Nucleic Acids Res, 1993 Mar 11, 21(5), 1199 - 203 Interaction of Escherichia coli ribosomal protein S7 with 16S rRNA; Dragon F et al.; The interaction between Escherichia coli ribosomal protein S7 and 16S rRNA was investigated using in vitro synthesized RNA transcripts . It was shown by nitrocellulose membrane filtration that RNA transcripts corresponding to the 3' major domain (nucleotides 926-1393) and to the lower half of this domain (nucleotides 926-986/1219-1393) bound S7 with the same affinity as 16S rRNA . A series of deletion mutants of the DNA coding for the lower half of the 3' major domain were constructed and the corresponding RNA fragments were assayed for their capacity to bind S7 . A minimal domain of 108 nucleotides which can still efficiently bind S7 was thus obtained . In this domain, the 1304-1308/1329-1333 irregular helix and the 1351-1371 irregular hairpin were found to contain important determinants for S7 binding. Biochim Biophys Acta, 1993 Mar 10, 1176(1-2), 90 - 4 Superoxide dismutase and thioredoxin restore defective p34cdc2 kinase activation in mouse two-cell block; Natsuyama S et al.; We recently showed that superoxide dismutase (SOD), a free radical scavenger, and thioredoxin, a potent protein disulfide reductase, release mouse two-cell stage developmental block in vitro . To elucidate the mechanism underlying the two-cell block and the effects of these enzymes, we studied the chronological changes in the kinase activity and the immunoblotting pattern of p34cdc2, a key regulator of the cell cycle, during the first and second cell cycles of the mouse embryonic development . In vivo embryos were freshly collected at fixed times, and in vitro embryos cultured from the pronuclear stage were also sampled with the same time-course . A marked elevation of p34cdc2 kinase activity was observed in vivo at 27-32 and 51 h after an injection of human chorionic gonadotropin . These times coincide with the M-phases of embryo cleavage . In vitro embryos showed high kinase activity during the M-phase of the first cleavage, but this activity was not elevated during the second cell cycle . The addition of recombinant human SOD (200 micrograms/ml) or thioredoxin from Escherichia coli (500 micrograms/ml) to the medium enabled kinase activation with a time course similar to that of in vivo embryos . The immunoblotting patterns suggested the dephosphorylation of p34cdc2 at the M-phase of the first and the second cleavages in vivo . Although p34cdc2 was dephosphorylated at the M-phase of the first cleavage and then rephosphorylated for embryos cultured in vitro, the second dephosphorylation was not observed during the second cell cycle . The addition of SOD or thioredoxin permitted the dephosphorylation at the M-phases of both the first and the second cleavage . These results suggest that one of the chief causes of two-cell block in vitro is the impairment in p34cdc2 dephosphorylation, recently shown to be catalyzed by the cdc25 homologue . This impairment is thought to be due to oxidative stress, because both SOD and thioredoxin are known to play a defensive role against it. Biochemistry, 1993 Mar 9, 32(9), 2423 - 30 Alignment of disulfide bonds of the extracellular domain of the interferon gamma receptor and investigation of their role in biological activity; Stuber D et al.; The extracellular ligand binding domain of the human interferon gamma receptor includes eight cysteine residues forming four disulfide bonds . Only the nonreduced protein binds interferon gamma . We investigated the alignment of the disulfide bonds, using an enzymatically deglycosylated form of a soluble interferon gamma receptor, produced in baculovirus-infected insect cells . The soluble receptor was digested with endoproteinase Glu-C and proteinase K, and the proteolytic fragments were characterized by amino acid sequence analysis and mass spectrometry . It was found that four consecutive disulfide bonds are formed between residues Cys60-Cys68, Cys105-Cys150, Cys178-Cys183, and Cys197-Cys218 . We also investigated the role of the disulfide bonds in biological activity of the receptor, using site-directed mutagenesis and by exchanging the cysteine residues for serines . The mutated proteins were expressed in Escherichia coli and analyzed for ligand binding capacity on protein blots . The assays showed that all disulfide bonds are essential for full ligand binding capacity . Double or quadruple mutations at cysteine residues 60 and 68, and residues 178, 183, 197, and 218, respectively, resulted in complete loss of the activity, whereas double mutations at residues 105 and 150, 178 and 183, and 197 and 218, respectively, resulted in a residual activity about 1 order of magnitude lower than that of the wild type . The specific antibodies gamma R38 and gamma R99 detected conformational epitopes stabilized by disulfide bonds involving cysteine residues 60 and 68, and 178 and 183, respectively. Biochemistry, 1993 Mar 9, 32(9), 2194 - 201 Binding of a protein-tyrosine phosphatase to DNA through its carboxy-terminal noncatalytic domain; Radha V et al.; The noncatalytic domain of a non-receptor-type protein-tyrosine phosphatase (the T-cell phosphatase or PTP-S) isolated from a rat spleen cDNA library shows homology with the basic domains of transcription factors Fos and Jun {Swarup, G., Kamatkar, S., Radha, V., & Rema, V . (1991) FEBS Lett . 280,65-69} . We have expressed this phosphatase in Escherichia coli under the control of T7 promoter . The PTP-S gene product expressed in E . coli shows protein-tyrosine phosphatase activity and binds to DNA at pH 7.4 as determined by DNA affinity chromatography, Southwestern blotting, and gel retardation methods . The carboxy-terminal region of this phosphatase was fused with glutathione S-transferase by constructing expression vectors . Experiments using fusion proteins with glutathione S-transferase suggest that the carboxy-terminal 57 amino acids of PTP-S are sufficient for DNA binding . Deletion of the C-terminal 57 amino acids of PTP-S protein abolished its DNA binding property, as determined by Southwestern blotting, but not its enzymatic activity . This suggests that the C-terminal 57 amino acids are essential for the DNA binding function of this protein but not for its enzymatic activity . Another non-receptor-type protein-tyrosine phosphatase, PTP-1, when expressed in enzymatically active form in E . coli did not bind to DNA . These results suggest that a nontransmembrane protein-tyrosine phosphatase, PTP-S, binds to DNA in vitro through its carboxy-terminal noncatalytic region. FEBS Lett, 1993 Mar 8, 318(3), 331 - 4 Dephosphorylation of human p34cdc2 kinase on both Thr-14 and Tyr-15 by human cdc25B phosphatase; Honda R et al.; In mammalian cells, p34cdc2 kinase undergoes phosphorylation at threonine-14, tyrosine-15 and threonine-161 in the S and G2 phases of the cell cycle . At the onset of mitosis, the kinase becomes dephosphorylated at threonine-14 and tyrosine-15, resulting in activation . Cdc25 phosphatase has been shown to dephosphorylate tyrosine-15 in vitro, but whether it also does at threonine-14 remains unclear . In this study, we have found that human cdc25B phosphatase dephosphorylates both threonine-14 and tyrosine-15 but not threonine-161. FEBS Lett, 1993 Mar 8, 318(3), 310 - 2 Characterization of the alternative oxidase protein in the yeast Hansenula anomala; Sakajo S et al.; The cyanide-resistant respiratory pathway is induced by respiratory inhibitors in the yeast Hansenula anomala . A monoclonal antibody against the alternative oxidase in the higher plant Sauromatum guttatum cross-reacted with a 36-kDa mitochondrial protein induced by antimycin A in H . anomala and with a protein encoded by a cDNA which was previously cloned for an antimycin A-inducible mRNA in the yeast . There was a similarity in the amino acid sequence between the cDNA-encoded protein and the plant alternative oxidase protein . We propose that the 36-kDa mitochondrial protein encoded by the cDNA is a component of alternative oxidase in H . anomala. FEBS Lett, 1993 Mar 8, 318(3), 297 - 300 v-Ha-Ras insertion/deletion mutants with reduced protease-inhibitory activity have no transforming activity; Sawada T et al.; We have purified 26 insertion/deletion mutants of v-Ha-ras oncogene products produced by Escherichia coli and investigated their protease-inhibitory activity toward papain and cathepsins B and L . Ki values for papain were relatively similar among the mutants, however, those for