the two subunits of luciferase revealed considerable homology among all of the strains of X . luminescens and with the DNA of other species of luminous bacteria, but none with the nonluminous Xenorhabdus species . Gross DNA alterations such as insertions, deletions, or inversions do not appear to be involved in the generation of dim variants known as secondary forms. J Bacteriol, 1990 Oct, 172(10), 5758 - 66 Autoregulation of Escherichia coli purR requires two control sites downstream of the promoter; Rolfes RJ et al.; The expression of Escherichia coli purR, which encodes the pur regulon repressor protein, is autoregulated . Autoregulation at the level of transcription requires two operator sites, designated purRo1 and purRo2 (O1 and O2) . Operator O1 is in the region of DNA between the transcription start site and the site for translation initiation, and O2 is in the protein-coding region . The repressor protein binds noncooperatively to O1 with a sixfold-higher affinity than to O2, and saturation of O1 by the repressor precedes saturation of O2 . Both O1 and O2 function in the two- to threefold autoregulation in vivo, as determined by measurement of beta-galactosidase and mRNA from purR-lacZ translational fusions . Of all the genes thus far known to be regulated by the Pur repressor, only purR employs a two-operator mechanism. J Bacteriol, 1990 Oct, 172(10), 5732 - 41 Cloning and characterization of RNA polymerase core subunits of Chlamydia trachomatis by using the polymerase chain reaction; Engel JN et al.; Taking advantage of sequence conservation of portions of the alpha, beta, and beta' subunits of RNA polymerase of bacteria and plant chloroplasts, we have designed degenerate oligonucleotides corresponding to these domains and used these synthetic DNA sequences as primers in a polymerase chain reaction to amplify DNA sequences from the chlamydial genome . The polymerase chain reaction products were used as a probe to recover the genomic fragments encoding the beta subunit and the 5' portion of the beta' subunit from a library of cloned murine Chlamydia trachomatis DNA . Similar attempts to recover the alpha subunit were unsuccessful . Sequence analysis demonstrated that the beta subunit of RNA polymerase was located between genes encoding the L7/L12 ribosomal protein and the beta' subunit of RNA polymerase; this organization is reminiscent of the rpoBC operon of Escherichia coli . The C . trachomatis beta subunit overproduced in E . coli was used as an antigen in rabbits to make a polyclonal antibody to this subunit . Although this polyclonal antibody specifically immunoprecipitated the beta subunit from Chlamydia-infected cells, it did not immunoprecipitate core or holoenzyme . Immunoblots with this antibody demonstrated that the beta subunit appeared early in infection. J Bacteriol, 1990 Oct, 172(10), 5610 - 6 Interaction between the min locus and ftsZ; Bi E et al.; In Escherichia coli, distinct but similar minicell phenotypes resulting from mutation at the minB locus and increased expression of ftsZ suggested a possible interaction between these genes . A four- to fivefold increase in FtsZ resulting from increased gene dosage was found to suppress the lethality of minCD expressed from the lac promoter . Since increased MinCD did not affect the level of FtsZ, this suggested that MinCD may antagonize FtsZ to inhibit its cell division activity . This possibility was supported by the finding that alleles of ftsZ isolated as resistant to the cell division inhibitor SulA were also resistant to MinCD . Among the ftsZ(Rsa) alleles, two appeared to be completely resistant to MinCD as demonstrated by the lack of an effect of MinCD on cell length and a minicell phenotype observed in the absence of a significant increase in FtsZ . It was shown that SulA inhibits cell division independently of MinCD. J Bacteriol, 1990 Oct, 172(10), 5586 - 92 Characterization of a promoter and a transcription terminator of Spiroplasma melliferum virus SpV4; Stamburski C et al.; Spiroplasma virus 4 (SpV4) is an isometric virus with single-stranded, circular DNA infecting the helical mollicute Spiroplasma melliferum, a honeybee pathogen . Previous studies in our laboratory led to the determination of the base sequence of the SpV4 DNA . Nine open reading frames and three promoterlike sequences (P1, P2, and P3) were identified . An inverted repeat leading to the formation of a hairpin structure on the transcription product was also found and predicted to be a transcription terminator (T) . We have now studied the in vivo transcription of the SpV4 genome by Northern (RNA) blot analysis of the total RNAs extracted from SpV4-infected spiroplasma cells . Transcripts of 7.8, 4.4, 3.4, and 2.7 kilobases (kb) were detected . The 3.4-kb RNA was the major transcript . The 5' and 3' ends of this transcript were determined by S1 mapping and primer extension . Characterization of the 3' end by S1 mapping showed that the 3.4-kb transcript terminates within the stretch of uridine residues following the hairpin structure of terminator T . Characterization of the 5' end by S1 mapping indicated that transcription proceeds from a newly recognized promoter, P0, located 36 nucleotides upstream of P1 . Primer extension resulted in two cDNA signals . The short cDNA was probably a primer extension artifact due to the presence of a hairpin structure on the transcript . When reverse transcriptase stopped at this hairpin or read through, the short or the long cDNA, respectively, was obtained . The size of the long cDNA identified P0 as the transcription promoter . Promoter P0 was also shown to be functional in Escherichia coli . Indeed, when inserted upstream of the chloramphenicol acetyltransferase gene of a promoter selection vector, it promoted transcription of this gene . As in the case of S . melliferum, two cDNAs were obtained by primer extension, the longer cDNA identifying P0 as the promoter. J Bacteriol, 1990 Oct, 172(10), 5555 - 62 Trigger factor depletion or overproduction causes defective cell division but does not block protein export; Guthrie B et al.; Trigger factor is an abundant cytosolic protein of Escherichia coli which can stabilize proOmpA for in vitro translocation across inner membrane vesicles . The gene encoding E . coli trigger factor was isolated and sequenced, allowing construction of strains in which the expression of trigger factor is readily regulated . We found no defect in the in vivo rate of synthesis or secretion of proOmpA in trigger factor-depleted cells . The primary physiological defect in trigger factor-depleted or -overproducing cells is an enrichment of filamented cells . Filamentation of the trigger factor-overproducing strain is suppressed by a multicopy plasmid expressing the essential division gene ftsZ, suggesting that trigger factor has an important role in cell division. J Bacteriol, 1990 Oct, 172(10), 5523 - 30 Regulatory interactions between phospholipid synthesis and DNA replication in Caulobacter crescentus; Loewy B et al.; Several Caulobacter crescentus mutants with lesions in phospholipid biosynthesis have DNA replication phenotypes . A C . crescentus mutant deficient in glycerol 3-phosphate dehydrogenase activity (gpsA) blocks phospholipid synthesis, ceases DNA replication, and loses viability in the absence of a glycerol phosphate supplement . To investigate the interaction between membrane synthesis and DNA replication during a single cell cycle, we moved the gpsA mutation into a synchronizable, but otherwise wild-type, strain . The first effect of withholding supplement was the cessation of synthesis of phosphatidylglycerol, a major component of the C . crescentus membrane . In the absence of glycerol 3-phosphate, DNA replication was initiated in the stalked cell at the correct time in the cell cycle and at the correct site on the chromosome . However, after replication proceeded bidirectionally for a short time, DNA synthesis dropped to a low level . The cell cycle blocked at a distinct middivision stalked cell, and this was followed by cell death . The "glycerol-less" death of the gpsA mutant could be prevented if the cells were treated with novobiocin to prevent the initiation of DNA replication . Our observations suggest that the processivity of C . crescentus replication requires concomitant phospholipid synthesis and that cell death results from incomplete replication of the chromosome. EMBO J, 1990 Oct, 9(10), 3369 - 78 Mutants of the EcoRI endonuclease with promiscuous substrate specificity implicate residues involved in substrate recognition; Heitman J et al.; The EcoRI restriction endonuclease cleaves DNA molecules at the sequence GAATTC . We devised a genetic screen to isolate EcoRI mutants with altered or broadened substrate specificity . In vitro, the purified mutant enzymes cleave both the wild-type substrate and sites which differ from this by one nucleotide (EcoRI star sites) . These mutations identify four residues involved in substrate recognition and catalysis that are different from the amino acids proposed to recognize the substrate based on the EcoRI-DNA co-crystal structure . In fact, these mutations suppress EcoRI mutants altered at some of the proposed substrate binding residues (R145, R200) . We argue that these mutations permit cleavage of additional DNA sequences either by perturbing or removing direct DNA-protein interactions or by facilitating conformational changes that allosterically couple substrate binding to DNA scission. J Infect Dis, 1990 Oct, 162(4), 974 - 7 Age-specific prevalence of antibody to enterotoxigenic Escherichia coli in Ecuadorian and German children; Brussow H et al.; Serum samples from 1397 Ecuadorian children 0-5 years of age were tested by ELISA for antibodies to enterotoxigenic Escherichia coli (ETEC) . A gradual prevalence and titer increase was seen for IgG antibodies to heat-labile enterotoxin in children 6-18 months old; 90% prevalences were reached in the second year of life . At this age less than 10% of West German children showed IgG antibodies to heat-labile enterotoxin . In Ecuador age-related ETEC-specific antibody titer increases correlated with age-related prevalence of diarrheal disease . On the other hand, pooled lipopolysaccharide from different ETEC-associated O serogroups could not be used as a seroepidemiologic marker of ETEC infections. Infect Immun, 1990 Oct, 58(10), 3442 - 4 Association of rns homologs with colonization factor antigens in clinical Escherichia coli isolates; Caron J et al.; rns is a trans-acting positive regulatory factor required for expression of the colonization factor antigen II (CFA/II) antigens CS1 and CS2 (J . Caron, L . M . Coffield, and J . R . Scott, Proc . Natl . Acad . Sci . USA 86:963-967, 1989) . All 35 CFA/II-positive strains hybridized with a rns gene probe, as did all 10 CFA/I strains and all 4 CS4 strains . Hybridization with rns was detected in 25% of non-enterotoxigenic Escherichia coli strains and was not detected in enteric pathogens with low G + C content. Infect Immun, 1990 Oct, 58(10), 3380 - 7 Legionella pneumophila htpAB heat shock operon: nucleotide sequence and expression of the 60-kilodalton antigen in L . pneumophila-infected HeLa cells; Hoffman PS et al.; A 60-kilodalton (kDa) immunodominant antigen of Legionella pneumophila is a heat shock protein (HSP) of the GroEL class of HSPs . The gene (htpB) coding the 60-kDa protein was localized to a 3.2-kilobase DNA fragment of L . pneumophila cloned into pUC19 (pSH16) (P . S . Hoffman, C . A . Butler, and F . D . Quinn, Infect . Immun . 57:1731-1739, 1989) . The nucleotide sequence of the DNA fragment cloned into M13 confirmed two open reading frames, htpA and htpB, that code for proteins of 96 and 548 amino acids, respectively . A consensus heat shock promoter sequence upstream of the start of htpA was identified, and no obvious promoter sequences were detected upstream of htpB . Amino acid sequence comparison studies revealed that the L . pneumophila HtpB protein exhibited 76% homology with the 65-kDa protein of Mycobacterium tuberculosis and 85% homology with both GroEL of Escherichia coli and HtpB of Coxiella burnetii . A comparison of the amino acid sequences among these proteins revealed several regions of nearly absolute sequence conservation, with the variable regions occurring in common areas . The purified L . pneumophila 60-kDa protein was antigenic for human T lymphocytes . Indirect fluorescent antibody studies indicated that the 60-kDa protein may be located in the periplasm or expressed on the surface by intracellular bacteria, suggesting that a stress-related mechanism may be involved in the expression of this immunodominant antigen. J Virol, 1990 Oct, 64(10), 4709 - 17 Analysis of mutations in the integration function of Moloney murine leukemia virus: effects on DNA binding and cutting; Roth MJ et al.; The 3' terminus of the pol gene of Moloney murine leukemia virus encodes the integration (IN) protein, required for the establishment of the integrated provirus . A series of six linker insertion mutations and two single-base substitutions were generated within the region encoding the IN protein . Mutations were initially generated within an Escherichia coli plasmid expressing the IN protein, and the resulting variants were assayed for DNA-binding activity . Mutations which altered conserved cysteine residues within a potential DNA finger-binding motif resulted in lower or variable DNA binding, which appeared to be the result of variable protein folding . Upon renaturation, these proteins were able to nonspecifically bind DNA in a manner similar to that of the other mutant IN proteins and the parent . When reconstructed back into full-length virus, seven of the eight mutations were lethal . All mutants produced a stable IN protein in virions and mediated normal conversion of the retroviral RNA to its three DNA forms . Fine-structure analysis of the linear double-stranded viral DNA indicated that all seven lethal alterations within the IN protein blocked the formation of the 3' recessed termini that normally precedes integration. Mol Endocrinol, 1990 Oct, 4(10), 1506 - 14 The production, purification, and bioactivity of recombinant bovine trophoblast protein-1 (bovine trophoblast interferon); Klemann SW et al.; Bovine trophoblast protein-1 (bTP-1) is a 172-amino acid interferon- alpha that has a role in maternal recognition of pregnancy in cattle . Here we describe production of bTP-1 by recombinant procedures in Escherichia coli . A bTP-1 gene was constructed which lacked the codons representing the signal sequence and provided a Met initiation codon ahead of the TGT codon encoding Cys1 of the mature protein . This construct was placed under the control of the Trp promoter within the expression vector pTrp2 . Expression occurred optimally in E . coli D112 in the absence of tryptophan and in the presence of 0.5% acid-hydrolyzed casein (casamino acids) when 0.5 mM indole acetic acid was included in the medium . The bTP-1 was deposited in inclusion bodies and accounted for as much as 27% of the total cellular protein . The inclusion bodies were isolated by differential centrifugation and washed . The bTP-1 was solubilized by use of guanidinium-HCI and 2-mercaptoethanol and allowed to renature in air . Final purification was achieved by anion exchange chromatography on DEAE-cellulose . The yield of purified product, which had an antiviral activity greater than 10(8) international reference units/mg, was approximately 20 mg/liter . The recombinant bTP-1 was relatively stable to freeze-thawing and frozen storage, and could induce the production of an acidic protein of 70,000 mol wt in cultured explants of endometrium prepared from ewes on day 13 of the estrous cycle . The latter protein is a characteristic product of interferon-alpha action on uterine tissue.