cathepsins B and L varied up to 10-fold . Among them, four mutants, 1-48 LIR 54-189, 1-110 LIS 112-189, 1-130 PDQ 146-189 and 1-155 LIR 166-189, showed significant reduction in the inhibitory activity toward cathepsin L and these four mutants have lost transforming activity toward NIH3T3 mouse fibroblasts . However, some other mutants also showed no transforming activity in spite of possession of the potent protease-inhibitory activity, suggesting that the protease-inhibitory activity of Ras might be necessary but not sufficient for its biological activity. Science, 1993 Mar 5, 259(5100), 1415 - 20 Molecular matchmakers; Sancar A et al.; Molecular matchmakers are a class of proteins that use the energy released from the hydrolysis of adenosine triphosphate to cause a conformational change in one or both components of a DNA binding protein pair to promote formation of a metastable DNA-protein complex . After matchmaking the matchmaker dissociates from the complex, permitting the matched protein to engage in other protein-protein interactions to bring about the effector function . Matchmaking is most commonly used under circumstances that require targeted, high-avidity DNA binding without relying solely on sequence specificity . Molecular matchmaking is an extensively used mechanism in repair, replication, and transcription and most likely in recombination and transposition reactions, too. J Mol Biol, 1993 Mar 5, 230(1), 41 - 50 Release factor-dependent false stops are infrequent in Escherichia coli; Jorgensen F et al.; We have estimated the frequency of release factor dependent events in which a sense codon is mistakenly translated as a stop codon . We refer to this event as a "false stop" . In order to facilitate the measurement of false stop freqeuncies we have used a plasmid expression system to increase individually the cellular levels of release factor (RF) I a and of release factor (RF) 2 . We were then able to measure the loss of translational processivity with the aid of a lacZ processivity assay at different concentrations of the release factors . We find that a 30- to 40-fold increase of the RF1 concentration reduces lacZ processivity from 0.6 to 0.3 . Assuming that the processivity loss is due only to false stops and that the RF1 overproduction data can be extrapolated back linearly to the normal RF1 concentration in the cell, this corresponds to a false stop frequency close to 10(-5) per codon in the presence of normal amounts of RF1 . Furthermore, a threefold increase of the RF2 concentrations had no measurable effect on the processivity of the lacZ gene . Our data suggest that false stops are relatively infrequent compared to the incidence of other translation errors. J Mol Biol, 1993 Mar 5, 230(1), 161 - 73 In vivo thermodynamic analysis of repression with and without looping in lac constructs . Estimates of free and local lac repressor concentrations and of physical properties of a region of supercoiled plasmid DNA in vivo; Law SM et al.; A strong-binding primary (O1) lac operator located 100 to 200 base-pairs (bp) upstream from a lac promoter control region reduces expression from a lac promoter controlled by a weaker-binding (Oc) lac operator between 3 and 20-fold on a multicopy plasmid in E . coli . We attribute this effect to loop formation in which a thermodynamically stable complex is formed between bidentate lac repressor tetramers and the O1 and Oc operators . A thermodynamic model for repression is developed to interpret these data in terms of the composite effects of free lac repressor concentration and of local repressor concentration (from looping) at the Oc site . The local repressor concentration is found to vary periodically with the distance in base-pairs between the O1 and the Oc operators, ranging from 2 to 20-fold larger than the free concentration (i.e . the bulk thermodynamic activity) of repressor in this F'Iq overproducing strain (estimated to be approximately less than 0.5 microM) . The amplitude of the periodic variation in expression and in local concentration appears to decrease with increasing interoperator distance in the range examined . Quantitative analysis of the looping data provides estimates of the physical properties of the intervening DNA region in vivo . For distances in the range 127 to 197 bp, the periodicity of modulation is uniformly 11.28(+/- 0.04) bp, which we interpret as the helical repeat of this region of supercoiled plasmid DNA in vivo . Possible origins of this altered helical repeat include the global linking deficit of the supercoiled DNA and any local linking deficit induced by divergent transcription from promoters bracketing the interoperator region . DNA cyclization analysis yields an apparent in vivo persistence length of this interoperator region of 64(+/- 26) A (which is approximately 15% of the in vivo result) and an in vivo torsional rigidity constant of 1.1(+/- 0.1) x 10(-19) erg cm, which is at the lower end of the range of values found in vitro. J Mol Biol, 1993 Mar 5, 230(1), 1 - 5 On the role of the P-site in leftward ribosome frameshifting at a hungry codon; Kolor K et al.; Previous work characterized ribosomal frameshifting within the sequence C UUC AAG provoked by lysyl-tRNA limitation . The ribosome frameshift is one base to the left of the AAG lysine codon, as shown by dotted overlining above . We now show that the frequency of this leftward ribosome frameshift is strongly influenced by the identity of the bases two, three and four positions to the left of the actual frameshift site . The nature of these influences coincides exactly with the possibilities of base-pairing between the sequence and the anticodon of the P-site peptidyl-tRNA when shifted one base to the left just upstream of the frameshift site . We conclude that a peptidyl shift in the P-site is intimately involved in leftward frameshifting in the adjacent A site when it codes for an aminoacyl-tRNA in short supply. Biochim Biophys Acta, 1993 Mar 5, 1162(1-2), 84 - 8 Introduction of Ca(2+)-binding amino-acid sequence into the T4 lysozyme; Leontiev VV et al.; The 51-62 loop of T4 phage lysozyme was altered by site-directed mutagenesis to obtain maximal homology with the typical EF-hand motif . A Ca(2+)-binding site was designed and created by replacing both Gly-51 and Asn-53 with aspartic acid . The mutant T4 lysozyme (G51D/N53D) was expressed in Escherichia coli . The activity of the G51D/N53D-mutant was about 60% of that of the wild-type protein . This mutant can bind Ca2+ ions specifically, while the effective dissociation constant was essentially greater than that of the EF-hand proteins . Stability of the G51D/N53D-mutant apo-form to urea- or temperature-induced denaturation was the same as that of the wild-type protein . In the presence of Ca2+ ions in solution the stability of the mutant T4 phage lysozyme was less than that of the wild-type protein . It is suggested that the binding of Ca2+ by the mutant is accompanied by the considerable conformational changes in the 'corrected' loop, which can lead to the Ca(2+)-induced destabilization of the protein. Biochim Biophys Acta, 1993 Mar 5, 1162(1-2), 35 - 9 Resistance of archaebacterial aEF-1 alpha.GDP against denaturation by heat and urea; Masullo M et al.; The elongation factor aEF-1 alpha, isolated as aEF-1 alpha.GDP from the thermoacidophilic archaebacterium Sulfolobus solfataricus, exchanges GDP for {3H}GDP at a rate which reaches a maximum at 95 degrees C . The rate constants at different temperatures of the heat inactivation of aEF-1 alpha.GDP are considerably lower compared to those referred to Escherichia coli EF-Tu.GDP . The Tm values determined for both aEF-1 alpha.GDP and EF-Tu.GDP are 97 and 53 degrees C, respectively . The addition of GDP during the heat treatment protects significantly EF-Tu.GDP but only slightly aEF-1 alpha.GDP . The ability of aEF-1 alpha.GDP to exchange GDP for {3H}GDP is impaired at 70 degrees C by urea at concentrations which are greater compared to those required to inactivate E . coli EF-Tu.GDP at 45 degrees C; apparently both factors are not protected by GDP against inactivation by urea. J Biol Chem, 1993 Mar 5, 268(7), 5323 - 31 Mutations in the helix-turn-helix motif of the Escherichia coli UvrA protein eliminate its specificity for UV-damaged DNA; Wang J et al.; The Escherichia coli UvrA protein possesses a stretch of amino acids, 494 to 513, that matches the consensus sequence of the helix-turn-helix motif of many sequence-specific DNA binding proteins . It also has two zinc finger motif regions and two ATP binding sites . To study the potential roles of both helix-turn-helix and zinc finger motifs in the functioning of UvrA protein, random mutations were created in these motif regions by degenerate oligonucleotide-directed mutagenesis . Using this method, 12 single substitution mutants (eight in the helix-turn-helix motif region, one in the N-terminal zinc finger region, and three in the C-terminal zinc finger region) were isolated that failed to confer UV resistance in the E . coli strain deleted of the uvrA gene . One "hyper" UV-resistant mutant, G275A, was identified that conferred significantly more UV resistance than the wild type in the MH1-delta A strain . To further investigate the mechanism of failure of these mutant UvrA proteins to support nucleotide excision repair, two mutant UvrA proteins, G502D and V508D, were selected for purification and characterization, since they carry mutations at the positions offered as the putative constellation for the helix-turn-helix motif . The binding affinity of these two mutants for nonirradiated plasmid DNA was unaffected by the mutations . Both mutant proteins exhibited substantial ATPase activity, and together with the UvrB protein, they were capable of generating positively supercoiled plasmid DNA from the relaxed form in the presence of ATP and bacterial topoisomerase I . However, both mutant proteins failed to respond to UV damage in the filter binding assay and were incapable of forming 2 x SSC-resistant nucleoprotein complexes with UvrB protein on UV-irradiated plasmid DNA . Taking these properties together, it appears that the mutations in the helix-turn-helix motif region impaired the UvrA protein's ability to recognize UV damage, while its other activities were largely unaffected . Interestingly, ERCC-3, a human DNA repair protein, also has a similar helix-turn-helix motif . Given the highly conserved nature of repair proteins in general, this observation raises the possibility that both procaryotes and eucaryotes might use similar mechanisms to recognize damaged sites in their genomes. J Biol Chem, 1993 Mar 5, 268(7), 5209 - 19 Hexokinase II mRNA and gene structure, regulation by insulin, and evolution; Printz RL et al.; A DNA segment that is highly conserved in glucokinase (hexokinase IV) and hexokinase I cDNA was used to identify specific cDNAs in a library prepared from rat adipose tissue mRNA . Some of these cDNAs were identified as being hexokinase I cDNA . Others, although similar to both the glucokinase and hexokinase I cDNAs, were unique . Two of these unique cDNAs overlapped and contained an open reading frame that encoded a protein of 103 kDa which, when expressed in Escherichia coli, had kinetic properties characteristic of hexokinase II . The entire hexokinase II mRNA sequence and the exon-intron structure of the hexokinase II gene were determined . A single transcription initiation site and two distinct termination sites account for the two observed hexokinase II RNA species of 5500 and 4400 nucleotides that were detected when either of the cDNAs was used as a hybridization probe against poly(A)+ RNA isolated from rat adipose tissue . Hexokinase II mRNA was decreased in adipose tissue from diabetic rats, but was restored by insulin treatment to levels found in nondiabetic control rats . Insulin also induced hexokinase II mRNA in two adipose cell lines (3T3-F442A and BFC-1B) and two skeletal muscle cell lines (C2C12 and L6) . In L6 cells, this increase was accounted for by a corresponding increase of hexokinase II gene transcription . Comparison of the structures of the hexokinase II and glucokinase genes support the hypothesis that the 100-kDa hexokinase arose by gene duplication and tandem ligation of a 50-kDa glucokinase-like ancestral gene. J Biol Chem, 1993 Mar 5, 268(7), 5201 - 8 Membrane topology and biogenesis of eukaryotic signal peptidase; Shelness GS et al.; The signal peptidase complex (SPC) is a hetero-oligomeric membrane protein containing subunits of 12, 18, 21, 22/23, and 25 kDa . The 18- and 21-kDa subunits are mammalian homologs of SEC11 protein, which is necessary for signal peptide processing and cell viability in the yeast Saccharomyces cerevisiae . The functional and/or structural contributions of the 12-, 22/23-, and 25-kDa subunits to SPC activity have not yet been elucidated . To explore the structure of SPC subunits and their relationships to signal peptide processing and protein translocation, we have examined their endoplasmic reticulum (ER) membrane topology and biogenesis . Signal peptidase activity and SPC subunits are resistant to protease treatment in intact and detergent-solubilized membranes . Heat-denatured SPC subunits and SPC subunits translated in vitro are, however, protease sensitive, suggesting that the assembly of the oligomeric complex confers protease resistance . To define the membrane topology of SPC subunits, both wild-type subunits and subunit fusion proteins containing additional sites for N-linked glycosylation were assembled into microsomal membranes in vitro . Despite the presence of multiple hydrophobic domains, each subunit is anchored to the ER membrane by a single amino-terminal transmembrane domain in an Ncytoplasmic Cexoplasmic (type II) orientation . This topology places the bulk of the protein mass in the ER lumen and positions a putative serine-containing active site domain in SPC 18 and 21 at the same relative distance from the membrane as the analogous region in Escherichia coli leader peptidase . These studies have also revealed that, in spite of the temporal and perhaps physical association of the SPC with the process of protein translocation, SPC subunits integrate into the ER membrane by a signal recognition particle-dependent pathway and, hence, rely on the existence of a preformed translocation apparatus for their own membrane assembly. J Biol Chem, 1993 Mar 5, 268(7), 5178 - 81 Iron specificity of the Fur-dependent regulation of the biosynthesis of the manganese-containing superoxide dismutase in Escherichia coli; Privalle CT et al.; The Fur protein, which regulates iron uptake in Escherichia coli, also represses the biosynthesis of the manganese-containing superoxide dismutase (MnSOD) . A strain of E . coli bearing a lacZ fusion to the aerobactin operon was used to compare the metal specificities of the regulation of MnSOD and of aerobactin by Fur . Iron, but not manganese, acted as a corepressor of the Fur-dependent inhibition of MnSOD biosynthesis . The iron-mediated inhibition of MnSOD biosynthesis was dependent upon an intact fur locus, indicating that the effect of iron is mediated by the fur gene product . The suppression of the accumulation of MnSOD by iron, but not by manganese, was not due to destabilization of the MnSOD polypeptide by iron . Thus this effect of iron was also seen in a sodA::lacZ operon fusion in which the production of beta-galactosidase was regulated by the sodA promoter . In contrast, both iron and manganese served as corepressors of aerobactin biosynthesis . It thus appears that the effectiveness of specific metal cations to act as corepressors with Fur varies with the gene being regulated by the Fur-metal complex. J Biol Chem, 1993 Mar 5, 268(7), 5048 - 54 Expression in Escherichia coli of the cloned flavin-containing monooxygenase from pig liver; Lomri N et al.; The amino acid sequence of pig liver flavin-containing monooxygenase (PgLFMO) was determined by nucleotide analysis of cDNA clones isolated using synthetic oligonucleotide probes . Most of the clones isolated were missing the 5'-noncoding region and the first seven codons including the putative initiation codon ATG . A full-length cDNA clone, PgLFMO1, was obtained by extending the 5'-end sequence to include an ATG initiation codon and the codons corresponding to the 5'-end using the polymerase chain reaction . Restriction sites for EcoRI and SalI were incorporated at the 5'- and 3'-ends of the cDNA, respectively, by polymerase chain reaction to provide a full-length cDNA clone, pTrcPgLFMO1 . Escherichia coli strain NM522 was transformed with pTrcPgLFMO1 and the PgLFMO was expressed under the control of the Trc promoter . Recombinant PgLFMO isolated from the bacterial lysates was purified to homogeneity and found to have N- and S-oxygenation kinetic properties similar to those of the native enzyme . Stereoselective S-oxygenation of (+)- and (-)-4-bromophenyl-1,3-oxathiolane or 2-methyl-1,3-benzodithiole by the expressed