(ABSTRACT TRUNCATED AT 250 WORDS) Biosci Rep, 1990 Oct, 10(5), 461 - 7 Expression of a human proenkephalin A cDNA in Escherichia coli; Monstein HJ; A 1000 base pair cDNA coding for the entire human proenkephalin A(proA) polypeptide was subcloned into the multifunctional pMPV 2911/M E . coli vector . The recombinant plasmid was found to express an approximately 30 kDa prohormone, which was recognized by a Met-Arg6-Phe2 antibody, directed against the C-terminal part of the enkephalin A prohormone . The expression of human proenkephalin A cDNA should thus permit the rapid purification of unfused recombinant enkephalin A prohormone, which itself may provide a model substrate to identify endoproteolytic processing activities. Mol Gen Genet, 1990 Oct, 224(1), 81 - 90 The osmZ (bglY) gene encodes the DNA-binding protein H-NS (H1a), a component of the Escherichia coli K12 nucleoid; May G et al.; A class of trans-acting mutations, which alter the osmoregulated expression of the Escherichia coli proU operon, maps at 27 min on the chromosome in a locus we have called osmZ . Mutations in osmZ are allelic to bglY, pilG and virR, affect gene expression, increase the frequency of the site-specific DNA inversion mediating fimbrial phase variation, stimulate the formation of deletions, and influence in vivo supercoiling of reporter plasmids . We have cloned the osmZ+ gene, mapped it at 1307 kb of the E . coli restriction map, identified its gene product as a 16 kDa protein, and determined the nucleotide sequence of the osmZ+ gene . The deduced amino acid sequence for OsmZ predicts a protein of 137 amino acid residues with a calculated molecular weight of 15,530 . The primary sequence of OsmZ is identical to that of H-NS (H1a), a DNA-binding protein that affects DNA topology and is known to be associated with the bacterial nucleoid . Thus, osmZ is the structural gene for the H-NS (H1a) protein . The nucleotide sequence of osmZ is almost identical to that of hns; however, hns was incorrectly located at 6.1 min on the E . coli linkage map . Increased osmZ gene dosage leads to cell filament formation, altered gene expression, and reduced frequency of fimbrial phase variation . Our results suggest that the nucleoid-associated DNA-binding protein H-NS (H1a) plays a critical role in gene expression and in determining the structure of the genetic material. Mol Gen Genet, 1990 Oct, 224(1), 1 - 9 Linear multimer formation of plasmid DNA in Escherichia coli hopE (recD) mutants; Niki H et al.; The hopE mutants of Escherichia coli, which cannot stably maintain a mini-F plasmid during cell division, have mutations in the recD gene coding for subunit D of the RecBCD enzyme (exonuclease V) . A large amount of linear multimer DNA of mini-F and pBR322 plasmid accumulates in these hopE mutants . The linear multimers of plasmid DNA in the hopE (recD) mutants accumulate in sbc+ genetic backgrounds and this depends on the recA+ gene function . Linear plasmid multimers also accumulated in a recBC xthA triple mutant, but not an isogenic xth A mutant or an isogenic recBC mutant . The recBC xth A mutant is defective in the conjugative type of recombination . Linear plasmid multimers were not detected in the recBC strain . We propose models to account for linear multimer formation of plasmids in various mutants. Circ Shock, 1990 Oct, 32(2), 101 - 12 Reduced microvascular adrenergic receptor activity due to opioids in endotoxin shock; Baker CH et al.; Arteriolar sensitivity to norepinephrine is decreased in endotoxin shock, and sympathetic activity appears altered . We have tested involvement of arteriolar opioid receptors in the response to endotoxin . Cremaster muscle arteriolar reactivity of anesthetized rats was studied using videomicroscopy . Escherichia coli endotoxin (6 mg/kg, i.v., LD100) was infused over a 1-hr period . Mean arterial pressure (Pm), frequency/diameter curves of A1, A2, and A3 arterioles to lumbar sympathetic nerve stimulation (1-16 Hz), and plasma velocity were obtained in group I at control, 30 min, and 90 min postendotoxin and in group II at control and during i.v . infusion of the opiate antagonist naltrexone (0.5 mg/kg/min) at 30 and 90 min postendotoxin . Frequency-diameter curves (percentage of control diameter) were significantly (P less than 0.05) shifted to the right postendotoxin in group I indicating reduced response to lumbar sympathetic stimulation or norepinephrine from the nerve terminals . In group II rats receiving naltrexone after endotoxin, the frequency-diameter curves were significantly (P less than 0.05) shifted to the left indicating enhanced vasoconstriction to lumbar sympathetic stimulation in comparison to control and to group I postendotoxin curves . Postendotoxin, Pm and plasma velocities decreased progressively in group I but were not changed from control in group II . Since opiate receptor blockade during endotoxin shock enhances adrenergic responses of arterioles, opiate receptor stimulation appears to suppress adrenergic receptors. Vaccine, 1990 Oct, 8(5), 438 - 40 Protection of guinea-pigs against foot-and-mouth disease virus by immunization with a PhoE-FMDV hybrid protein; Agterberg M et al.; A hybrid protein was constructed containing two antigenic determinants of the structural protein VP1 of foot-and-mouth disease virus, inserted in a cell surface-exposed region of Escherichia coli outer membrane protein PhoE . Immunization of guinea-pigs with partially purified protein resulted in high levels of neutralizing antibodies and complete protection against challenge with the virus. J Gen Virol, 1990 Oct, 71 ( Pt 10), 2291 - 9 The complete nucleotide sequence of enterovirus type 70: relationships with other members of the picornaviridae; Ryan MD et al.; Enterovirus type 70 (EV70) is the causative agent of acute haemorrhagic conjunctivitis and may also give rise to a rare neurological complication closely resembling poliomyelitis . The complete nucleotide sequence of the genome of EV70 has been determined from cDNA cloned in Escherichia coli . The genome consists of a 5' non-coding region of 726 nucleotides (nt), a long open reading frame of 6582 nt and a 3' non-coding region of 82 nt prior to the poly(A) tract . Comparison of the nucleotide sequence and the predicted amino acid sequence of the polyprotein with those published for other enteroviruses reveals sufficiently high similarity to predict antigenic regions and polyprotein cleavage sites . The P1 region of EV70 is as similar to those of the entero- as to those of the rhinoviruses, whereas the P2 and P3 regions are more closely related to the coxsackie B and swine vesicular disease viruses than other entero- or rhinoviruses. Biochem J, 1990 Oct 1, 271(1), 59 - 66 Role of cysteine residues in ribonuclease H from Escherichia coli . Site-directed mutagenesis and chemical modification; Kanaya S et al.; The role of the three cysteine residues at positions 13, 63 and 133 in Escherichia coli RNAase H, an enzyme that is sensitive to N-ethylmaleimide {Berkower, Leis & Hurwitz (1973) J . Biol . Chem . 248, 5914-5921}, was examined by using both site-directed mutagenesis and chemical modification . Novel aspects that were found are as follows . First, none of the cysteine residues is required for activity . Secondly, chemical modification of either Cys-13 or Cys-133 with thiol-blocking reagents inactivates the enzyme, but that of Cys-63 does not . Thus the sensitivity of E . coli RNAase H to N-ethylmaleimide arises not from blocking of the thiol group but from steric hindrance by the modifying group incorporated at either Cys-13 or Cys-133. Proc Natl Acad Sci U S A, 1990 Oct, 87(19), 7574 - 8 Genetic analysis of membrane protein topology by a sandwich gene fusion approach; Ehrmann M et al.; We describe a cloning vector that allows the construction of phoA sandwich fusions in which mature alkaline phosphatase is inserted into target proteins . In contrast to previous fusions obtained using the TnphoA transposon, the entire amino acid sequence of the target protein is present in the fusion product . We have constructed a series of sandwich fusions of alkaline phosphatase to the multispanning cytoplasmic membrane protein MalF . Despite the fact that the alkaline phosphatase was tethered to MalF at both its N and its C terminus, the enzyme exhibited high activity when it was fused to a periplasmic domain of the membrane protein . Cells harboring an alkaline phosphatase sandwich fusion to the end of the first membrane-spanning segment of MalF exhibited both MalF and alkaline phosphatase activity . When alkaline phosphatase was inserted into a cytoplasmic domain of MalF, its specific activity was very low . Our results suggest that the alkaline phosphatase activity of phoA sandwich fusions provides a more sensitive monitor than previous methods of the cellular localization of the domain of the target protein to which the enzyme is fused . Thus, the sandwich fusion approach can give a more accurate picture of membrane protein topology. J Bacteriol, 1990 Oct, 172(10), 6129 - 34 Mutational analysis of the Escherichia coli glpFK region with Tn5 mutagenesis and the polymerase chain reaction; Lupski JR et al.; Transposon Tn5 mutagenesis of the Escherichia coli chromosome was used to isolate 21 independent insertion mutations conferring an altered colony color phenotype on MacConkey-glycerol plates . The polymerase chain reaction was used to map 16 of these Tn5 insertions within the glpFK region at 88 min . The most polar Tn5 insertion was shown by nucleotide sequencing to be in the proposed glpF open reading frame . The data suggest that the glpF and glpK genes are in an operon with a bent DNA segment (BENT-6) involved in transcriptional regulation of this operon. J Bacteriol, 1990 Oct, 172(10), 6127 - 8 The gdhA gene is located at 38.6 minutes on the Escherichia coli map; Kim SY et al.; We report here that the gdhA gene of Escherichia coli, which encodes the NADP-specific glutamate dehydrogenase, is located at 38.6 min on the map . We have confirmed this location by showing linkage with three Tn10 insertions that are linked to the aroD, pheS, and ansA loci, by complementation by a restriction-mapped lambda clone, and by showing correspondence between the restriction maps of the chromosome and the cloned and sequenced gdhA gene. J Bacteriol, 1990 Oct, 172(10), 6122 - 6 Functional expression of Desulfovibrio vulgaris Hildenborough cytochrome c3 in Desulfovibrio desulfuricans G200 after conjugational gene transfer from Escherichia coli; Voordouw G et al.; Plasmid pJRDC800-1, containing the cyc gene encoding cytochrome c3 from Desulfovibrio vulgaris subsp . vulgaris Hildenborough, was transferred by conjugation from Escherichia coli DH5 alpha to Desulfovibrio desulfuricans G200 . The G200 strain produced an acidic cytochrome c3 (pI = 5.8), which could be readily separated from the Hildenborough cytochrome c3 (pI = 10.5) . The latter was indistinguishable from cytochrome c3 produced by D . vulgaris subsp . vulgaris Hildenborough with respect to a number of chemical and physical criteria. J Bacteriol, 1990 Oct, 172(10), 6112 - 21 Identification and expression of the Escherichia coli fdhD and fdhE genes, which are involved in the formation of respiratory formate dehydrogenase; Schlindwein C et al.; The two closely linked fdhD and fdhE genes of Escherichia coli are required for the formation of active membrane-bound phenazine methosulfate-linked formate dehydrogenase (FDH-PMS) . Both genes were isolated from a cosmid library . Restriction endonuclease analysis associated with Mu dII1734 insertion mutagenesis indicated that the two genes were separated by at least 4 kilobases and transcribed in opposite orientations . Initial experiments indicate that the region between the two genes seems not to be essential to FDH-PMS activity . fdhD and fdhE were expressed either in maxicells or from the T7 promoter-polymerase system . They were shown to encode proteins with approximate Mr 30,500 and 32,000, respectively . Both proteins appeared in the soluble fraction and were not recognized by an FDH-PMS-specific antiserum . Therefore, neither fdhD nor fdhE plays a structural role in the formation of FDH-PMS . Expression of a phi(fdhD-lacZ) operon fusion was decreased about threefold by aerobiosis but was indifferent to other effectors tested . It was unaffected by pfl, chlA, selA, and fnr mutations . Expression of a phi(fdhE-lacZ) operon fusion was slightly induced by nitrate . This induction, requiring the presence of functional chl and fnr alleles, was mediated via nitrate metabolism . Transcription of phi(fdhE-lacZ) fusion was fully dependent on wild-type sel alleles . This might suggest the participation of fdhE in the synthesis of the selenopolypeptide of FDH-PMS. J Bacteriol, 1990 Oct, 172(10), 6020 - 5 Requirement for terminal cytochromes in generation of the aerobic signal for the arc regulatory system in Escherichia coli: study utilizing deletions and lac fusions of cyo and cyd; Iuchi S et al.; Escherichia coli has two terminal oxidases for its respiratory chain: cytochrome o (low O2 affinity) and cytochrome d (high O2 affinity) . Expression of the cyo operon, encoding cytochrome o, is decreased by anaerobic growth, whereas expression of the cyd operon, encoding cytochrome d, is increased by anaerobic growth . We show by the use of lac gene fusion that the expressions of cyo and cyd are under the control of the two-component arc system . In a cyo+ cyd+ background, expression of phi(cyo-lac) is higher when the organism is grown aerobically than when it is grown anaerobically . A mutation in either the sensor gene arcB or the pleiotropic regulator gene arcA almost abolishes the anaerobic repression . In the same background, expression of phi(cyd-lac) is higher under anaerobic growth conditions than under aerobic growth conditions . A mutation in arcA or arcB lowers both the aerobic and anaerobic expressions, suggesting that ArcA plays an activating role instead of the typical repressing role . Under aerobic growth conditions, double deletions of cyo and cyd lower phi(cyo-lac) expression but enhance phi(cyd-lac) expression . The double deletions also prevent elevated aerobic induction of the lct operon (encoding L-lactate dehydrogenase), another target operon of the arc system . In contrast, these deletions do not circumvent aerobic repression of the nar operon (encoding the anaerobic respiratory enzyme nitrate reductase) under the control of the pleiotropic fnr gene product . It thus appears that ArcB senses the presence of O2 by level of an electron transport component in reduced form or that of an nonautoxidizable compound linked to the process by a redox reaction, whereas Fnr senses O2 by a different mechanism. J Bacteriol, 1990 Oct, 172(10), 6010 - 9 Cloning and mutagenesis of genes encoding the cytochrome bd terminal oxidase complex in Azotobacter vinelandii: mutants deficient in the cytochrome d complex are unable to fix nitrogen in air; Kelly MJ et al.; The genome of Azotobacter vinelandii contains DNA sequences homologous to the structural genes for the Escherichia coli cytochrome bd terminal oxidase complex . Two recombinant clones bearing cydA- and cydB-like sequence were isolated from an A . vinelandii gene library and subcloned into the plasmid vector pACYC184 . Physical mapping demonstrated that the cydA- and cydB-like regions in A . vinelandii are contiguous . The cydAB and flanking DNA was mutagenized by the insertion of Tn5-B20 . Mutations in the cydB-hybridizing region resulted in the loss of spectral features associated with cytochromes b595 and d . A new locus, cydB, encoding cytochromes b595 and d in A . vinelandii is proposed . A second region adjacent to cydB was also involved in expression of the cytochrome bd complex in A . vinelandii, since mutations in this region resulted in an increase in the levels of both cytochrome b595 and cytochrome d . The regions involved in expression of the cytochrome bd complex and cydB are transcribed in the same direction . Mutants deficient in cytochromes b595 and d were unable to grow on N-deficient medium when incubated in air but could fix nitrogen when the environmental O2 concentration was reduced to 1.5% (vol/vol) . It is proposed that the branch of the respiratory chain terminated by the cytochrome bd complex supports the high respiration rates required for the respiratory protection of nitrogenase. J Bacteriol, 1990 Oct, 172(10), 5956 - 60 Mutations that affect Tn5 insertion into