PgLFMO showed some differences from that of the native enzyme, however . These data indicate that active PgLFMO was expressed in E . coli but that the enzyme action was distinct from the native enzyme. J Biol Chem, 1993 Mar 5, 268(7), 5040 - 7 The TyrR protein of Escherichia coli, analysis by limited proteolysis of domain structure and ligand-mediated conformational changes; Cui J et al.; The TyrR protein of Escherichia coli K12 is a homodimer containing 513 amino acids/subunit . This protein is important in the transcriptional regulation of several genes whose protein products catalyze steps in aromatic amino acid biosynthesis or transport . Methods were developed for efficiently purifying the TyrR protein to apparent homogeneity . We analyzed the pattern of cleavage of the TyrR protein by trypsin, either in the absence of ligands or in the presence of saturating levels of L-tyrosine, ATP, or poly(dI-dC) . At low (1:200 ratio by weight) trypsin levels, in the absence of ligands, two major digestion products accumulated . These were polypeptides of 22 and 31 kDa, shown to contain amino acid residues 1-190 and 191-467, respectively . The pattern of trypsin cleavage was unaffected by tyrosine . In the presence of ATP, an intermediate species of 53 kDa, probably containing amino acid residues 1-467, was observed . The kinetics of appearance of the 53-kDa species were consistent with a role for ATP in accelerating the hydrolysis of the R467-F468 peptide bond . The 53-kDa polypeptide underwent further tryptic hydrolysis to yield fragments of 22 and 31 kDa . When both tyrosine and ATP were present, the rate of formation of the 22- and 31-kDa fragments was more rapid than in the absence of these ligands . It appears that when both ligands are bound, the rates of hydrolysis of peptide bonds R190-Q191 and R467-F468 are both enhanced . Additional limited proteolysis experiments suggested that polypeptide segment 191-467 contains ATP binding site(s), and that the rate of cleavage of peptide bonds R190-Q191 and R467-F468 is altered when the TyrR protein interacts with poly(dI-dC), an analog of target DNA . Our results reveal the presence of two major structural domains within the TyrR protein . The first domain (amino acid residues 1-190) is extremely resistant to hydrolysis by trypsin . The second domain (residues 191-467), which is likely to contain ATP-binding site(s), is homologous to several other transcriptional activators specific for promoters responsive to the sigma 54 form of RNA polymerase . The remainder of the TyrR protein (residues 468-513) contains the operator recognition elements, probably arranged in the form of a helix-turn-helix motif . This polypeptide segment was not detected as a discrete tryptic hydrolysis product. J Biol Chem, 1993 Mar 5, 268(7), 4880 - 8 Kinetic and regulatory mechanisms for (Escherichia coli) homoserine dehydrogenase-I . Equilibrium isotope exchange kinetics; Wedler FC et al.; Isotope exchange kinetics at chemical equilibrium were used to probe the mechanisms of substrate binding and regulatory behavior of homoserine dehydrogenase-I from Escherichia coli . At pH 9.0, 37 degrees C, Keq = 100 (+/- 20) for the catalyzed reaction: L-aspartate-beta-semialdehyde + NADPH + H+ = L-homoserine + NADP+ . Saturation curves for the exchange reactions, {14C}L-homoserine <--> L-aspartate-beta-semialdehyde and {3H}NADP+ <--> NADPH were observed as a function of different reactant-product pairs, varied in constant ratio at equilibrium . The NADP+ <--> NADPH exchange rate was inhibited upon variation of pairs involving L-aspartate-beta-semialdehyde and L-homoserine, consistent with preferred order random binding of cofactors before amino acids . Optimal rate constants, derived by simulations of equilibrium isotope exchange kinetics data with the ISOBI program, indicate faster dissociation of amino acids than cofactors from the central complexes but nearly equal rates for association of cofactors and amino acids to free enzyme . Rate limitation of net turnover in both directions is determined by dissociation of cofactor from the E-cofactor complex . The allosteric modifier, L-threonine, produces distinctive perturbations of the saturation curves for isotope exchange, which were analyzed systematically with the ISOBI program . The best fit to the data was obtained by L-threonine inhibiting catalysis between the central complexes without altering substrate association-dissociation rates . Simulations also showed that rate-limiting catalysis suppresses the kinetic inhibition effects that are characteristic of preferred order substrate binding, producing patterns typical for a (rapid equilibrium) random kinetic scheme. J Biol Chem, 1993 Mar 5, 268(7), 4775 - 9 Kinetic analysis of a mutant (His107-->Tyr) responsible for human carbonic anhydrase II deficiency syndrome; Tu C et al.; The replacement His107-->Tyr is a cause of carbonic anhydrase II deficiency syndrome in humans (Venta, P . J., Welty, R . J., Johnson, T . M., Sly, W . S., and Tashian, R . E . (1991) Am . J . Hum . Genet . 49, 1082-1090) . We have prepared this mutant of human carbonic anhydrase II by site-directed mutagenesis and expressed it in Escherichia coli . The mutant was too unstable to purify; however, we were able to stabilize and store it at 4 degrees C in cell lysates containing 1-4 mg/ml bovine serum albumin . The concentration of this mutant in the lysate was determined by titration with the tight-binding inhibitor ethoxzolamide . The stability in this preparation was sufficient to determine that this mutant of carbonic anhydrase II has kcat/Km and apparent pKa for the hydration of CO2 equivalent to that of wild-type HCA II . The maximum velocity of CO2 hydration, which is dependent on the rate of proton transfer between enzyme and solution, was 3-fold smaller than for HCA II suggesting that the proton transfer pathway in the mutant is slightly less efficient than in wild type . Preliminary conformational energy calculations show that the replacement of His107 with the larger residue Tyr results in considerable distortion of the cavity surrounding site 107 and in the loss of at least two hydrogen bonds. J Biol Chem, 1993 Mar 5, 268(7), 4728 - 33 The baculovirus Autographa californica encodes a protein tyrosine phosphatase; Sheng Z et al.; The genome of the baculovirus Autographa californica encodes a 19-kDa protein (BVP) containing an active site sequence motif ((I/V)HCXAGXXR(S/T)G) that characterizes a large family of protein tyrosine phosphatases (PTPs) . The baculoviral protein was expressed in Escherichia coli and purified so that its enzymatic properties could be examined . We have demonstrated that recombinant BVP has intrinsic protein tyrosine phosphatase activity . Like VH1, a PTP encoded by vaccinia virus, BVP also dephosphorylates seryl or threonyl residues . However, the similarity of BVP to VH1 or the catalytic domains from PTPs of eukaryotic origin is restricted to a small region surrounding the active site motif . In contrast, the similarity of BVP to two putative PTPs encoded by the CDC14 gene of Saccharomyces cerevisiae and a gene of unknown function from Caenorhabditis elegans extends throughout its sequence . We postulate that BVP and its two homologs constitute a unique subfamily that may differ from other PTPs in having a specialized function, mode of regulation, or substrate preference. J Biol Chem, 1993 Mar 5, 268(7), 5069 - 76 Overexpression in Escherichia coli and affinity purification of chick kidney ferredoxin; Tang C et al.; Vertebrate ferredoxins are 12-14-kDa iron-sulfur proteins, some of which transfer electrons to mitochondrial cytochrome P450s . The function of many of these cytochrome P450s is to catalyze stereospecific hydroxylation of endogenous steroids . As part of our interest in the kidney mitochondrial 1 alpha-hydroxylation of 25-hydroxyvitamin D3, we have constructed an expression plasmid coding for a fusion protein containing the chick kidney ferredoxin . We subcloned chick kidney ferredoxin cDNA, obtained from our vitamin D-deficient chick kidney library by polymerase chain reaction (Brandt, M . E., Gabrik, A . H., and Vickery, L . E . (1991) Gene (Amst.) 97, 113-117) into Qiagen's pQE9, which contains an N-terminal 6xHis tag (peptide sequence for 6 adjacent histidines present in the recombinant proteins) . The coding sequence was preceded by a factor Xa cleavage site . The resulting plasmid, pQTcFdx, was overexpressed in Escherichia coli, and the soluble fusion protein was