pBR322: importance of local DNA supercoiling; Lodge JK et al.; The major hot spot of transposon Tn5 insertion in plasmid pBR322 (hot spot I) is in the promoter for the tetracycline resistance gene (tet) . We made a series of pBR322 derivatives with mutations in and around this promoter and assayed their effects on insertion of Tn5 into hot spot I . Those mutations which reduced transcription from the tet promoter also reduced the frequency of insertion into hot spot I . Transcription and translation of tet are thought to cause the formation of paired domains of negative and positive supercoiling in pBR322 . An amber codon in tet, 345 base pairs from hot spot I, decreases the negative supercoiling of the DNA segment containing hot spot I because it terminates translation of tet prematurely . We report here that this amber mutation also reduces insertion into hot spot I . These results suggest that the ability of Tn5 to insert into its major hot spot in pBR322 depends directly on negative supercoiling of the target DNA. J Bacteriol, 1990 Oct, 172(10), 5938 - 48 Organization of dimethyl sulfoxide reductase in the plasma membrane of Escherichia coli; Sambasivarao D et al.; Dimethyl sulfoxide reductase is a trimeric, membrane-bound, iron-sulfur molybdoenzyme induced in Escherichia coli under anaerobic growth conditions . The enzyme catalyzes the reduction of dimethyl sulfoxide, trimethylamine N-oxide, and a variety of S- and N-oxide compounds . The topology of dimethyl sulfoxide reductase subunits was probed by a combination of techniques . Immunoblot analysis of the periplasmic proteins from the osmotic shock and chloroform wash fluids indicated that the subunits were not free in the periplasm . The reductase was susceptible to proteases in everted membrane vesicles, but the enzyme in outer membrane-permeabilized cells became protease sensitive only after detergent solubilization of the E . coli plasma membrane . Lactoperoxidase catalyzed the iodination of each of the three subunits in an everted membrane vesicle preparation . Antibodies to dimethyl sulfoxide reductase and fumarate reductase specifically agglutinated the everted membrane vesicles . No TnphoA fusions could be found in the dmsA or -B genes, indicating that these subunits were not translocated to the periplasm . Immunogold electron microscopy of everted membrane vesicles and thin sections by using antibodies to the DmsABC, DmsA, DmsB subunits resulted in specific labeling of the cytoplasmic surface of the inner membrane . These results show that the DmsA (catalytic subunit) and DmsB (electron transfer subunit) are membrane-extrinsic subunits facing the cytoplasmic side of the plasma membrane. J Bacteriol, 1990 Oct, 172(10), 5795 - 802 General organization of the conjugal transfer genes of the IncW plasmid R388 and interactions between R388 and IncN and IncP plasmids; Bolland S et al.; The complete conjugal transfer gene region of the IncW plasmid R388 has been cloned in multicopy vector plasmids and mapped to a contiguous 14.9-kilobase segment by insertion mutagenesis . The fertility of the cloned region could still be inhibited by a coresident IncP plasmid . The transfer region has been dissected into two regions, one involved in pilus synthesis and assembly (PILW), and the other involved in conjugal DNA metabolism (MOBW) . They have been separately cloned . PILW also contains the genes involved in entry exclusion . MOBW contains oriT and the gene products required for efficient mobilization by PILW . MOBW plasmids could also be mobilized efficiently by PILN, the specific pilus of the IncN plasmid pCU1, but not by PILP, the specific pilus of the IncP plasmid RP1. J Bacteriol, 1990 Oct, 172(10), 5706 - 13 The CytR repressor antagonizes cyclic AMP-cyclic AMP receptor protein activation of the deoCp2 promoter of Escherichia coli K-12; Sogaard-Andersen L et al.; We have investigated the regulation of the Escherichia coli deoCp2 promoter by the CytR repressor and the cyclic AMP (cAMP) receptor protein (CRP) complexed to cAMP . Promoter regions controlled by these two proteins characteristically contain tandem cAMP-CRP binding sites . Here we show that (i) CytR selectively regulated cAMP-CRP-dependent initiations, although transcription started from the same site in deoCp2 in the absence or presence of cAMP-CRP; (ii) deletion of the uppermost cAMP-CRP target (CRP-2) resulted in loss of CytR regulation, but had only a minor effect on positive control by the cAMP-CRP complex; (iii) introduction of point mutations in either CRP target resulted in loss of CytR regulation; and (iv) regulation by CytR of deletion mutants lacking CRP-2 could be specifically reestablished by increasing the intracellular concentration of CytR . These findings indicate that both CRP targets are required for efficient CytR repression of deoCp2 . Models for the action of CytR are discussed in light of these findings. J Bacteriol, 1990 Oct, 172(10), 5686 - 9 Pyrophosphatase is essential for growth of Escherichia coli; Chen J et al.; The ppa gene for inorganic pyrophosphatase is essential for the growth of Escherichia coli . A recombinant with a chromosomal ppa::Kanr lesion and a temperature-sensitive replicon with a ppa+ gene showed a temperature-sensitive growth phenotype, and a mutant with the sole ppa+ gene under control of the lac promoter showed inducer-dependent growth . When the lacp-ppa mutant was subcultured without inducer, the pyrophosphatase level decreased, the PPi level increased, and growth stopped . Cellular PPi reached 16 mM about 6 h after growth arrest without loss of cell viability. EMBO J, 1990 Oct, 9(10), 3241 - 52 Structural diversity and evolution of human receptor-like protein tyrosine phosphatases; Krueger NX et al.; Protein tyrosine phosphatases (PTPases), together with protein tyrosine kinases, regulate the tyrosine phosphorylation that controls cell activities and proliferation . Previously, it has been recognized that both cytosolic PTPases and membrane associated, receptor-like PTPases exist . In order to examine the structural diversity of receptor-like PTPases, we isolated human cDNA clones that cross-hybridized to a Drosophila PTPase cDNA clone, DPTP12, under non-stringent hybridization conditions . The cDNA clones thus isolated included LCA and six other novel receptor-like PTPases, named HPTP alpha, beta, gamma, delta, epsilon, and zeta . The cytoplasmic regions of HPTP alpha and epsilon are highly homologous, and are composed of two tandemly duplicated PTPase-like domains . The extracellular regions of HPTP alpha and epsilon are, respectively, 123 amino acids and 27 amino acids, and do not have obvious similarity to any known protein . The cytoplasmic region of HPTP beta contains only one PTPase domain . The extracellular region of HPTP beta, which is 1599 amino acids, is composed of 16 fibronectin type-III repeats . HPTP delta is very similar to leukocyte common antigen related molecule (LAR), in both the extracellular and cytoplasmic regions . Partial sequences of HPTP gamma and zeta indicate that they are highly homologous and contain two PTPase-like domains . The PTPase-like domains of HPTP alpha, beta and delta expressed in Escherichia coli had tyrosine phosphatase activities. J Virol, 1990 Oct, 64(10), 5008 - 18 Structure-function studies of the herpes simplex virus type 1 DNA polymerase; Haffey ML et al.; The analysis of the deduced amino acid sequence of the herpes simplex virus type 1 (HSV-1) DNA polymerase reported here suggests that the polymerase structure consists of domains carrying separate biological functions . The HSV-1 enzyme is known to possess 5'-3'-exonuclease (RNase H), 3'-5'-exonuclease, and DNA polymerase catalytic activities . Sequence analysis suggests an arrangement of these activities into distinct domains resembling the organization of Escherichia coli polymerase I . In order to more precisely define the structure and C-terminal limits of a putative catalytic domain responsible for the DNA polymerization activity of the HSV-1 enzyme, we have undertaken in vitro mutagenesis and computer modeling studies of the HSV-1 DNA polymerase gene . Sequence analysis predicts that the major DNA polymerization domain of the HSV-1 enzyme will be contained between residues 690 and 1100, and we present a three-dimensional model of this region, on the basis of the X-ray crystallographic structure of the E . coli polymerase I . Consistent with these structural and modeling studies, deletion analysis by in vitro mutagenesis of the HSV-1 DNA polymerase gene expressed in Saccharomyces cerevisiae has confirmed that certain amino acids from the C terminus (residues 1073 to 1144 and 1177 to 1235) can be deleted without destroying HSV-1 DNA polymerase catalytic activity and that the extreme N-terminal 227 residues are also not required for this activity. J Virol, 1990 Oct, 64(10), 4948 - 56 Novel transcription from the Epstein-Barr virus terminal EcoRI fragment, DIJhet, in a nasopharyngeal carcinoma; Gilligan K et al.; Transcription of Epstein-Barr virus (EBV) genes in epithelial tissue, one of the two principal cell types infected by EBV, is not well characterized . EBV transcription in a nasopharyngeal carcinoma established in nude mice, C15, has been analyzed by using strand-specific RNA probes and sequence analysis of a C15 cDNA library . In C15, two equally abundant mRNAs of 3.7 and 2.8 kilobases (kb) are encoded by the sequences that encode latent membrane protein (LMP) . Hybridization with probes specific for the 3' end of the LMP mRNA to Northern (RNA) blots and sequence analysis of cDNAs representing the messages indicated that the 3.7- and 2.8-kb mRNAs are 3' coterminal . Sequence analysis of additional cDNAs revealed an mRNA that is spliced identically to the LMP mRNA but is initiated 5' to the promoter for LMP . A probe representing the sequences contained within the cDNA which are 5' to the LMP promoter identified the 3.7-kb mRNA in C15 and a low-abundance 3.7-kb mRNA in B95-8 RNA . These data indicate that transcription of the LMP-encoding sequences is complex and that LMP can be expressed from an additional RNA in both nasopharyngeal carcinoma and lymphoid cells . Hybridization with BamHI-A identified a predominant 4.8-kb mRNA and two less abundant larger-molecular-weight mRNAs transcribed in C15 . These mRNAs are consistently expressed in all passages in nude mice of the C15 tumor . Hybridization with strand-specific probes and sequence analysis of three cDNAs revealed that these mRNAs are transcribed from left to right . Sequence analysis of cDNAs representing the 3' end of the mRNAs identified an open reading frame that could potentially encode a protein of 174 amino acids . In situ hybridization of a 35S-labeled RNA probe homologous to the BamHI-A cDNA to tissue sections revealed that the BamHI-A mRNA is not focally expressed and is transcribed in all cells within the C15 tumor . Linear forms of EBV DNA were not detected in any of the C15 tumors, and replicative viral antigens have not been detected . These data suggest that the C15 tumor represents a latently infected tumor and that the transcription from BamHI-A, which is expressed in all cells, is not associated with virus replication. J Virol, 1990 Oct, 64(10), 4625 - 31 Functional analysis of the internal translation initiation site of foot-and-mouth disease virus; Kuhn R et al.; Mutagenesis of the large untranslated sequence at the 5' end of the genome of foot-and-mouth disease virus revealed that a region of approximately 450 nucleotides preceding the open reading frame of the viral polyprotein is involved in the regulation of translation initiation at two internal start sites . Variations in two domains of this region reduced the translation efficiency up to 10-fold, whereas an intermediate segment seemed to be less essential . A pyrimidine-rich sequence preceding the start codon was most sensitive in that conversion of single pyrimidine residues to purines decreased the translation efficiency strongly . The data are in agreement with a recently proposed general structural model for the internal ribosome entry site of the cardiovirusaphthovirus subgroup of picornaviruses (E . V . Pilipenko, V . M . Blinov, B . K . Chernov, T . M . Dmitrieva, and V . I . Agol, Nucleic Acids Res . 17:5701-5711, 1989) . They suggest, however, that this model represents only a core structure for the internal entry of ribosomes and that foot-and-mouth disease virus and other members of the picornaviruses need additional regulatory RNA elements for efficient translation initiation. FEMS Microbiol Lett, 1990 Oct, 60(1-2), 225 - 7 Electrotransfection of Escherichia coli with lambda gt10 DNA; Powell R et al.; We have used electroporation to introduce lambda gt10 DNA into E . coli C600 (Electrotransfection) . We obtained approximately 10(7) pfu (plaque forming units) per micrograms of lambda gt10 DNA . This frequency is 100-fold higher than the maximum reported for classical calcium chloride-induced transfection . We have also compared electrotransfection with in vitro packaging in cloning experiments using relatively small amounts (less than or equal to 10 ng) of DNA . Our results slow that electrotransfection can generate approximately 1000-fold more plaques than in vitro packaging at these concentrations. Avian Dis, 1990 Oct-Dec, 34(4), 941 - 3 Differentiation of pathogenic and nonpathogenic Escherichia coli isolated from poultry; Panigrahy B et al.; Twenty-one isolates of Escherichia coli recovered from chickens and turkeys were evaluated for pathogenicity in 1-week-old chicks . Fifteen produced coli-septicemia (pathogenic) and six were innocuous (nonpathogenic) . Both pathogenic and nonpathogenic E . coli were tested for their ability to selectively absorb Congo red (CR) dye incorporated into agar medium . Eight of 15 pathogenic E . coli (somatic antigen types O1, O78, O11, O88, and OX9) absorbed the dye and produced red colonies (CR+) between 48 to 72 hours of incubation . All serotypes of E . coli with homologous somatic antigen O78 were CR+, while those of O2 antigen were CR- (white colonies) . Five of six nonpathogenic E . coli also were CR+ . In contrast to pathogenic E . coli, however, nonpathogenic isolates absorbed CR early, between 18 to 24 hours of incubation . Although CR dye binding did not correlate well with pathogenicity, it may be an identifiable property of some serotypes of E . coli. Mol Gen Genet, 1990 Oct, 224(1), 129 - 35 Use of recA803, a partial suppressor of recF, to analyze the effects of the mutant Ssb (single-stranded DNA-binding) proteins in vivo and in vitro; Madiraju MV et al.; We examined the possibility that the ssb-1 and ssb-113 mutants exert some of their effects by interfering with the normal function of wild-type RecF protein . Consistent with this possibility, we found that recA803, which partially suppresses recF mutations, also partially suppresses both ssb mutations, as detected by an increase in UV resistance . No evidence was obtained for suppression of the defect in lexA regulon inducibility caused by the ssb mutations . Consequently we suggest that suppression occurs by increasing recombinational repair . In vitro tests of Ssb mutant and wild-type proteins revealed that the single-stranded DNA dependent ATPase activity of RecA protein is more susceptible to inhibition than the joint-molecule-forming activity . All three Ssb proteins inhibit the ATPase activity of RecA wild-type protein almost completely while under similar conditions they inhibit the joint-molecule-forming activity only slightly . Both activities of RecA803 protein were found to be less inhibited by the three Ssb proteins than those of RecA wild-type protein . This is consistent with the suppressing ability of recA803 . We found no evidence to contradict the previously proposed hypothesis that ssb-1 affects recombinational repair by acting as a weaker form of Ssb protein . We found, however, only very weak evidence that Ssb-113 protein interferes directly with recombinational repair so that the possibility that it interferes with a normal function of RecF protein must remain open. Proc Natl Acad Sci U S A, 1990 Oct, 87(20), 