purified from the cell lysate in one step by Ni(II)-nitrilotriacetic acid-agarose chromatography . We obtained 7-10 mg of greater than 99% homogeneous fusion protein from a 1-liter culture and 4-6 mg of mature ferredoxin cleaved by factor Xa . The fusion protein possessed an absorption spectrum and an electron paramagnetic resonance spectrum quantitatively indistinguishable from those published for ferredoxin purified from adrenal glands and placenta or expressed in E . coli with another vector . The fusion protein was active in supporting the 1 alpha-hydroxylation of 25-hydroxyvitamin D3 in a reconstitution assay of a solubilized, partially purified preparation of cytochrome P450 from vitamin D-deficient chick kidney . We conclude that the procedure described here is an efficient way to produce and purify vertebrate ferredoxin; the {2Fe-2S} cofactor is assembled in vivo and effectively incorporated into the fusion protein in E . coli; slight alterations at the N terminus do not alter incorporation of the {2Fe-2S} cofactor or the biological activity of ferredoxin, and post-translational modifications, such as phosphorylation, are not an absolute requirement for ferredoxin electron transporting activity . The recombinant ferredoxin can be used for physical studies and other structure-function studies. J Biol Chem, 1993 Mar 5, 268(7), 5220 - 6 Chaperonin-mediated reconstitution of the phytochrome photoreceptor; Grimm R et al.; The native phytochrome photoreceptor was purified to homogeneity from etiolated seedlings of oat (Avena sativa L.) and used for renaturation experiments . Light scattering measurements showed that the GroEL molecular chaperone interacts with non-native phytochrome to suppress aggregation of the refolding polypeptide, following its dilution from a chaotrope . The binary complex formed between non-native phytochrome and GroEL was stable and could be isolated by size exclusion chromatography . Discharge of the photoreceptor from GroEL was obtained with 2 mM MgATP, although 6 mM adenosine 5'-O-(3-thiotriphosphate) was also effective . Phytochrome released from GroEL with MgATP was found primarily in the form of 124-kDa monomers, as judged by size exclusion chromatography and nondenaturing gel electrophoresis, although dimers and other oligomeric forms were also observed . The reconstituted dimers, and other oligomeric forms, preferentially cross-reacted with a monoclonal antibody that recognizes native-like epitopes . In vitro folding reactions, using chemically denatured phytochrome, revealed that successful reconstitution of the photoreceptor required the presence of the GroEL chaperonin and MgATP under the conditions tested . Reconstitution in the presence of GroEL yielded phytochrome that could exhibit photoreversibility between the red-light absorbing and far-red absorbing forms. J Mol Biol, 1993 Mar 5, 230(1), 11 - 4 Suppression of loss-of-function mutations in Escherichia coli ribonuclease P RNA (M1 RNA) by a specific base-pair disruption; Morse DP et al.; A plasmid encoding ribonuclease P RNA of Escherichia coli (M1 RNA) was mutagenized with hydroxylamine in vitro and defective rnpB genes were identified by screening in an in vivo suppression assay . Defective rnpB sequences were mutagenized with a second round of hydroxylamine to restore activity . We report here that conversion of the C32.G48 base-pair of RNase P RNA to either C.A or U.G restored activity to defective rnpB genes bearing a variety of spatially distinct primary mutations . Disruption of this base-pair in an otherwise wild-type rnpB sequence increased the growth rate of the indicator strain E . coli FS101, consistent with the opening of C32.G48 during in vivo assembly of or catalysis by RNase P. Biochemistry, 1993 Mar 2, 32(8), 2082 - 9 Colicin Ia inserts into negatively charged membranes at low pH with a tertiary but little secondary structural change; Mel SF et al.; Colicin Ia, a member of the channel-forming family of colicins, inserts into model membranes in a pH- and lipid-dependent fashion . This insertion occurs with single-hit kinetics, requires negatively charged lipids in the target membrane, and increases in rate as the pH is reduced below 5.2 . The low-pH requirement does not act by inducing a secondary structural change in colicin Ia, which remains 66% +/- 4% alpha-helical between pHs 7.3 and 3.1 as determined by circular dichroism . The secondary structure also remains unchanged between pHs 7.3 and 4.2 in the hydrophobic environment provided by the detergent octyl beta-D-glucopyranoside (beta-OG) . However, at pH 3.1 in the presence of beta-OG, an 11% +/- 3% decrease in the alpha-helical content is observed . Further, beta-OG induces a change in tryptophan fluorescence and an altered pattern of proteolytic digestion, indicative of a tertiary structural changes . This suggests that colicin Ia undergoes a tertiary but little or no secondary structural change in its transition from a soluble to a transmembrane protein. Biochemistry, 1993 Mar 2, 32(8), 2024 - 30 Overexpression, purification, and mechanistic study of UDP-N-acetylenolpyruvylglucosamine reductase; Benson TE et al.; The recently isolated Escherichia coli murB gene (Pucci et al., 1992) has been cloned into an expression vector and the encoded UDP-N-acetylenolpyruvylglucosamine reductase (EC 1.1.1.158) was overproduced to about 10% of soluble cell protein . The encoded 38-kDa protein has been purified to near homogeneity . It was found to be a monomer and to contain stoichiometric amounts of bound FAD which is reducible in catalytic turnover . The enzyme utilizes the 4-pro-S hydrogen of NADPH to reduce the enolpyruvyl group of UDP-N-acetylglucosamine enolpyruvate to the lactyl ether in UDP-N-acetylmuramic acid . NMR analysis of products from 2H2O and 4S-{2H}NADPH incubations establishes that a hydride from NADPH via E.FADH2 is transferred to the beta-methyl of the 3-O-lactyl moiety and a proton from solvent to the alpha-carbon of the lactyl moiety of UDP-N-acetylmuramic acid . A mechanism for this unusual enolether reduction in bacterial cell wall assembly is proposed. Biochemistry, 1993 Mar 2, 32(8), 2104 - 10 Radiolytic footprinting . Beta rays, gamma photons, and fast neutrons probe DNA-protein interactions; Franchet-Beuzit J et al.; Ionizing radiations induce numerous damages in DNA, especially strand breaks . The hydroxyl radical OH., produced by the radiolysis of water, is mainly responsible for this effect . The fact that strand breakage occurs at all nucleotides and that bound proteins may locally radioprotect DNA at the binding site lead us to develop a radiolytic footprinting method to study DNA-protein interactions . Three different radiations were used: beta rays, gamma photons, and fast neutrons . In order to validate this technique, three well-known interaction systems were tested: the lac repressor-lac operator of Escherichia coli, the cyclic AMP receptor protein (CRP) of E . coli and its specific site in the lac regulation region, and the core nucleosome . Radiolytic footprinting gives results similar to those obtained by more classical probes: DNase I, complexes of orthophenanthroline (OP) and copper, complexes of ethylenediaminetetraacetate ion (EDTA) and iron, and UV light . For the same system (lac repressor), irradiation with either gamma photons or fast neutrons gives identical results. Biochemistry, 1993 Mar 2, 32(8), 2013 - 23 Site-directed mutagenesis of conserved cysteine residues within the beta subunit of Escherichia coli nitrate reductase . Physiological, biochemical, and EPR characterization of the mutated enzymes; Augier V et al.; We have used site-directed mutagenesis to alter the ligands to the iron-sulfur centers of Escherichia coli nitrate reductase A . The beta subunit of this enzyme contains four Cys groups which are thought to accommodate the single {3Fe-4S} center and the three {4Fe-4S} centers involved in the electron-transfer process from quinol to nitrate . The third Cys group (group III) contains a Trp at a site occupied by a Cys residue in typical ferredoxin arrangements or in the DmsB subunit of dimethyl sulfoxide (DMSO) reductase . In an attempt to determine the coordination site of the different iron-sulfur centers in the amino acid sequence, we have changed the Trp of group III to Cys, Ala, Phe, and Tyr and the first Cys residue of groups II-IV to Ala and Ser . Physiological, biochemical, and EPR studies were performed on the mutated enzymes . Substitution of Ala for either Cys184, Cys217, or Cys244 results in the full loss of all four iron-sulfur centers present in the wild-type enzyme . These inactive enzymes still possess the alpha,beta, and gamma polypeptides associated in a membrane-bound complex . These Cys have important structural roles and are very likely involved in the coordination of the iron-sulfur centers . Substitution of Cys184 with a Ser residue produces an enzyme containing the four iron-sulfur centers, but displaying reduced activity . EPR studies suggest that Cys184 is a ligand of the {4Fe-4S} center whose midpoint potential is -200 mV in the native enzyme . All substitutions performed in this study on Trp220 lead to mutant enzymes harboring the four iron-sulfur centers and a nitrate reductase activity close to that of the wild-type . In spite of the high similarity between the NarH and DmsB subunits, the Trp220-->Cys substitution does not allow the conversion of the {3Fe-4S} center of the nitrate reductase into a {4Fe-4S} center . Therefore, Trp220 does not seem to play any major role in the beta subunit. Mol Gen Mikrobiol Virusol, 1993 Mar-Apr, (2), 21 - 7 {Features of expression of the pertussis toxin operon under the control of its own and heterologous promotors in Bordetella bronchiseptica cells}; Govorun VM et al.; Expression of the cloned operon encoding the pertussis toxin synthesis under the control of operons own vir-dependent promoter or vir-independent promoter of Escherichia coli origin was studied . Proteins produced by the recombinant strains have been characterized . The pertussis toxin operon was shown to express under the control of both homologous and heterologous promoters in Bordetella bronchiseptica cells . Use of the lac-promoter increases the yield of produced toxin twofold . Copy number of operon in the cell does not influence the level of toxin production . The synthesized protein can be transported into the culture medium . The biological and physico-chemical properties of the protein are similar to the ones of the natural pertussis toxin . Bordetella bronchiseptica strain producing the toxin with decreased toxic activity has been obtained . Thus, a simple system for cellular expression of the toxin operon was constructed in Bordetella bronchiseptica . It permits one to construct new strains producing nontoxic derivatives of the pertussis toxin for construction of nonreactogenic vaccines. Hum Mol Genet, 1993 Mar, 2(3), 265 - 71 Rapid physical mapping of YAC inserts by random integration of I-Sce I sites; Colleaux L et al.; We have developed a novel strategy, based on the random insertion by homologous recombination of artificial I-Sce I sites within mammalian repetitive DNA sequences, which should greatly facilitate the high resolution physical mapping of large DNA fragments cloned in YAC . A set of transgenic yeast strains containing appropriately spaced I-Sce I sites within the YAC insert defines a series of nested physical intervals against which new genes, clones or DNA fragments can be mapped by simple hybridisation . Sequential hybridisation using such a series of nested YAC fragments as probes can also allow the rapid sorting of phage or cosmid libraries into contigs . This approach, which has been applied to a YAC containing a 460 kb insert from the mouse X chromosome, may also have applications for the restriction mapping of large genomic segments, mapping of exons and the search for homologous genes. Pediatr Neurol, 1993 Mar-Apr, 9(2), 127 - 30 Ventriculitis in infants: diagnosis by color Doppler flow imaging; Tatsuno M et al.; A color Doppler flow imaging technique was used to study the dynamics of cerebrospinal fluid (CSF) in infants with meningitis . Eight infants with bacterial meningitis (6) or aseptic meningitis (2) were studied with color Doppler imaging of CSF flow for a total of 18 times . In 2 infants with bacterial meningitis, Doppler sonograms of CSF flow were obtained in the aqueduct during the acute stage . The CSF flow demonstrated a to-and-fro motion which was synchronized with cardiac pulsations and respiration . The detection of CSF flow on color Doppler flow imaging in the aqueduct may indicate the existence of ventriculitis . Color Doppler flow imaging is useful for the evaluation of CSF flow dynamics in infants. Am J Vet Res, 1993 Mar, 54(3), 379 - 86 Effects of intra-articularly administered endotoxin on clinical signs of disease and synovial fluid tumor necrosis factor, interleukin 6, and prostaglandin E2 values in horses; Hawkins DL et al.; In each of 4 horses, sterile synovitis was induced by intra-articular injection of 3 micrograms of Escherichia coli endotoxin (lipopolysaccharide, LPS) into one antebrachiocarpal joint; an equal volume (2 ml) of phosphate-buffered saline solution (PBSS) was injected into the opposite, control carpus . Blood and 1.5 ml of synovial fluid were obtained at postinjection hours (PIH) 0, 2, 4, 8, 12, 18, 42, 66, and 144 . Synovial fluid sample collection was accomplished by use of an indwelling, intra-articular catheter through PIH 12, and by arthrocentesis subsequently . Joint fluid samples were analyzed for cell counts, protein concentration, cytologic variables, and tumor necrosis factor (TNF), interleukin 6 (IL-6), and prostaglandin E2 (PGE2) values . Tumor necrosis factor and IL-6 activities and WBC count were also measured in blood . To monitor local inflammation, skin temperature of each carpus was imaged, using a thermographic scanner prior to each sample collection time . Horses had minimal systemic effects . Mean (+/- SEM) rectal temperature increased significantly to 39.02 +/- 0.15 C only at PIH 18 after intra-articular injection of LPS . One horse had signs of mild depression from PIH 7 to 18, but its vital signs did not change appreciably . Each horse had mild signs of discomfort in the LPS-injected limb from PIH 1 to 3 until PIH 8 to 10 . Mean peak surface temperature of the LPS-injected carpi was significantly higher than that of control carpi from PIH 8 to 144 (P < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS) Med Microbiol Immunol (Berl), 1993 Mar, 182(1), 1 - 12 In vivo effects of intravascularly applied Escherichia coli hemolysin: dissociation between induction of granulocytopenia and lethality in monkeys; Vagts D et al.; The effects of intravascular application of endotoxin-depleted Escherichia coli hemolysin (HlyA) was studied in rabbits and monkeys . In rabbits, bolus application of HlyA calculated to effect final blood levels of approximately 2-3 HU/ml (200-300 ng/ml) caused an acute fall of polymorphonuclear blood leukocytes to less than 20% of starting levels within 5 min . Additionally, platelet counts dropped to approximately 30% of starting levels, whereas lymphocyte counts varied considerably and seldom fell to less than 50% . Nine out ten animals that received 2-4 HU/ml toxin died within 90 min post application . These animals presented with signs of acute respiratory failure and post mortem inspection of the internal organs revealed hemorrhagic pulmonary edema . Other internal organs appeared unaffected . Application of less than 1 HU/ml HlyA was never fatal (n = 9), and only transient leukopenia was noted . Monkeys presented with a remarkable and different response . Two animals were repeatedly given HlyA at high doses ranging from 3 to 10 HU/ml . Both animals developed selective granulocytopenia, but following a short, transient drop in blood pressure they showed no severe clinical signs of cardiovascular or pulmonary malfunction . Histological examinations revealed accumulation of polymorphonuclear granulocytes in both animals in liver, lung and spleen . Very high leukocyte elastase levels were measured in one animal over a period of 1.5 h . The present results demonstrate a remarkable tolerance of monkeys towards the leukocidal effects of E . coli hemolysin . Lethality in rabbits must be due to additional effects of the toxin, possibly on cells in the pulmonary vasculature . Neither pulmonary sequestration of granulocytes nor massive release of elastase from these cells is in itself sufficient to provoke pulmonary dysfunction in monkeys. J Photochem Photobiol B, 1993 Mar, 17(3), 219 - 28 The photo repair of pyrimidine dimers by DNA photolyase and model systems; Heelis PF et al.; Pyrimidine dimers are eliminated from DNA by a number of different mechanisms known as DNA repair . Photoreactivation, the reversal of the harmful effects of short wavelength radiation by subsequent exposure to longer wavelengths, is one such mechanism . In