8130 - 4 Genetic organization of the cellulose synthase operon in Acetobacter xylinum; Wong HC et al.; An operon encoding four proteins required for bacterial cellulose biosynthesis (bcs) in Acetobacter xylinum was isolated via genetic complementation with strains lacking cellulose synthase activity . Nucleotide sequence analysis indicated that the cellulose synthase operon is 9217 base pairs long and consists of four genes . The four genes--bcsA, bcsB, bcsC, and bcsD--appear to be translationally coupled and transcribed as a polycistronic mRNA with an initiation site 97 bases upstream of the coding region of the first gene (bcsA) in the operon . Results from genetic complementation tests and gene disruption analyses demonstrate that all four genes in the operon are required for maximal bacterial cellulose synthesis in A . xylinum . The calculated molecular masses of the proteins encoded by bcsA, bcsB, bcsC, and bcsD are 84.4, 85.3, 141.0, and 17.3 kDa, respectively . The second gene in the operon (bcsB) encodes the catalytic subunit of cellulose synthase . The functions of the bcsA, bcsC, and bcsD gene products are unknown . Bacterial strains mutated in the bcsA locus were found to be deficient in cellulose synthesis due to the lack of cellulose synthase and diguanylate cyclase activities . Mutants in the bcsC and bcsD genes were impaired in cellulose production in vivo, even though they had the capacity to make all the necessary metabolic precursors and cyclic diguanylic acid, the activator of cellulose synthase, and exhibit cellulose synthase activity in vitro . When the entire operon was present on a multicopy plasmid in the bacterial cell, both cellulose synthase activity and cellulose biosynthesis increased . When the promoter of the cellulose synthase operon was replaced on the chromosome by E . coli tac or lac promoters, cellulose production was reduced in parallel with decreased cellulose synthase activity . These observations suggest that the expression of the bcs operon is rate-limiting for cellulose synthesis in A . xylinum. FEBS Lett, 1990 Oct 1, 271(1-2), 227 - 30 Expression of subunit III of the ATP synthase from spinach chloroplasts in Escherichia coli; Burkovski A et al.; Expression of subunit III of the ATP synthase from spinach chloroplasts in Escherichia coli has been achieved . Although the protein is inserted into the bacterial cytoplasmic membrane, formation of a functional Fo complex was not observed. Arch Biochem Biophys, 1990 Oct, 282(1), 125 - 31 Mutagenesis of the a subunit of the F1F0-ATP synthase from Escherichia coli in the region of Asn-192; Vik SB et al.; Site-directed cassette mutagenesis was used to generate a series of amino acid substitutions in the a subunit of the Escherichia coli F1F0-ATP synthase . The following substitutions for Asn-192 were analyzed and shown to inhibit partially the ATP-dependent proton translocation without disrupting F1-F0 interactions: Leu, Val, Pro, Ser, Thr, and Arg . A group of multiple substitutions at residues Gln-181, Asn-184, and His-185 had no significant effect on ATP synthase function, as judged by growth yields, or by assays of ATP-dependent proton translocation, indicating that this region of the a subunit is not involved in function . Three double mutants were constructed in order to assess the independence of residues Asn-192, Glu-196, and Asn-214 . Results of proton translocation assays of membranes from cells containing these double mutations are consistent with the interpretation that each of these residues is involved with proton movement, and that residues Asn-192 and Glu-196 may be coupled . Finally, the relationship between the mechanism of proton translocation by the E . coli ATP synthase and the chloroplast enzyme was probed by constructing variants of the E . coli a subunit containing several features of homologous chloroplast proteins . It was determined that these chloroplast features, in the region of Glu-196 of the E . coli a subunit,, were detrimental to ATP synthase function. J Bacteriol, 1990 Oct, 172(10), 5602 - 9 Analysis of ftsZ mutations that confer resistance to the cell division inhibitor SulA (SfiA); Bi E et al.; In Escherichia coli, the ftsZ gene is thought to be an essential cell division gene . Several dominant mutations that make lon mutant cells refractory to the cell division inhibitor SulA, sulB9, sulB25, and sfiB114, have been mapped to the ftsZ gene . DNA sequence analysis of these mutations and the sfiB103 mutation confirmed that all of these mutations mapped within the ftsZ gene and revealed that the two sulB mutations were identical and by selection for resistance to higher levels of SulA, contained a second mutation within the ftsZ gene . We therefore propose that these mutations be redesignated ftsZ(Rsa) for resistance to SulA . A procedure involving mutagenesis of ftsZ cloned on low-copy-number vectors was used to isolate three additional ftsZ(Rsa) mutations . DNA sequence analysis of these mutations revealed that they were distinct from the previously isolated mutations . One of these mutations, ftsZ3(Rsa), led to an altered FtsZ protein that could no longer support cell growth but still conferred the Rsa phenotype in the presence of ftsZ+ . In addition to being resistant to SulA, all ftsZ(Rsa) mutations also conferred resistance to a LacZ-FtsZ hybrid protein (ZZ) . One possibility is that FtsZ functions as a multimer and that FtsZ(Rsa) mutant proteins have an increased ability for multimerization, making them resistant to SulA and ZZ. Carcinogenesis, 1990 Oct, 11(10), 1771 - 4 A new assay for O6-alkylguanine-DNA-alkyltransferase to determine DNA repair capacities using lambda-phage DNA as substrate; Klein S et al.; One O6-methylguanine (O6-meG) was introduced into each BamHI site of lambda-phage DNA as a substrate for the determination of the DNA repair protein O6-alkylguanine-DNA-alkyltransferase . A new assay using as the detection group 32P-labeled phosphate introduced at the 3' position of the modified nucleoside by incorporation of 32P-labeled TTP in the 3'-neighboring position proved highly sensitive: 10(-16) mol of the DNA lesion was still easily detectable . This DNA, which has greater than 1000 bp represents a good model for cellular DNA and was used as a substrate to measure the individual repair capacities for O6-meG in human lymphocytes of 20 healthy male and female donors . There were great inter-individual variations in the activity of this repair protein in man . The influences of age, sex or smoking behavior on the repair capacity of O6-meG were negligible. Aliment Pharmacol Ther, 1990 Oct, 4(5), 457 - 64 Comparison of three regimens in the management of acute gastroenteritis in infants; McClean P et al.; Sixty infants were randomly assigned to one of three groups on admission to hospital with a diagnosis of gastroenteritis . After rehydration, Group A received a low-lactose, low-fat feed (HN25) in full strength; Group B were regarded on to a conventional formula (SMA); Group C received a hydrolysed soya and collagen feed (Prejomin) in full strength . All feeds were continued for 5 days . The median duration of loose stools from starting the feed was 24 hours in Group A, compared to 119 hours and 95 hours in Groups B and C, respectively . Group A showed a mean percentage increase in weight of 2.34%, Group B showed a mean loss of 1.45%, and Group C a mean increase of 0.15% . These differences were statistically significant . Recovery from gastroenteritis is hastened by the use of a low-lactose, low-fat feed in the initial post-rehydration phase of the disease. Mol Endocrinol, 1990 Oct, 4(10), 1580 - 91 Cloning and expression of the cDNA for a Drosophila insulin-degrading enzyme; Kuo WL et al.; We have previously identified and characterized a metalloproteinase from Drosophila that cleaves insulin and transforming growth factor-alpha, but not epidermal growth factor, at physiological concentrations . On the basis of enzymatic properties and substrate specificity, this enzyme was identified as the Drosophila homolog of the mammalian insulin-degrading enzyme (IDE) . We now report the cloning and sequencing of the cDNA coding for the Drosophila IDE (dIDE) . Northern blot analysis indicates that the dIDE is translated from a 3.6-kilobase transcript similar in size to one of the two human IDE transcripts . The gene for the dIDE has been mapped to chromosome 3L (77B) . The sequence of the dIDE is very similar to that of the human IDE, and both enzymes share limited but significant identity with the bacterial metalloproteinase protease III . Indirect studies based upon inhibitors, degradation products, and microinjected antibodies have suggested that the IDE can initiate cellular insulin degradation in mammalian cells . To determine whether dIDE expressed in mammalian cells can also degrade insulin, we transfected the cDNA into murine NIH3T3 cells . Extracts of the transfected cells showed increased insulin-degrading activity, demonstrating that the dIDE can be functionally expressed in mammalian cells . These results indicate that the properties of the IDE are evolutionarily conserved. Biull Eksp Biol Med, 1990 Oct, 110(10), 415 - 6 {Mutagenic effects of ionized plasma light flux}; Stupin IV et al.; The effect of ion plasma light flux on the genomes of auxotrophic Escherichia coli strain and Drosophilla melanogaster has been examined . Essentially no mutagenic effect was found in doses close to therapeutic ones. Cell Growth Differ, 1990 Oct, 1(10), 455 - 62 A ubiquitous nuclear protein stimulates the DNA-binding activity of fos and jun indirectly; Abate C et al.; The protooncogenes c-fos and c-jun encode nuclear proteins (fos and jun, respectively) that function cooperatively as a heterodimeric protein complex in the regulation of gene transcription . These proteins dimerize via a structural motif known as the leucine zipper and bind to activator protein-1 sites via a conserved domain that is rich in basic amino acids . Previously, we demonstrated that while fos and jun polypeptides expressed in Escherichia coli dimerize efficiently, they exhibit only a low level of DNA-binding activity . Here we show that the DNA-binding activity of fos-jun heterodimers and jun-jun homodimers is stimulated dramatically by a ubiquitous nuclear protein . This protein does not appear to participate in the DNA-protein complex, and it does not affect the specificity of the interaction with DNA . These results suggest that a nuclear protein regulates the DNA-binding activity of fos and jun indirectly. J Interferon Res, 1990 Oct, 10(5), 541 - 7 Interferons-alpha and -gamma inhibit interleukin-1 beta-stimulated osteoclast-like cell formation in long-term human marrow cultures; Kurihara N et al.; Immune cell products can have major effects on bone remodeling . Cytokines, such as interleukin-1 (IL-1), are potent stimulators of bone resorption, whereas interferon-gamma (IFN-gamma) inhibits bone resorption stimulated by these factors . Bone resorption is a result of either increased numbers of osteoclasts, increased activity of individual osteoclasts, or both . Recently, we have shown that human recombinant IFN-gamma is a potent inhibitor of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3)-induced formation of multinucleated cells (MNC) that express the osteoclast phenotype . However, it is unknown if other IFNs share this capacity to inhibit MNC formation . Therefore, we tested the effects of natural IFNs-alpha and -gamma and Escherichia coli-derived recombinant human IFN-gamma on MNC formation and on granulocyte-macrophage (CFU-GM) colony formation (an early osteoclast precursor) in human marrow cultures treated with IL-1 beta or CSF-GM, respectively, to determine their effects on human osteoclast formation . Each type of IFN inhibited CFU-GM colony formation similarly in a dose-dependent fashion, with 50% inhibition seen at 64-250 U/ml . Natural or recombinant IFN-gamma inhibited IL-1 beta-stimulated MNC formation in a dose-dependent manner with an ID50 of 1-16 U/ml . IFN-alpha was more potent than IFN-gamma, with an ID50 of less than 1 U/ml . Furthermore, both IFN-alpha and -gamma inhibited fusion of precursors of these multinucleated cells . These data demonstrate that IFNs-alpha and -gamma inhibit MNC formation by inhibiting the growth of precursors for these cells as well as their subsequent fusion. AIDS Res Hum Retroviruses, 1990 Oct, 6(10), 1169 - 75 Expression in Escherichia coli and purification of human immunodeficiency virus type 1 capsid protein (p24); Ehrlich LS et al.; Capsid protein (p24;CA) of human immunodeficiency virus type 1 (HIV-1) was synthesized in Escherichia coli strain BL21 (DE3) using a plasmid encoding a truncated HIV-1 gag/pol gene . The plasmid, which contained a mutation in the frameshift region, expressed viral proteinase (PR), a pol gene product, in the gag reading frame, resulting in efficient processing of mature CA and other gag-related products . The expressed CA is soluble, recognized by monoclonal antibodies directed against HIV CA and has an N-terminal sequence identical to that of CA purified from HIV . Purification was done under mild conditions where coexpressed HIV PR retained enzymatic activity . Milligram quantities of 90% pure CA protein were obtained after chromatography on DEAE cellulose followed by facilitated aggregation of the CA in the unbound fraction . The precipitated CA was readily dissolved in low ionic strength aqueous buffer . Gel exclusion chromatography results indicated that, in solution, CA existed in oligomeric form. FEBS Lett, 1990 Oct 1, 271(1-2), 128 - 30 Low temperature cultivation of Escherichia coli carrying a rice lipoxygenase L-2 cDNA produces a soluble and active enzyme at a high level; Shirano Y et al.; Using a T7 RNA polymerase promoter system, rice lipoxygenase L-2 cDNA was expressed in E . coli as a fusion protein with 18 amino acid residues at the amino terminal end of the original enzyme . Incubation at 37 degrees C for 3 h in the presence of the inducer resulted in the production of inactive lipoxygenase . However, when induction was carried out at 15 degrees C for 16 h, active lipoxygenase, amounting to 3% of the total soluble protein, was produced . The enzyme was purified by ammonium sulfate precipitation and Mono-Q column chromatography to homogeneity at a yield of 80% . Expression of this protein should permit future site-directed mutagenesis of the gene and crystallization of the enzyme. Biochem J, 1990 Oct 1, 271(1), 139 - 45 Isolation and characterization of lipoylated and unlipoylated domains of the E2p subunit of the pyruvate dehydrogenase complex of Escherichia coli; Ali ST et al.; The dihydrolipoamide acetyltransferase subunit (E2p) of the pyruvate dehydrogenase complex of Escherichia coli has three highly conserved and tandemly repeated lipoyl domains, each containing approx . 80 amino acid residues . These domains are covalently modified with lipoyl groups bound in amide linkage to the N6-amino groups of specific lysine residues, and the cofactors perform essential roles in the formation and transfer of acetyl groups by the dehydrogenase (E1p) and acetyltransferase (E2p) subunits . A subgene encoding a hybrid lipoyl domain was previously shown to generate two products when overexpressed, whereas a mutant subgene, in which the lipoyl-lysine codon is replaced by a glutamine codon, expresses only one product . A method has been devised for purifying the three types of independently folded domain from crude extracts of E . coli, based on their pH-(and heat-)stabilities . The domains were characterized by: amino acid and N-terminal sequence analysis, lipoic acid content, acetylation by E1p, tryptic peptide analysis and immunochemical activity . This has shown that the two forms of domain expressed from the parental subgene are lipoylated (L203) and unlipoylated (U203) derivatives of the hybrid lipoyl domain, whereas the mutant subgene produces a single unlipoylatable domain (204) containing the Lys-244----Gln substitution. Proc Natl Acad Sci U S A, 1990 Oct, 87(19), 7492 - 6 Direct evidence for translational regulation by leader RNA and Tat protein of human immunodeficiency virus type 1; SenGupta DN et al.; Translational effects of the RNA leader and Tat protein of human immunodeficiency virus type 1 (HIV-1) were investigated in rabbit reticulocyte lysate . Hybrid RNA species with natural or mutated HIV-1 leader fused to human interferon- gamma mRNA were produced in vitro from recombinant plasmids . HIV-1 leader RNA was found to inhibit translation through two mechanisms . A 3-fold trans-inhibition of translation was demonstrated by mixing hybrid HIV-1 leader RNA with indicator interferon mRNA . By comparison, HIV-1 leader caused a 50-fold cis-inhibition in lysate in which two trans-inhibitory factors, double-stranded RNA-dependent protein kinase and (2'-5')oligoadenylate synthetase, were suppressed . In contrast, purified HIV-1 Tat protein produced in Escherichia coli enhanced by 4-fold translation from HIV-1 leader-interferon mRNA but not from interferon mRNA lacking HIV sequences or from total poly(A)+ RNA . Translation of mRNA containing either a single base substitution in the loop of the "trans-acting responsive" sequence (TAR) or an alternative stem-loop in TAR was nevertheless stimulated by Tat . The enhancement of translation by Tat was largely due to relief of cis-inhibition, since the effect was found even in lysate in which double-stranded RNA-dependent protein kinase was inhibited with 2-aminopurine . These results suggest that translation is an important level of control in the replication cycle of HIV-1. J Exp Med, 1990 Oct 1, 172(4), 1115 - 25 Inflammatory lipid mediator generation elicited by viable hemolysin-forming Escherichia coli in lung vasculature; Grimminger F et al.; Escherichia coli hemolysin, a transmembrane pore-forming exotoxin, is considered an important virulence factor for E . coli-related extraintestinal infections and sepsis . The possible significance of hemolysin liberation for induction of inflammatory lipid mediators was investigated in isolated rabbit lungs infused with viable bacteria (concentration range, 10(4)-10(7)/ml) . Hemolysin-secreting E . coli (E . coli-Hly+), but not an E . coli strain that releases an inactive form of the exotoxin, induced marked lung leukotriene (LT) generation with predominance of cysteinyl LTs . Eicosanoid synthesis was not inhibited in the presence of plasma with toxin-neutralizing capacity . Pre-application of 2 x 10(8) human granulocytes, which sequestered in the lung microvasculature, caused a severalfold increase in leukotriene generation in response to E . coli-Hly+ challenge both in the absence and presence of plasma . Data are presented indicating neutrophil-endothelial cell cooperation in arachidonic acid lipoxygenase metabolism as an underlying mechanism . We conclude that liberation of hemolysin from viable E . coli induces marked lipid mediator generation in lung vasculature, which is potentiated in the presence of neutrophil sequestration and may contribute to microcirculatory disturbances during the course of severe infections. J Bacteriol, 1990 Oct, 172(10), 6035 - 41 DNA sequence of the purC gene encoding 5'-phosphoribosyl-5-aminoimidazole-4-N-succinocarboxamide synthetase and organization of the dapA-purC region of Escherichia coli K-12; Tiedeman AA et al.; 5'-Phosphoribosyl-5-aminoimidazole-4-N-succinocarboxamide synthetase (EC 6.3.2.6), encoded by the purC gene of Escherichia coli K-12, catalyzes the synthesis of 5'-phosphoribosyl-5-aminoimidazole-4-N-succinocarboxamide from 5'-phosphoribosyl-5-aminoimidazole-4-carboxylic acid . The mature protein, as deduced from the purC structural gene sequence, contains 237 amino acids and has a calculated Mr of 26,998 . The control region of the purC gene was identified by primer extension mapping of the 5' end of the purC mRNA . The purC control region contains a binding site for and is regulated by the purine repressor, the product of the purR gene . An unusual feature of the 5' untranslated region of the purC mRNA is the presence of a repetitive extragenic palindrome sequence normally found in intercistronic or 3' untranslated regions . The DNA sequence was extended 1.281 kilobases upstream of the purC structural gene and overlapped with the previously determined dapA sequence . Termination of transcription from the dapA-purC intercistronic region may occur within the -35 region of the purC control region . The purC gene has been positioned on the E . coli restriction map and is transcribed in a counterclockwise direction. J Bacteriol, 1990 Oct, 172(10), 5871 - 6 Involvement of the histidine protein (HPr) of the phosphotransferase system in chemotactic signaling of Escherichia coli K-12; Grubl G et al.; It is known that in mutants of Escherichia coli lacking the histidine protein (HPr) of the carbohydrate: phosphotransferase system, all substrates of the system can be taken up in the presence of the fructose-regulated HPr-like protein FPr (gene fruF) . Although this protein fully substituted for HPr in transport and phosphorylation, we found that it was not able to complement efficiently for HPr in mediating chemotaxis toward phosphotransferase system substrates . Furthermore, transport activity and chemotaxis could be genetically dissected by the exchange of single amino acids in HPr . The results suggest a specific role of HPr in chemotactic signaling . We propose a possible link of signal transduction pathways for phosphotransferase system- and methyl chemotaxis protein-dependent substrates via HPr. J Bacteriol, 1990 Oct, 172(10), 5697 - 705 Characterization of the minimum replicon of the broad-host-range plasmid pTF-FC2 and similarity between pTF-FC2 and the IncQ plasmids; Dorrington RA et al.; The nucleotide sequence of a 3,202-base-pair fragment which contained the minimum region required for replication of the broad-host-range plasmid, pTF-FC2, has been determined . At least five open reading frames and a region that affected the host range were identified . Proteins corresponding in size and location to four of the five open reading frames were produced in an in vitro transcription-translation system . The predicted amino acid sequences of two of the proteins were aligned with those of the RepA and RepC proteins of the broad-host-range IncQ plasmid RSF1010 and found to be 43 and 60% homologous, respectively . Despite this similarity, neither the RepA nor the RepC protein of the IncQ plasmid was able to complement mutations in the pTF-FC2 repA and repC genes . Although there was a considerable amount of DNA homology between pTF-FC2 and RSF1010 in the oriV region and the region coding for the RepA and RepC proteins, no other homology between the two plasmids at either the DNA or protein level could be detected. EMBO J, 1990 Oct, 9(10), 3171 - 8 Analysis of the substrate binding sites of human galactosyltransferase by protein engineering; Aoki D et al.; An expression vector, pIN-GT, encoding the soluble form of beta 1,4-galactosyltransferase (GT) has been constructed from human GT cDNAs and the pIN-III-ompA2 expression vector . Escherichia coli strain SB221 harboring the pIN-GT plasmid produces and secretes a fusion protein consisting of the ompA signal and GT . The expression of GT was detected by assaying enzymatic activity as well as by Western blotting using anti-GT antibodies . The recombinant GT was purified to homogeneity by N-acetylglucosamine-Sepharose affinity chromatography . The NH2-terminal peptide sequence of purified GT confirmed the cleavage site of the fusion protein by bacterial signal peptidase . This expression system was utilized to produce mutant forms of GT in order to identify specific amino acids involved in substrate binding sites . Photoaffinity labeling of GT with UDP-galactose analog, 4-azido-2-nitrophenyluridylylpyrophosphate (ANUP), followed by cyanogen bromide (CNBr) cleavage revealed that ANUP bound to a fragment of GT composed of amino acid residues from Asp276 to Met328 . Within this peptide segment, Tyr284, Tyr287, Tyr309, Trp310 and Trp312 were separately substituted into Gly and Tyr287 into Phe by site-directed mutagenesis . Enzymatic activity assay showed drastic reduction of the activity in all of the mutants except that Tyr287----Phe remained as active as wild-type GT . Kinetic studies of the mutated GT showed that Tyr284, Tyr309 and Trp310 are critically involved in the N-acetyglucosamine binding and Tyr309 is involved in UDP-galactose binding as well.(ABSTRACT TRUNCATED AT 250 WORDS) Int J Exp Pathol, 1990 Oct, 71(5), 717 - 25 Recombinant gamma interferon causes neutrophil migration mediated by the release of a macrophage neutrophil chemotactic factor; Ribeiro RA et al.; A dose-dependent neutrophil migration was observed following the injection of purified (Hu IFN-gamma) or recombinant (rIFN-gamma) human gamma interferon into rat peritoneal cavities . This finding contrasts with their inability to cause chemotaxis in vitro in the Boyden chamber . Neutrophil migration into peritoneal cavities and subcutaneous air pouches induced by both preparations of interferon was abolished by pretreatment of the animals with dexamethasone . IFN-gamma-induced neutrophil migration was enhanced when the macrophage population of the peritoneal cavities was increased by previous injection of thioglycollate and reduced by peritoneal lavage . Macrophage monolayers pretreated either with rIFN-gamma or with lipopolysaccharide from E . coli release into the supernatant a factor that stimulates neutrophil recruitment in animals treated with dexamethasone . Dexamethasone blocked this release but did not affect the neutrophil recruitment induced by this factor . These results suggest that IFN-gamma-induced neutrophil migration in vivo may be mediated by the release from resident macrophages of a neutrophil chemotactic factor and that dexamethasone blockade of neutrophil recruitment by IFN-gamma is due to inhibition of the release of this factor. Mol Cell Biol, 1990 Oct, 10(10), 5532 - 5 Fos and jun cooperate in transcriptional regulation via heterologous activation domains; Abate C et al.; The products of c-fos and c-jun (Fos and Jun) function in gene regulation by interacting with the AP-1 binding site . Here we have examined the contribution of Fos and Jun toward transcriptional activity by using Fos and Jun polypeptides purified from Escherichia coli . Fos contained a transcriptional activation domain as well as a region which exerted a negative influence on transcriptional activity in vitro . Moreover, distinct activation domains in both Fos and Jun functioned cooperatively in transcriptional stimulation . Thus, regulation of gene expression by Fos and Jun results from an integration of several functional domains in a bimolecular complex. J Mol Recognit, 1990 Oct-Dec, 3(5-6), 204 - 7 Specific isolation by anhydrotrypsin-agarose chromatography of a recombinant protein tagged with an affinity tail arginine at the C-terminus; Hirabayashi J et al.; A characteristic property of anhydrotrypsin, i.e., its ability to strongly bind C-terminal arginine, proved to be useful as a tool for specific enrichment of a recombinant protein . An arginine tail was introduced at the C-terminus, for example, of a human beta-galactoside-binding lectin by site-directed mutagenesis . The resulting mutant recombinant lectin, which retained sugar-binding activity as high as the wild-type lectin, became recognizable by anhydrotrypsin . It was adsorbed on an anhydrotrypsin-agarose column and eluted with benzoylglycylarginine . The added arginine tail could be specifically removed by carboxypeptidase B . When E . coli lyzate containing the mutant lectin was applied to the column more than 10-fold enrichment of the mutant lectin was attained . This procedure should be generally applicable and may be advantageous because the addition of a single arginine residue may have minimal effect on the structure and function of the target protein. J Vet Diagn Invest, 1990 Oct, 2(4), 334 - 7 Mycoplasma iowae species-specific DNA probe; Zhao SH et al.; A Mycoplasma iowae (MI) species-specific DNA probe (designated pMI-2) of 6.0 kbp (kilobase pairs) was isolated from an MI strain I-695 genomic library prepared in plasmid pUC8 and Escherichia coli strain JM83 . When labeled with {32}P by nick translation, the probe hybridized in dot blot assays with 6 reference strains and 8 field isolates of MI but not with 16 other known species of avian mycoplasmas . The pMI-2 probe detected a minimum of 1.5 ng of MI strain I-695 chromosomal DNA . Under identical conditions of hybridization, the probe did not hybridize with a high concentration (200 ng) of M . gallisepticum or M . synoviae chromosomal DNA. Photochem Photobiol, 1990 Oct, 52(4), 897 - 901 Growing Escherichia coli mutants deficient in riboflavin biosynthesis with non-limiting riboflavin results in sensitization to inactivation by broad-spectrum near-ultraviolet light (320-400 nm); Lloyd RE et al.; Two mutants of Escherichia coli unable to synthesize riboflavin were grown with limiting (2 micrograms ml-1) and non-limiting (10 micrograms ml-1) concentrations of riboflavin . These riboflavin auxotrophs when grown to exponential phase with non-limiting riboflavin are more sensitive to broad spectrum near-ultraviolet light (NUV, 320-400 nm) inactivation than when they are grown with limiting riboflavin . Exponential phase cells of the riboflavin auxotrophs grown with limiting riboflavin are sensitized when irradiated in saline supplemented with riboflavin . This suggests that extracellular riboflavin is important as a NUV sensitizer when intracellular levels of riboflavin are reduced . The concentration of riboflavin in crude extracts from exponentially growing cells correlates well with the sensitivity of these mutants to NUV inactivation . The level of riboflavin supplementation has little effect on the NUV sensitivity of the parental strain. Photochem Photobiol, 1990 Oct, 52(4), 757 - 60 Photochemical modification of lac repressor--III . Mutant I12X86 versus wild-type repressor; Spodheim-Maurizot M et al.; Photodestruction of the two tryptophan (TRP) residues of the core of the wild-type Escherichia coli lac repressor has already been used as a probe in the study of interactions of the repressor with DNA and effectors . The good correlation between phenomena occurring in the core (photodestruction of TRP residues, effectors binding) and at the headpieces (DNA specific and non-specific binding) can be understood in terms of allosteric behavior of the protein . In the present study, the same approach is applied to a repressor with peculiar binding properties, the I12X86 mutant . The photodestruction of TRP residues of this tight binding repressor, bearing two different amino acids as compared to the wild-type one (Ser 61----Leu, Pro 3----Tyr) indicates a probably subtle (since not detected by classical spectroscopic methods) difference of structure of the entire protein and confirms the similarity between specific and non-specific binding of this mutant repressor to DNA, observed by other methods. Jpn J Antibiot, 1990 Oct, 43(10), 1667 - 73 {Investigation on clinical efficacy and passage into ascites of cefminox in diffuse peritonitis associated with infantile acute appendicitis}; Itagaki K et al.; Cefminox (CMNX), one of newly developed cephamycin antibiotics, was administered to 7 cases of diffuse peritonitis associated with infantile acute appendicitis to determine its concentrations in the blood, the appendiceal tissue and the purulent ascites and simultaneously to investigate its clinical efficacy . CMNX was intravenously injected at a dose of 20 mg/kg (at a maximum amount of 1.0 g/body) before operation and intravenously by bolus injection or by drip infusion twice daily after operation for 3 to 11 days in a total dose of 2.36 to 14.06 g . Fourteen strains of bacteria were isolated from the purulent ascites: Escherichia coli was isolated from 6 cases but superinfections in 5 cases . MICs of CMNX against these isolated organisms were at or lower than 3.13 micrograms/ml for 12 out of 14 strains . CMNX penetrated into the appendiceal tissue and the purulent ascites very well . The concentrations in the pus reached higher than those in the tissue in about 1 hour after administration and were found to reach as high as 9.63 micrograms/ml in 5 hours after administration . Its clinical efficacies were excellent in 4 cases, good in 2 cases and poor in 1 case . No subjective or objective adverse reactions were observed nor any abnormalities were found in laboratory examinations. Sci China B, 1990 Oct, 33(10), 1210 - 7 Isolation, characterization and expression of the complementary DNA for human tumor necrosis factor (TNF-alpha); Xiao L et al.; A cDNA for human TNF-alpha (615bp) was isolated by means of polymerase chain reaction (PCR) using first strand cDNA from PMA-induced HL-60 cells as template . The result from sequencing the 615 bp cDNA fragment indicated that it corresponded to the entire sequence of mature human TNF coding region . Direct expression of mature human TNF was achieved using a plasmid pHT-1 constructed by ligation of the cDNA and a synthetic DNA . The IPTG-induced bacterial product (hTNF) showed cytotoxicity to mouse L-929 cells . The TNF activity was further identified by neutralization of a specific monoclonal antibody against human TNF-alpha . Approximately 80,000 units of activity were detected per ml of culture at A600 = 2. Bioorg Khim, 1990 Oct, 16(10), 1348 - 54 {Hybridase cleavage of RNA . II . Automatic synthesis of mixed oligonucleotide probes}; Romanova EA et al.; Several oligo(ribodeoxyribo)nucleotides to be used as probes in the RNase H-induced hydrolyses of 5S ribosomal RNA E . coli were synthesized on the Victoria-4M automatic gene synthesizer by the phosphoramidite approach, which allows for introducing ribonucleotides into any position of the oligomer . 