photoreactivation, the enzyme DNA photolyase utilises light in order to catalyse the cleavage of the cyclobutane ring of the pyrimidine dimer . The results of recent studies of E . coli DNA photolyase and model systems using techniques such as steady state and flash photolysis, time resolved fluorescence and photo CIDNP are surveyed . A mechanism is proposed for the in vitro reaction of E . coli DNA photolyase which involves photoreduction of the FAD radical cofactor followed by electron donation to the dimer from the excited singlet state of reduced FAD. Biochem Mol Biol Int, 1993 Mar, 29(4), 687 - 94 Electrophoretic analysis of endotoxin-activated gelation reaction of Carcinoscorpius rotundicauda amoebocyte lysate; Ho B et al.; Electrophoretic analysis of a 60 min reaction between E . coli endotoxin and the amoebocyte lysate showed that the coagulation reaction was complete by 15 min, with the conversion of coagulogen (21 kDa) to coagulin (17 kDa) . Coincident with this observation was the maximal activities at 15 min, of Factor C and proclotting enzyme . On agitation of the coagulin gel clots, bioactive endotoxin was recovered . Densitometric scan of the electrophoretically-resolved proteins showed that the sum of coagulogen and coagulin remained almost constant at various time intervals of the coagulation reaction . Electrophoresis serves as a convincing and visually discernible method of studying the kinetics of coagulation, and defining the onset and completion of gelation . Furthermore, it is a useful means of examining the integrity of fresh lysate preparations based on the presence or absence of the 17 kDa coagulin band. Plant Mol Biol, 1993 Mar, 21(6), 1191 - 4 In vivo suppression of phytochrome aggregation by the GroE chaperonins in Escherichia coli; Edgerton MD et al.; When expressed in Escherichia coli, a truncated form of phytochrome (oat PHYA AP3 residues 464-1129) self associates to form a series of products ranging in size from monomers to aggregates of greater than 20 subunits . When these same phytochrome sequences are coexpressed with the chaperonins GroEL and GroES, the truncated phytochrome migrates as a native-like dimer in size exclusion chromatography and no higher-order aggregates were detected . GroEL and GroES inhibition of phytochrome aggregation in E . coli presumably occurs via the suppression of folding pathways leading to incorrectly folded phytochrome. Plant Mol Biol, 1993 Mar, 21(6), 1069 - 76 Co-transcription pattern of an introgressed operon in the maize chloroplast genome comprising four ATP synthase subunit genes and the ribosomal rps2; Stahl DJ et al.; Several examples of the introduction of a gene from one gene complex into another (introgression) are found when chloroplast RP gene clusters are compared to those in Escherichia coli or cyanobacteria . Here we describe the transcript pattern of one such cluster from maize (Zea mays) that includes the genes for 4 subunits of the thylakoid ATP synthase (atpI, H, F, A) and the rps2 gene . Twelve transcript species covering the size range from 7,000 to 800 nt were identified in RNA isolated from dark-grown and greening maize seedlings, and several of them were characterized by reverse transcription analysis . A major species of 6,200 nt, with its 5' end at 181 nt upstream of the initiating ATG of rps2, contained the transcripts of all the 5 genes . Two further sets of transcripts having their 5' ends ca . 120 and 50 nt upstream of the initiation codons of the atpI and atpH genes were also identified . Thus, this plastid gene cluster in maize is functionally organized as an operon with additional regulatory features to allow for increased accumulation of mRNAs for the thylakoid components. Bioorg Khim, 1993 Mar, 19(3), 286 - 92 {Preparation, isolation, and study of mutant forms of ribosomal protein L7/L12}; Todorova RT; Three mutant forms of the ribosomal protein L7/L12 with Ser1, Met14 and Met26 substituted by the Tyr residue were constructed for studying the protein's N-terminal domain . Three point mutations were introduced into the L7/L12 gene by means of the phage M13mp18 system, the mutant genes were expressed in Escherichia coli cells, and methods of the proteins' purification were developed . The mutant proteins L7/L12 are very close, in structure and properties to the wild type protein and represent suitable objects for the 1H-NMR study of the N-terminal domain. Mol Biol (Mosk), 1993 Mar-Apr, 27(2), 429 - 35 {Three mutant forms of ribosomal protein L7/L12}; Todorova RT; Three mutant forms of the ribosomal protein L7/L12 with Ser1, Met14, and Met26 replacements with Tyr were constructed for studying the N-terminal domain of the protein . Three point mutations in the gene L7/L12 on the phage M13mp18 were generated . Recombinant plasmids containing the mutant genes were constructed on the base of expression vector pKK223-3 and plasmid pUC19 . The mutant proteins were expressed in Escherichia coli cells, and methods of their purification were developed . It was found that the mutant proteins L7/L12 do not differ from the wild-type protein L7/L12 and represent a suitable object for 1H-NMR study of the L7/L12 protein . The three mutant proteins bind with the E . coli ribosome. Mol Biol (Mosk), 1993 Mar-Apr, 27(2), 392 - 401 {Analysis of reporter gene expression at different segments of the vaccinia virus genome}; Timiriasova TM et al.; Two reporter genes: the firefly Photinus pyralis luciferase gene and the Escherichia coli beta-galactosidase gene were used for construction and characterization of the five unique recombinant vaccinia strain LIVP viruses expressing these genes in three nonessential regions of the virus genome . We give comparative characteristics of beta-galactosidase and luciferase activities in experiments of transient expression and expression dynamics of reporter genes by different stable recombinant viruses . Both genes are expressed with high efficiency independent on the sites of virus genome localization . It is shown that the TK-, HA- and N-regions of vaccinia virus DNA may be used for inserting foreign genes. Mol Biol (Mosk), 1993 Mar-Apr, 27(2), 299 - 304 {Immunochemical analysis of Escherichia coli expression products of I3 and A2 genes of vaccinia virus strain L-IVL genome}; Chikaev NA et al.; Genes I3 and A2 of the vaccinia virus strain L-IVP DNA were cloned into bacterial expressing vectors . The monospecific antisera to the expression products of these genes in E . coli were obtained . By means of immunochemical cross-analysis two polypeptides of equal electrophoretic mobility were found in the virion preparations in the band of the major envelope protein p35 . The major of them is the product of gene I3, and the minor is encoded by gene A2. Klin Padiatr, 1993 Mar-Apr, 205(2), 78 - 82 {Surfactant therapy in severe neonatal respiratory failure--multicenter study--III . Surfactant therapy in 41 premature infants < 34 weeks with suspected congenital infection (case-control analysis)}; Brehmer U et al.; As a part of a multicenter study (424 patients, 16 hospitals) 41 ventilated very low birth weight infants (24-33 gestational weeks, 640-1560 g), who were suspected to have connatal pneumonia, were treated with a bovine surfactant (Alveofact, 50-200 mg/kgBW) . Using a case control design they were compared with 41 patients having IRDS . There were no differences between the collectives with regard to the acute response to surfactant, the clinical course, and the outcome at the 28 . d of life . Accidental treatment with natural surfactant in very immature neonates suffering from severe respiratory distress due to connatal pneumonia resp . connatal pneumonia accompanying IRDS seems not to harm these patients. Nat Genet, 1993 Mar, 3(3), 229 - 34 Direct in vivo gene transfer to ependymal cells in the central nervous system using recombinant adenovirus vectors; Bajocchi G et al.; To evaluate the potential for adenovirus-mediated central nervous system (CNS) gene transfer, the replication deficient recombinant adenovirus vectors Ad.RSV beta gal (coding for beta-galactosidase) and Ad-alpha 1AT (coding for human alpha 1-antitrypsin) were administered to the lateral ventricle of rats . Ad.RSV beta gal transferred beta-galactosidase to ependymal cells lining the ventricles whereas Ad-alpha 1AT mediated alpha 1-antitrypsin secretion into the cerebral spinal fluid for 1 week . These observations, together with beta-galactosidase activity in the globus pallidus and substantia nigra following stereotactic