2'-Hydroxy function was protected by tert-butyldimethylsilyl group whose hydrophobicity simplified isolation of the oligonucleotides by reverse-phased HPLC. Bioessays, 1990 Oct, 12(10), 473 - 7 Mismatch repair in mammalian cells; Heywood LA et al.; A vital process in maintaining a low genetic error rate is the removal of mismatched bases in DNA . The importance of this process in E . coli is demonstrated by the 100-1000 fold increase in mutation frequency observed in cells deficient in this repair system . Mismatches can arise as a consequence of recombination, errors in replication and as a result of spontaneous chemical deamination, the latter process resulting in an estimated twelve T:G mismatches per genome per day in mammalian cells . Recent studies, discussed here, provide evidence for the existence of specific mismatch repair systems in mammalian and human cells. Biochimie, 1990 Oct, 72(10), 735 - 43 Conformation in solution of yeast tRNA(Asp) transcripts deprived of modified nucleotides; Perret V et al.; A synthetic gene of yeast aspartic acid tRNA with a promoter for phage T7 RNA polymerase was cloned in Escherichia coli . The in vitro transcribed tRNA(Asp) molecules are deprived of modified nucleotides and retain their aspartylation capacity . The solution conformation of these molecules was mapped with chemical structural probes and compared to that of fully modified molecules . Significant differences in reactivities were observed in Pb2+ cleavage of the RNAs and in modification of the bases with dimethyl sulphate . The most striking result concerns C56, which becomes reactive in unmodified tRNA(Asp), indicating the disruption of the C56-G19 base pair involved in the D- and T-loop interaction . The chemical data indicate that unmodified tRNA(Asp) transcripts possess a relaxed conformation compared to that of the native tRNA . This conclusion is confirmed by thermal melting experiments . Thus it can be proposed that post-transcriptional modifications of nucleotides in tRNA stabilize the biologically active conformations in these molecules. Mol Microbiol, 1990 Oct, 4(10), 1779 - 83 Characterization of FapR, a positive regulator of expression of the 987P operon in enterotoxigenic Escherichia coli; Klaasen P et al.; Expression of the 987P gene cluster is activated by the adjacent IS1 element of an STpa transposon . Nucleotide sequence analysis of the 987P-DNA region contiguous with this IS1 element revealed the presence of an open reading frame designated fapR, encoding a basic protein of 260 amino acid residues with a molecular mass of 30,349 Daltons . The gene product, FapR, possesses similarity to a number of positive regulators of gene expression: VirF, Rns, AppY and EnvY . Moreover, a 43-amino-acid residue sequence in the C-terminal part of FapR is similar to the C-terminal domain of AraC, RhaR, and RhaS . Expression of fapR is dependent on the adjacent IS1 element . The FapR protein appears to be required for activation of the silent promoter of the fimbrial subunit gene, fapC. Mol Microbiol, 1990 Oct, 4(10), 1753 - 63 Molecular genetic analysis of an FNR-dependent anaerobically inducible Escherichia coli promoter; Bell AI et al.; From the effects of 13 deletions and three linker-scanner mutations at the Escherichia coli nirB promoter we have located sequences necessary for FNR-dependent induction of activity by anaerobiosis and further nitrite-dependent stimulation of expression . We describe a nirB promoter derivative that allows the cloning of 'cassettes' carrying different FNR-binding sequences and experiments in which a number of point mutations were introduced into these sequences . FNR-dependent stimulation of expression from the nirB promoter is critically dependent on the location of the FNR-binding site, and deletion or insertion of one base pair is sufficient to disrupt promoter function . We have transferred a number of cassette FNR-binding sequences from the nirB promoter to the unrelated melR promoter . The insertion of FNR-binding sequences at the melR promoter is sufficient to confer fnr-dependency on expression . However expression from these hybrid promoters is not as efficiently repressed during aerobic growth, suggesting that the function of bound FNR is dependent on the sequence context of the FNR-binding sequence. Arch Roum Pathol Exp Microbiol, 1990 Oct-Dec, 49(4), 297 - 313 Characterization of colonization factor antigen CFA/I from an enterotoxigenic Escherichia coli strain (0128:H12); Dima VF et al.; CFA/I antigen was isolated and purified from E . coli, mutant 279 B-1-14, serotype 0128:H12, and had the following biochemical and biological features: a) amino-acid content was similar to that of purified antigen prepared from strain H10407; b) latex particles sensitization with purified CFA/I antigen produced bovine and human erythrocytes group A/II hemagglutination in carbohydrates presence; c) purified anti-CFA/I specific antibodies agglutinated CFA/I-positive enterotoxigenic E . coli strains; d) 3H-leucine-labelled CFA/I antigen adhered to rabbits intestinal mucosa at significant values; e) intestinal mucosa pretreating with purified CFA/I antigen, followed by 3H-leucine labelled enterotoxigenic bacteria infection, had a least 3 local effects: 1) intestinal mucosa protection against parental enterotoxigenic bacteria; 2) inhibition of CFA/I-positive bacteria adherence to intestinal mucosa; 3) release of approximately 96% intraluminally inoculated bacteria. Microb Pathog, 1990 Oct, 9(4), 285 - 91 A silent regulatory gene cfaD' on region 1 of the CFA/I plasmid NTP 113 of enterotoxigenic Escherichia coli; Gaastra W et al.; A DNA sequence, homologous to the cfaD gene of CFA/I region 2, was identified on CFA/I region 1 . This sequence is designated cfaD' . It differs from the cfaD gene in containing two deletions and a stop codon . The cfaD' sequence therefore can only encode a truncated CfaD-like protein . The CfaD protein may be a DNA binding protein and functions as a positive regulator of CFA/I fimbriae expression . A regulatory function for the cfaD' is not likely since deletion of the cfaD' sequence does not affect production of CFA/I fimbriae in E . coli K-12 strains . That the cfaD' sequence is present on CFA/I wild-type plasmids isolated from CFA/I strains of different serotypes, obtained at various geographical locations, suggests, however, that this DNA region is not completely without a function. Pflugers Arch, 1990 Oct, 417(2), 174 - 9 Effect of antisecretory factor on Escherichia coli STa enterotoxin-induced alkalinisation of pig jejunal acid microclimate; McEwan GT et al.; The effect of challenge by Escherichia coli STa enterotoxin on pig jejunal mucosal surface pH was investigated in vivo . Exposure to STa resulted in a rapid and reversible alkalinisation (P less than 0.001) of the jejunal mucosa from 6.27 +/- 0.11 (5) to 6.89 +/- 0.03 (5) . This action of STa is probably mediated through cyclic 3'5'-guanosine monophosphate (cGMP) since the 8-bromo analogue of cGMP induced the same effect as that observed after STa challenge . The action of STa on mucosal pH was partially inhibited by pre-administration of an antisecretory factor (ASF) preparation . The action of 8-bromo cGMP was unchanged by the presence of ASF . This implies that ASF inhibition occurs during the early stages of STa action prior to stimulation of guanylate cyclase . This effect of STa on the pig jejunal mucosal surface pH, or acid microclimate, may explain why weak acid supplementation of oral rehydration solutions can be ineffective in certain types of diarrhoeal disease. Mol Microbiol, 1990 Oct, 4(10), 1667 - 78 Functional importance of the Escherichia coli ribosomal RNA leader box A sequence for post-transcriptional events; Theissen G et al.; To shed more light on the controversial findings concerning the functional participation of the highly conserved nut-like leader box A sequence element in ribosomal RNA transcription antitermination we have carried out a mutational study . We have substituted the box A and combined this mutation with several deletions comprising the rRNA leader elements box B, box C and the tL region . The mutations are located within the genuine rrnB operon cloned on multicopy plasmids . We determined the effects of the mutations on cell growth, rRNA accumulation and ribosomal subunit stoichiometry . Cells transformed with the mutated plasmids were affected in their growth rate, and showed a surprising deficiency of the promoter-proximal 16S compared to the 23S RNA, indicative of a post-transcriptional degradation event . Accordingly, we could demonstrate a reduced amount of free 30S relative to 50S ribosomal subunits in exponentially growing cells . Similar stoichiometric aberrations in the ribosome pool were detected in conditionally Nus factor-defective strains . The results show that the leader box A sequence within rRNA operons has important post-transcriptional functions for 16S RNA stability and ribosomal subunit stoichiometry . A model is proposed, describing the biogenesis and quality control of ribosomes based on rRNA leader and Nus-factor interactions . It is compatible with the previously observed effects of box A in antitermination. J Appl Physiol, 1990 Oct, 69(4), 1315 - 22 Effect of LY171883 on endotoxin-induced lung injury in pigs; Olson NC et al.; We evaluated the role of sulfidopeptide leukotrienes as mediators of endotoxin-induced respiratory failure in pigs . Escherichia coli endotoxin (055-B5) was infused intravenously into anesthetized 10- to 14-wk-old pigs at 5 micrograms/kg the 1st h followed by 2 micrograms.kg-1.h-1 for 3 h in the presence and absence of LY171883, a specific leukotriene D4 (LTD4)/LTE4 receptor antagonist . Endotoxin caused hemoconcentration, granulocytopenia, decreased cardiac index, systemic hypotension, pulmonary hypertension, increased pulmonary vascular resistance, bronchoconstriction, hypoxemia, increased permeability of the alveolar-capillary membrane, pulmonary edema, and increased plasma concentrations of thromboxane B2 (TxB2), prostaglandin F2 alpha (PGF2 alpha), and 6-keto-PGF1 alpha . LY171883 did not modify endotoxin-induced cardiopulmonary and hematologic abnormalities, except for a modest attenuation of pulmonary hypertension (at 1 h) and increased pulmonary vascular resistance (at 1-2 h) . Ex vivo stimulation of whole blood with calcium ionophore caused large increases in plasma concentrations of TxB2, PGF2 alpha, and LTB4 . These increases were not significantly modified in blood derived from pigs treated with LY171883, indicating no inhibition of cyclooxygenase or 5-lipoxygenase . We conclude that LTD4 and LTE4 are not important mediators of endotoxin-induced lung injury in anesthetized pigs, although they may contribute modestly to pulmonary vasoconstriction. Circ Shock, 1990 Oct, 32(2), 123 - 32 Beta-adrenergic drug therapy in newborn canine endotoxic shock; Goto M et al.; The mortality of septic shock remains high in newborns . Although the effectiveness of adrenergic drug therapy continues to be controversial, adrenergic drugs have been used for the treatment of newborn endotoxic shock . To elucidate the effects of beta-adrenergic drugs on the fulminant hemodynamic deterioration of newborn endotoxic shock, newborn dogs (2-10-day-old, 264-800 g) were given Escherichia coli lipopolysaccharide (LPS; 10 mg/kg iv) and treated with isoproterenol (0.1 micrograms/kg/min) or dopamine (5 micrograms/kg/min) infusion from 5 to 120 min after LPS injection . Isoproterenol attenuated the effects of LPS by increasing the mean arterial pressure (32 +/- 2 vs . 13 +/- 1 mmHg at 120 min), cardiac output (183 +/- 29 vs . 118 +/- 23 ml/min/kg at 120 min), and the survival time (5.3 vs 2.9 hr) . However, dopamine did not improve the hemodynamic deterioration . As dopamine-beta-hydroxylase activity in the blood was significantly lower in newborn dogs than in adult dogs, inadequate response of newborn dogs to dopamine was thought to be in part due to enzymatic immaturity. Vet Microbiol, 1990 Oct, 25(1), 55 - 65 Nine DNA probes for detection of toxin and adhesin genes in Escherichia coli isolated from diarrhoeal disease in animals; Woodward MJ et al.; DNA gene probes specific for genes encoding heat labile enterotoxin (LTI), heat stable enterotoxins (STIa, STII), vero cytotoxins (VT1, VT2), and adhesins K88 (F4), K99(F5), F41 and 987P(F6) were used to examine 873 isolates of E . coli from cases of diarrhoea (680 from pigs, 187 from cattle and six from sheep) . A total of 188 were toxin gene positive and of these 84 belonged to the classical ETEC serogroups . Of the other 104 toxin gene positive strains, 80 hybridized with the VT2 probe of which 34 were from cases of porcine post-weaning diarrhoea belonging to serogroup 0138:K81 and 22 were untypable strains from cattle. Immunology, 1990 Oct, 71(2), 295 - 300 The secretory antibody response in milk and bile against fimbriae and LPS in rats monocolonized or immunized in the Peyer's patches with Escherichia coli; Dahlgren UI et al.; The homing of lymphoid cells to mucosa-associated lymphoid tissue is, amongst other factors, influenced by the nature of the antigen used to induce an immune response . To study this phenomenon we have monocolonized rats with a type 1 fimbriated Escherichia coli O6K13H1 strain and compared the secretory antibody response to colonization with the primary and secondary response obtained in rats immunized in the Peyer's patches (PP) . Samples were tested with respect to the titres of antibodies against two antigens present on the E . coli strain: O6 lipopolysaccharide (LPS) and type 1 fimbrial antigen . In the primary immunized animals, IgA anti-fimbrial antibodies were mainly seen in milk and IgA anti-LPS antibodies mostly found in bile . In the booster immunized, and in the monocolonized, animals there was a shift of the antibody response towards the bile . Thus anti-fimbrial antibodies appeared in milk at approximately the same or at a lower level than in bile and the IgA anti-LPS antibodies were almost completely absent in the milk . The IgG antibody response of the animals immunized in the PP was primarily confined to milk for both anti-LPS and anti-fimbrial antibodies, while the colonized animals responded with higher levels in bile than in milk . IgM antibodies were only seen in the milk, except in primary immunized animals in which biliary IgM antibodies also were found . The data illustrate that: (i) primary stimulated cells predestined to produce IgA anti-LPS antibodies home mainly to the intestine, while cells predestined to anti-fimbrial antibody production have a greater tendency to populate the mammary gland; (ii) after repeated antigen stimulation and maturation of the immune response the cells are directed from the mammary gland to the intestine . We thus conclude that the nature of the antigen and the stage of lymphocyte maturation influences the homing of the cells and the appearance of various antibodies in different secretions. Int J Radiat Biol, 1990 Oct, 58(4), 603 - 11 Determination of the constants of the Alper formula for single-strand breaks from kinetic measurements on DNA in aqueous solution and comparison with data from cells; Schulte-Frohlinde D et al.; On the basis of rate constants measured in aqueous solution for (i) DNA single-strand break (ssb) formation induced by OH radicals, (ii) prevention of ssb formation by reaction of DNA radicals with glutathione, and (iii) addition of O2 to DNA radicals, oxygen enhancement ratios (OER) and K values of the Alper equation have been calculated . The values obtained were compared with OER and K values determined for ssb formation in lambda DNA irradiated in Escherichia coli as a function of the oxygen concentration . Without adjustment of any parameter the two sets of data are similar when the corrected Alper formula is used . The results support the oxygen fixation-thiol repair model of Howard-Flanders and Alper, and indicate that under selected conditions DNA in aqueous solution may serve as a model system for DNA in cells. Infect Immun, 1990 Oct, 58(10), 3448 - 54 Effect of type 1 piliation on in vitro killing of Escherichia coli by mouse peritoneal macrophages; Keith BR et al.; Escherichia coli K-12 mutants possessing defined lesions affecting type 1 pilus production, receptor binding, or length were examined for their ability to resist killing by mouse peritoneal macrophages in vitro . Mutants were mixed pairwise at known ratios in wells containing macrophages, and after incubation, the ratio of the survivors was assayed . The difference in phagocytic killing between type 1 piliated cells and isogenic nonpiliated cells was significant, the piliated cells being approximately threefold more resistant . Pilus length had little effect upon survival, as the long-piliated mutants were no more resistant to killing than the normal-length parents . Interestingly, the receptor-binding function of type 1 pili was most important in effecting resistance, as mutants lacking the ability to bind receptor were killed as effectively as nonpiliated mutants . These data are consistent with the notion that pili actually impede killing by macrophages rather than serve as passive physical barriers to uptake. Infect Immun, 1990 Oct, 58(10), 3434 - 7 Genetically engineered S and F1C fimbriae differ in their contribution to adherence of Escherichia coli to cultured renal tubular cells; Marre R et al.; Escherichia coli K-12 strains producing S-fimbrial adhesins, F1C fimbriae, and mutagenized fimbriae were tested in a binding assay with a renal tubular cell line . S-fimbrial adhesins and F1C fimbriae mediated binding to tubular cells . The SfaA, SfaG, and SfaS subunits of S fimbriae contributed to attachment . Site-specific mutations in the sfaS gene reduced binding . The inhibition profile of F1C fimbriae resembled that of S fimbriae. Infect Immun, 1990 Oct, 58(10), 3178 - 82 Presence of K88-specific receptors in porcine ileal mucus is age dependent; Conway PL et al.; Ileal mucus and epithelial cells were isolated from newborn piglets that had never been fed and 35-day-old unweaned piglets . Both newborn and 35-day-old piglet mucus preparations supported growth of Escherichia coli Bd 1107/75 08, a K88-fimbriated porcine enterotoxigenic strain, equally well (i.e., generation times of 28 min were observed in both cases) . Adhesion of E . coli Bd 1107/75 08 to 35-day-old piglet ileal epithelial cells was, at most, 2 times that of the same strain to newborn piglet ileal epithelial cells; however, adhesion of E . coli Bd 1107/75 08 to 35-day-old piglet ileal mucus was 16 times that of the same strain to newborn piglet ileal mucus . The receptor in 35-day-old piglet ileal mucus was K88 specific, since it could be removed by purified K88ab fimbriae . Furthermore, adhesion of E . coli Bd 1107/75 08 to 35-day-old piglet ileal mucus was blocked by PAB10, a K88ab-, K88ac-, K88ad-specific monoclonal antibody . Although E . coli Bd 1107/75 08 traversed both newborn and 35-day-old piglet ileal mucus about equally well in vitro and bound well to underlying ileal epithelial cells after passing through newborn ileal mucus, it did not bind to ileal epithelial cells after passing through 35-day-old piglet ileal mucus . The data are discussed with respect to the role that K88-specific receptors present in newborn and ileal mucus might play in the pathogenesis of porcine enterotoxigenic E . coli strains which bear K88 fimbriae. J Clin Lab Immunol, 1990 Oct, 33(2), 69 - 73 Influence of human plasma high density lipoproteins from septic patients on different functions of normal human neutrophils; Jarstrand C et al.; High Density Lipoproteins (HDL) from three patients with E . coli sepsis contained high, low and no Serum Amyloid Protein (Apo SAA), respectively . Preincubation of neutrophils from healthy persons for half an hour with sepsis HDL as well as normal HDL increased the phagocytosis, the stimulated nitroblue tetrazolium reduction, the chemotaxis and the random migrations of these cells . However, for all these functions, lower values were obtained after incubation with sepsis HDL containing high amounts of apo SAA than with normal HDL . A qualitative change of HDL might thus in part be responsible for the decreased function of neutrophils noted during the acute phase of bacterial infections. Mol Microbiol, 1990 Oct, 4(10), 1645 - 52 Mutations produced by DNA polymerase III holoenzyme of Escherichia coli after in vitro synthesis in the absence of single-strand binding protein; Carraway M et al.; Single-stranded plasmid DNA, containing the mnt gene, was replicated in vitro with DNA polymerase III holoenzyme . Escherichia coli mutH bacteria, defective in mismatch repair, were transformed with the products of in vitro synthesis . Mutations in mnt were readily identified and 33 out of 65 isolates were single base changes including transition, transversion and frameshift mutations . The remaining 32 isolates were deletions of apparently random length and substitutions (deletion/insertions) . The intergenic deletions as well as the transition and frameshift mutations were identical to those previously isolated from mismatch repair-defective cells in vivo. Antimicrob Agents Chemother, 1990 Oct, 34(10), 2016 - 8 Nucleotide sequence and intracellular location of the product of the fosfomycin resistance gene from transposon Tn2921; Navas J et al.; An 863-base-pair DNA fragment containing the fosfomycin resistance gene from transposon Tn2921 was sequenced . Analysis of the sequence revealed the presence of a single open reading frame that encoded the FOS polypeptide of 16 kilodaltons from Tn2921 . Minicell fractionation studies showed that the FOS protein was located in the bacterial cytoplasm. Mol Microbiol, 1990 Oct, 4(10), 1737 - 43 Involvement of OmpF during reception and translocation steps of colicin N entry; Bourdineaud JP et al.; {125I}-colicin N binds to OmpF receptor sites (70,000 per cell) with an average Kassoc of 3.2 x 10(6) M-1 at 23 degrees C . Monoclonal antibody directed against a cell-surface-exposed epitope of OmpF is able to complete with the binding of the colicin in vitro and also to protect against colicin N in vivo . OmpF is an absolute requirement for colicin N uptake . OmpC cannot serve as a substitute for OmpF during translocation across the outer membrane under receptor bypass conditions, which is in contrast to colicin A . Colicin N does not cross-react with various monoclonal antibodies directed against colicin A. Mol Microbiol, 1990 Oct, 4(10), 1711 - 4 Distribution of msDNAs among serotypes of enteropathogenic Escherichia coli strains; Lim D et al.; A genetic element, called a retron, is present in certain Escherichia coli strains . It consists of genes for the production of a covalently linked DNA-RNA compound and a reverse transcriptase . The presence of a retron can be detected by testing for a satellite DNA band by polyacrylamide gel electrophoresis . This DNA band consists of the DNA portion of the DNA-RNA compound and is called msDNA (multicopy single-stranded DNA) . In a survey of intestinal E . coli isolates we detected msDNAs in classical enteropathogenic (EPEC) strains and in strains with aggregative adherence to tissue-culture cells (AA), but not in enteroinvasive (EIEC) and enterotoxigenic (ETEC) strains . Among 76 EPEC strains belonging to 14 different serotypes, msDNA was found to be present in 7 serotypes . In total, five different types of msDNA were found, although within each serotype, the msDNAs were the same . These results suggest that different retrons are clonally inherited. Mol Microbiol, 1990 Oct, 4(10), 1661 - 6 HeLa cell invasion by a strain of enteropathogenic Escherichia coli that lacks the O-antigenic polysaccharide; Riley LW et al.; The interaction with HeLa cells of an enteropathogenic Escherichia coli (EPEC) strain and its plasmid-cured derivative strain was examined . An O111:NM EPEC strain B171 harbours a 54 megadalton plasmid (pYR111) necessary for the expression of both localized adherence (LA) to HeLa cells and the O-repeating side chain of the lipopolysaccharide . Under light microscopy, the plasmid-cured derivative strain B171-4 was observed to interact with HeLa cells in a pattern distinct from LA . Transmission electron microscopy showed that the bacteria were internalized by HeLa cells . In contrast, strain B171 induced pedestal-like projections and invaginations of the plasma membrane, but was never completely internalized . A quantitative assay to determine the number of internalized bacteria revealed that strain B171-4 was internalized at levels 30-70-fold higher than those of avirulent E . coli strains . Cytochalasin B reduced the levels of internalization of both strain B171-4 and an enteroinvasive E . coli strain (E11), but did not affect LA by strain B171 . These results suggest that EPEC strain B171 may carry a specific chromosomally determined surface factor needed to initiate internalization by HeLa cells . However, a plasmid-determined factor alters the nature of this interaction; the combined effects of the chromosomal and plasmid determinants lead to the characteristic attachment of the bacteria in clusters on the surface of the eukaryotic cell. FEMS Microbiol Lett, 1990 Oct, 60(1-2), 117 - 22 Monoclonal antibodies against serotype specific and conserved epitopes in morphotype E Escherichia coli flagellins; Schoenhals G et al.; Monoclonal antibodies (mAbs) were used to examine the interrelationships between morphologically identical flagellar filaments from Escherichia coli H serotype strains belonging to morphotype E . Serotype specific mAbs recognised epitopes exposed on the surface of flagellar filaments from H1, H7, H23, H49 and H51, but were inaccessible to immunolabelling in H45 . Several mAbs which recognised conserved epitopes were also examined . mAb 7-56.1 recognised an epitope present in all morphotype E flagellins but not expressed on the filament surface . Similarly, mAb 1-5.1 recognised an internal epitope shared only by serotypes H1 and H12 . Serotype H23 expressed a surface epitope which was present but not surface exposed in H7, H1 and H45 filaments. Nippon Juigaku Zasshi, 1990 Oct, 52(5), 1023 - 7 Serotyping of O and pilus antigens of Escherichia coli strains isolated from chickens with coli-septicemia; Ike K et al.; One hundred and fifty one Escherichia coli strains were isolated from broiler chickens with coli-septicemia in Aichi (63 strains), Shizuoka (58 strains), and Kagoshima (30 strains) prefectures from 1980 to 1987, and their O and pilus antigens were serologically typed . One hundred and twenty five strains (82.8%) were typed into 23 O serogroups, and twenty six strains (17.2%) remained untypable . The predominant O serogroups were O2 (35 strains, 23.2%) and O78 (24 strains, 15.9%) . Distribution of O serogroup was different, depending on prefectures where they are isolated . In total, 109 strains (72.2%) possessed Type 1 and/or Fmsha pili (Type 1; 41 strains, Fmsha; 22 strains, and Type 1 and Fmsha; 46 strains), and 42 strains (27.2%) were non-piliated . All the strains lacked K88, K99, 987P, F41, and Att25 pili . The ratios of piliated strains to non-piliated ones were almost the same among the three prefectures . Strains possessing Type 1 pili showed variety of O antigens, but most of the strains with Fmsha pili belonged to O2 serogroup. J Cell Sci, 1990 Oct, 97 ( Pt 2), 317 - 24 Tailless keratins assemble into regular intermediate filaments in vitro; Hatzfeld M et al.; To study the influence of the non alpha-helical tail domain of keratins in filament formation, we prepared a truncated keratin 8 mutant, K8/tailless . Using site-directed in vitro mutagenesis we introduced a stop codon in the position coding for amino acid number 417 of the K8/wild-type sequence, thereby deleting 86 amino acids of the non alpha-helical tail domain but leaving the consensus sequence at the end of the rod domain intact . Expression of the truncated keratin 8 in Escherichia coli allowed us to purify the protein by a two-step procedure . The filament-forming capacity of the truncated K8 with wild-type K18 and K19 was analyzed using in vitro reconstitution . The in vitro assembly studies with K8/tailless and K18 wild-type indicate that the C-terminal tail domain of a type II keratin, including the homologous subdomain H2, is not required for filament formation . Moreover, reconstitution experiments with K8/tailless and K19, a naturally occurring tailless keratin I, show that the tail domains of type I as well as type II keratins are not an essential requirement for in vitro filament formation . Our results suggest that in vitro filament elongation does not depend on interactions between head and tail domains, although the tail domain might have a role in stabilization of intermediate filaments arising from certain keratin pairs. J Gen Microbiol, 1990 Oct, 136 ( Pt 10), 2013 - 20 Isolation of recombinant fragments of the major outer-membrane protein of Chlamydia trachomatis: their potential as subunit vaccines; Conlan JW et al.; Recombinant fragments of the major outer-membrane protein (MOMP) of Chlamydia trachomatis, expressed at high levels in Escherichia coli, were isolated and purified . Antisera to the recombinant proteins reacted preferentially with overlapping synthetic peptides covering the immunoaccessible variable segments of MOMP . These sera also reacted in a species-specific manner with the surface of intact infectious elementary bodies, and in a Chlamydia genus-specific manner in assays using denatured or bound chlamydial antigens . The ability of recombinant MOMP preparations to elicit antibody to the surface of chlamydial elementary bodies raises the possibility that these proteins may be useful for chlamydial vaccine development. AIDS, 1990 Oct, 4(10), 953 - 60 Neutralizing cross-reactive and non-neutralizing monoclonal antibodies to HIV-1 gp120; Akerblom L et al.; Amino acid sequences inducing neutralizing antibodies to HIV-1 were sought . Murine monoclonal antibodies (MAbs) were characterized by their reactivity with the envelope precursor gp160 or the Escherichia coli recombinant DNA products pB1 and pE3 representing the carboxy- and amino-terminal halves of mature envelope gp120 . Fine mapping of the MAb determinants was performed using defined 15-mer synthetic peptides spanning the entire envelope gp120 region of HIV-1 . One group of MAbs recognizes epitopes (amino acids 304-323) occurring in a small region with variable and conserved amino acid sequences of gp120 . These MAbs mediate neutralization of the HIV-1 strain HTLV-IIIB (HIV-1IIIB) which was used for immunization . Nine out of 11 primary HIV-1 isolates were neutralized well or moderately well . In addition, prominent serological reactivity was noted with peptide sequences of strains of various European or American origins, but not with two HIV-1 strains of African origin . The cross-reactivity contrasts with previously described type-specific reactions to other sequences of this region . The reactivity to the short conserved site GPGR with its flanking amino acids may explain the broad sequence cross-reactivity seen with our neutralizing MAbs . Two other MAbs recognize conserved epitopes (amino acids 79-103) situated in the amino-terminal region of gp120 . These MAbs did not neutralize HIV-1IIIB. J Neurosci Res, 1990 Oct, 27(2), 144 - 52 Isolation of cDNAs from a mouse astroglial cell line by a subtracted cDNA library; Rhyner TA et al.; Astrocytes belong to the glial cell population and represent a major subclass of the CNS . Although different subtypes of astrocytes have been described according to their morphological characteristics, biochemical markers of each subtype of astrocytes are not yet available . We have thus undertaken to compare gene expression pattern of different astroglial subtypes . In this study we have taken advantage of two astroglial cell clones derived from 8 day postnatal mouse cerebellar explants and which might be the in vitro equivalents of the velate protoplasmic (D19) and of the Golgi-Bergmann (C8S) astrocytes (Alliot and Pessac, Brain Res., 306: 283-291, 1984) . We have constructed a subtracted cDNA library derived from cytoplasmic poly(A)+ RNAs of the D19 cell line . This library was enriched 12-fold for D19 specific sequences by subtractive hybridization with an excess of cytoplasmic poly(A)+ RNAs purified from the C8S astroglial clone . This subtracted library was differentially screened with cDNA probes derived from D19 and C8S cell lines; both probes were subtracted with C8S poly(A)+ RNAs . Eight cDNA clones corresponding to transcripts overexpressed in D19 were selected . Three cDNAs encode for smooth muscle actin, one for fibronectin and one for polyadenylate binding protein . The three other gene products have not been previously reported . The in vivo distribution pattern of these sequences in various mouse adult tissues shows that all these transcripts are expressed in the cerebellum and/or in the brain. Genes Dev, 1990 Oct, 4(10), 1801 - 10 Pseudo-templated transcription by Escherichia coli RNA polymerase at a mutant promoter; Jacques JP et al.; A G----T mutation at the start-point of transcription of the phage P22 sar promoter (sar + 1T) causes a novel defect in promoter clearance by Escherichia coli RNA polymerase (RNAP) in vitro . Under standard transcription conditions, in the presence of high concentrations of all four NTPs, the predominant products from this promoter are poly(U) chains of varying length . Because the mutation creates a run of four T: A base-pairs from - 1 to +3 (TGTT----TTTT), we propose that synthesis of poly(U) is pseudo-templated by the A4 stretch on the template strand . G----A and G----C mutations at position +1 do not cause pseudo-templated transcription . Several molecules of poly(U) are produced and released per sar+1T promoter-polymerase complex without dissociation of RNAP from the template DNA . The exponential relationship between yield and size of individual poly(U) species indicates that there is a constant probability that another U residue will be added to the nascent chain . Presumably, pseudo-templated transcription occurs by a slippage (stuttering) mechanism like that proposed to explain certain kinds of RNA editing in eukaryotic viral mRNAs. Genes Dev, 1990 Oct, 4(10), 1790 - 800 Domains of initiator tRNA and initiation codon crucial for initiator tRNA selection by Escherichia coli IF3; Hartz D et al.; Initiation factors are used by Escherichia coli to select the initiator tRNA over elongator tRNAs during translation initiation . IF3 appears to "inspect" the anticodon end of the tRNA, probably along with the initiation codon . The anticodon stem and loop of the initiator tRNA, together with part of the initiation codon of the mRNA, can be thought of as a unit . Changes made in the anticodon stem, the anticodon loop, or the anticodon of an initiator tRNA fragment result in a loss of selection by IF3 in an in vitro assay for translation initiation . IF3 allows the selection of an initiator tRNA anticodon stem and loop fragment on GUG and UUG codons but does not select that tRNA fragment in response to AUU. Am J Trop Med Hyg, 1990 Oct, 43(4), 373 - 9 Bb65, a major immunoreactive protein of Bartonella bacilliformis; Knobloch J et al.; A 65 kDa protein (Bb65) has been identified as one of the major specific antigens of Bartonella bacilliformis, the causative agent of bartonellosis which is a bacterial infectious disease of inhabitants of the Andes . The gene encoding this antigen (7B2) was isolated from an expression library made directly from randomly generated fragments of B . bacilliformis genomic DNA using Bartonella antibodies raised in rabbits and sera of bartonellosis patients . The Bartonella 7B2 gene was expressed in Escherichia coli and the recombinant Bb65 protein was purified by column chromatography . Using polyclonal antibodies raised in rabbits, the antigen was shown to be present in all of 13 B . bacilliformis isolates from different Peruvian regions . Immune electron microscopy demonstrated the probable cytoplasmatic localization of Bb65 . When applied to enzyme immunoassays, Bb65 sensitively and specifically bound to IgG antibody of sera of bartonellosis patients, convalescents, and immunes from various Peruvian regions . IgM antibody was not recognized by Bb65, neither was IgG antibody circulating during the first 2 weeks of illness . The amino-terminal amino acid sequence of Bb65 was 53% homologous to the 65 kDa heat shock protein of Mycobacterium tuberculosis. Proc Natl Acad Sci U S A, 1990 Oct, 87(20), 8105 - 9 Cleavage of tRNA within the mature tRNA sequence by the catalytic RNA of RNase P: implication for the formation of the primer tRNA fragment for reverse transcription in copia retrovirus-like particles; Kikuchi Y et al.; The retrovirus-like particles of Drosophila are intermediates of retrotransposition of the transposable element copia . In these particles, a 39-nucleotide-long fragment from the 5' region of Drosophila initiator methionine tRNA (tRNA(iMet) is used as the primer for copia minus-strand reverse transcription . To function as primer for this reverse transcription, the Drosophila tRNA(iMet) must be cleaved in vivo at the site between nucleotides 39 and 40 . When a synthetic Drosophila tRNA(iMet) precursor was incubated with M1RNA, the catalytic RNA of Escherichia coli RNase P, other cleavages within the mature tRNA sequence were detected in addition to the efficient removal of the 5' leader sequence of this tRNA precursor . One of these cleavage sites is between nucleotides 39 and 40 of Drosophila tRNA(iMet) . Based on this result, we propose a model for formation of the primer tRNA fragment for reverse transcription in copia retrovirus-like particles. Mol Immunol, 1990 Oct, 27(10), 973 - 80 Mapping of epitopes on the 86 kDa subunit of the Ku autoantigen; Wen J et al.; Patients with rheumatic disorders develop autoantibodies to a nuclear protein antigen termed Ku . The Ku antigen has been identified as a non-histone DNA-binding protein complex composed of two polypeptides: 86 kDa and 70 kDa . Initial competition experiments with four monoclonal antibodies specific for the 86 kDa subunit of Ku protein suggested there was more than one epitope on this polypeptide . To determine the region for these epitopes, cDNA deletions were made from both 5' and 3' ends . The immunoreactivity of the expressed proteins was assayed by immunoblotting . A 40 amino acids region located near the C-terminus of the polypeptide between amino acids 667-708 was essential for binding the monoclonal and autoimmune antibodies . Within this region, we differentiated three epitopes for the monoclonal antibodies . The sequence critical for binding of the autoimmune antibodies was in the same region, between amino acids 689-708 . None of the four monoclonal antibodies blocked the binding of the autoantibodies . The implications of these results for the etiology of the anti-Ku autoantibody response are discussed. J Bacteriol, 1990 Oct, 172(10), 6042 - 7 Sequence and expression of the Escherichia coli recR locus; Yeung T et al.; The Escherichia coli RecR protein participates in a recombinational DNA repair process . Its gene is located in a region of chromosome that extends from 502 to 509 kilobases on the physical map and that contains apt, dnaX, orf12-recR, htpG, and adk . Most, if not all, of these are involved in nucleic acid metabolism . The orf12-recR reading frames consist of 935 base pairs and overlap by one nucleotide, with the 3' A of the orf12 termination codon forming the 5' nucleotide of the recR initiation codon . The orf12-recR promoter was located upstream of orf12 by sequence analysis, promoter cloning, and S1 nuclease protection analysis . The start point of transcription was determined by primer extension . The transcript 5' end contained a long, apparently untranslated region of 199 nucleotides . Absence of a detectable promoter specific for recR and the overlap of the orf12 and recR reading frames suggest that translation of recR is coupled to that of orf12 . By maxicell analysis, it was determined that both orf12 and recR are translated. EMBO J, 1990 Oct, 9(10), 3337 - 42 Isolation and structure of a cDNA expressing a mammalian 3-methyladenine-DNA glycosylase; O'Connor TR et al.; A cDNA plasmid expression library was constructed from the poly(A)+ mRNA of H4 cells, a rat hepatoma cell line . The library was introduced into Escherichia coli strain BH290 deficient in the repair of 3-methyladenine (3-meAde) residues in DNA . This DNA repair deficiency renders the stain phenotypically sensitive to treatment with alkylating agents . The cDNA library was screened for survivors to methylmethane sulfonate . BH290 cells hosting one of the plasmids, pAPDG10 (Alkylated N-Purine-DNA Glycosylase), from surviving cells had a sensitivity to MMS equivalent to that of the wild type strain . Crude extracts of BH290 cells harboring the pAPDG10 plasmid released 3-meAde and 7-methylguanine residues from DNA methylated with {methyl-3H}dimethylsulfate . The cDNA sequence of 993 bp inserted in pAPDG10 has a single open reading frame greater than 85 amino acids in length . The derived APDG protein sequence of 253 amino acids and the 3-meAde-DNA glycosylase II of E . coli coded for by the alkA gene have regions of conserved sequences . Analysis of the genomic DNA using Southern hybridization suggests that the APDG gene has a minimal size of 6.5-12 kb . Northern blot analysis shows that the transcript produced in H4 cells is also present in normal rat liver cells. J Biotechnol, 1990 Oct, 16(1-2), 49 - 55 A family of expression vectors based on the rrnB P2 promoter of Escherichia coli; Lukacsovich T et al.; We describe here the construction of a family of expression vectors, based on the P2 promoter of the Escherichia coli rrnB gene by removing regulatory sequences downstream of the Pribnow-box and replacing them with the lac operator . These vectors allow cloning of foreign genes in such a way that their products are synthesized either in the form of fusion proteins of different length, or without fusion partners, with or without the original translational initiation signals . One of the vectors contains a synthetic oligothreonine-coding sequence that helps to stabilize the product of the cloned gene . These vectors allow high-level regulated expression of foreign genes, even if their products are relatively short peptides. Biotechnology (N Y), 1990 Oct, 8(10), 945 - 9 Removal of a proteolytic activity associated with aggregates formed from expression of creatine kinase in Escherichia coli leads to improved recovery of active enzyme; Babbitt PC et al.; Expression of creatine kinase (CK) from a Torpedo californica electric organ cDNA in Escherichia coli results in an insoluble protein product with no detectable CK activity . Although this is a stable aggregate that can be isolated in an enriched form by centrifugation, initial attempts to generate enzyme activity by denaturing and refolding yielded only minute amounts of active protein . We find that these low recoveries are due to proteolysis of the CK during denaturation and refolding . While this proteolytic activity is not inhibited by either phenylmethanesulfonyl fluoride (PMSF) or EDTA, it can be largely removed from the CK aggregate by extraction with a detergent-containing buffer prior to denaturation . This treatment improves the recovery of active CK approximately 100-fold . We have also found similar proteolytic activity associated with the aggregate formed when a mutant of bovine pancreatic trypsin inhibitor (BPTI) is expressed in E . coli . Discovery of this proteolytic activity in two different expression systems suggests that it should be considered as a potential problem for recovery of active protein from other inclusion bodies as well. Agric Biol Chem, 1990 Oct, 54(10), 2611 - 7 Isolation of restriction-reduced mutants from Streptomyces; Kakinuma S et al.; Restriction-reduced mutants were isolated from Streptomyces rosa subsp . notoensis KA301 and S . tanashiensis strain Kala which produce the benzoisochromanequinone antibiotics nanaomycin and kalafungin, respectively . The mutants of S . rosa, which can be transformed with a multi-copy plasmid and in which the actinophage Pa16 can propagate, were selected . They were transformed with a single-copy plasmid propagated in S . lividans TK24, and with its modified plasmid propagated in the mutant at higher efficiency . The mutants of S . tanashiensis were selected by their capability to be transformed with a multi-copy plasmid . The efficiency of transformation with a single-copy plasmid propagated in S . lividans TK24 was low, but was much increased by heating the protoplasts at 42 degrees C for 15 min prior to the transformation . These mutants derived from both strains probably lack at least one of their restriction systems. Biotechnology (N Y), 1990 Oct, 8(10), 956 - 8 A novel screening system for yeast strains capable of secreting tissue plasminogen activator; Gill GS et al.; We have developed a simple screening procedure that allowed us to identify Saccharomyces cerevisiae strains able to secrete human tissue plasminogen activator (tPA) into the culture medium . The screen can be used to isolate more efficient secretor strains and to look for novel tPA analogs . Employing one of these strains to study the effect of glycosylation on secretion, we show that glycosylation in the catalytic domain of tPA plays an important role in folding and/or secretion of the molecule . Removing this glycosylation site resulted in a 3-5-fold reduction in the level of tPA secretion . We anticipate that this system will prove useful in studying yeast secretory pathway as well as structure-function relationships in the tPA molecule. Biochem Biophys Res Commun, 1990 Sep 28, 171(3), 1224 - 8 Catalase HPI influences membrane permeability in Escherichia coli following near-UV stress; Leven S et al.; The katG gene in Escherichia coli encodes catalase HPI, which is involved in membrane transport and protects the cell during oxidative stress . Hydrogen peroxide (H2O2) induces synthesis of HPI . We examined the role of HPI in membrane permeability (proline uptake) following exposure to near-ultraviolet radiation (NUV) . We found that NUV resulted in the same type of induction as H2O2 . KatG::Tn10 cells experienced a large drop in uptake after NUV exposure, and levels remained low following incubation . A strain carrying a katG+ plasmid, however, showed considerably less decrease in uptake after NUV, and uptake quickly resumed upon incubation . Further, in an srd mutant which lacks 4-thiouracil, NUV resulted in only a small drop in proline uptake, which was immediately resumed.
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