administration of Ad.RSV beta gal to the globus pallidus, suggest that adenovirus vectors will be useful for CNS gene therapy. Nat Genet, 1993 Mar, 3(3), 224 - 8 Transfer of a foreign gene into the brain using adenovirus vectors; Akli S et al.; The ability of a replication-deficient adenovirus vector to transfer a foreign gene into neural cells of adult rats in vivo has been analysed . A large number of neural cells (including neurons, astrocytes and ependymal cells) expressed an E . coli lacZ transgene for at least 45 days after inoculation of various brain areas . Injecting up to 3 x 10(5) pfu in 10 microliters did not result in any detectable cytopathic effects--these were only observed for very high titres of infection (> 10(7) pfu 10 microliters-1) . Adenovirus vectors therefore appear to be a promising means for in vivo transfer of therapeutic genes into the central nervous system. Biochem Mol Biol Int, 1993 Mar, 29(3), 499 - 510 Steroid induced single strand breaks in DNA mediated by active oxygen species and its biological consequences; Qadri SA et al.; A mutagenic steroidal derivative (3 beta-Acetoxy-5 alpha-Cholestano{6 alpha,5-d'}1'-3'oxathiolane-2' thione) structurally related to cholesterol caused strand scission and induced nicks in calf thymus, supercoiled pBR322 and single stranded M13 mp8 phage DNAs . S1 nuclease hydrolysis, reaction with pBR322 and M13 phage DNA as well as treatment of E . coil mutant strains and phage was used to evaluate the effect of test steroid on the DNA molecule . The strand scission/nicking of DNA by the test steroid was enhanced by some metal ions, especially the Cu(II) . Scavengers of active oxygen radical species significantly inhibited the S1 nuclease hydrolysis by the test steroid indicating the major role of active oxygen species in DNA strand scission and nicking . The steroid brought about the DNA degradation even in the absence of S1 nuclease . There was an appreciable reduction in the survival of steroid treated polA and lig mutants of E . coli K12 compared to the wild type strain . Phage on steroid treatment also lost its plaque forming units (P.F.U.) which was more pronounced in the polA and rec A background. Mol Gen Genet, 1993 Mar, 237(3), 407 - 11 A mutation in the dsbA gene coding for periplasmic disulfide oxidoreductase reduces transcription of the Escherichia coli ompF gene; Pugsley AP; An insertion mutation in the Escherichia coli dsbA gene, coding for periplasmic disulfide oxidoreductase, dramatically reduces the level of OmpF porin protein in the cell envelope . Studies with chromosomal ompF-lacZ operon and gene fusions indicate that this is due to reduced ompF transcription . Identical effects resulted from growth in medium containing the reducing agent dithiothreitol, but the combined effects of the reducing agent and dsbA were no greater than the effects of each individually . The dsbA mutation did not prevent normal regulation of ompF transcription by the local anaesthetic procaine or by osmolarity . OmpF does not contain a cysteine residue, and the sole cysteine residue in the cytoplasmic membrane regulator of ompF transcription, EnvZ, is predicted to be located on the cytoplasmic side of the membrane, and is therefore unlikely to be involved in the effects of the dsbA mutation . The effects of the dsbA mutation and of the reducing agent on ompF transcription may be due to the failure to from an essential disulfide bond in an as yet unidentified envelope protein that affects ompF transcription. Mol Gen Genet, 1993 Mar, 237(3), 395 - 9 Escherichia coli RuvA and RuvB proteins involved in recombination repair: physical properties and interactions with DNA; Shiba T et al.; Escherichia coli RuvA and RuvB proteins are encoded by an SOS-regulated operon, which is involved in DNA repair and recombination . RuvB has weak ATPase activity, which is enhanced by the addition of RuvA and DNA, and RuvA and RuvB in the presence of ATP promote branch migration at Holliday junctions . In this work, the physical states of RuvA and RuvB and their interactions with DNA were studied by sedimentation analysis and gel filtration chromatography . RuvA formed a stable tetramer in solution, which resisted dissociation by SDS at room temperature . RuvB formed a dimer in solution . When RuvA and RuvB were mixed, an oligomer complex was formed consisting of a tetrameric form of RuvA and a dimeric form of RuvB, and this complex bound to DNA . The maximal enhancement of the RuvB ATPase activity by RuvA was achieved at this stoichiometry in the presence of excess DNA. Mol Gen Genet, 1993 Mar, 237(3), 334 - 42 SSV1-encoded site-specific recombination system in Sulfolobus shibatae; Muskhelishvili G et al.; We present evidence for the existence of a conservative site-specific recombination system in Archaea by demonstrating integrative recombination of Sulfolobus shibatae virus SSV1 DNA with the host chromosome, catalysed by the SSV1-encoded integrase in vitro . The putative int gene of SSV1 was expressed in Escherichia coli yielding a protein of about 39 kDa . This protein alone efficiently recombined linear DNA substrates containing chromosomal (attA) and viral (attP) attachment sites; recombination with either negatively or positively supercoiled SSV1 DNA was less efficient . Intermolecular attA x attA and attP x attP recombination was also promoted by the SSV integrase . The invariant 44 bp "common attachment core" present in all att sites contained sufficient information to allow recombination, whilst the flanking sequences effected the efficiency . These features clearly distinguish the SSV1--encoded site--specific recombination system from others and make it suitable for the study of regulatory mechanisms of SSV1 genome--host chromosome interaction and investigations of the evolution of the recombination machinery. Mol Microbiol, 1993 Mar, 7(6), 937 - 46 In vitro transcription in Chlamydia psittaci and Chlamydia trachomatis; Mathews SA et al.; Extracts of Chlamydia psittaci and Chlamydia trachomatis were used to transcribe molecularly cloned chlamydial genes in vitro . The extracts were prepared by lysing reticulate bodies, obtaining the 10,000 x g centrifugation pellet, and eluting RNA polymerase from the pellet by treatment with 2M KCl to yield a fraction designated SS2 . Some in vitro transcription was initiated from non-chlamydial promoters and a small amount of transcription was from endogenous DNA template in SS2 . However, optimal transcription from exogenous templates required chlamydial promoter sequences, and primer extension analysis indicated that chlamydia promoter-specific in vitro transcription was initiated from the same start sites recognized in vivo . A monoclonal antibody that was generated against Escherichia coli sigma 70 and which immunologically cross-reacts with C . trachomatis sigma 66 inhibited in vitro transcription of vector and cloned chlamydial DNA, suggesting that transcriptional initiation in the SS2 fraction is mediated by sigma 66 . An in vitro transcription assay based on detection of transcripts of specific lengths was applied to the chlamydial system; this assay and others described here should be useful in defining chlamydial promoters and other transcriptional regulatory elements. Mol Microbiol, 1993 Mar, 7(6), 859 - 64 Involvement of the RNA polymerase alpha subunit C-terminal region in co-operative interaction and transcriptional activation with OxyR protein; Tao K et al.; The role of the alpha subunit of Escherichia coli RNA polymerase in transcription activation by the OxyR protein was investigated using in-vitro-reconstituted RNA polymerase containing alpha subunits carrying C-terminal truncations or an amino acid substitution . Mutant RNA polymerases failed to respond to transcription activation of the E . coli OxyR-dependent promoters . DNase I footprinting analysis indicates that the OxyR protein exerts a co-operative effect on the binding of wild-type RNA polymerase, but not the mutant RNA polymerases, to the katG promoter . Together, these results suggest that direct protein-protein contact between the OxyR protein and the C-terminal contact site I region of the RNA polymerase alpha subunit plays an essential role in transcription activation at the OxyR-dependent promoters. Mol Microbiol, 1993 Mar, 7(6), 847 - 57 Specific binding sites in the alcR and alcA promoters of the ethanol regulon for the CREA repressor mediating carbon catabolite repression in Aspergillus nidulans; Kulmburg P et al.; The CREA repressor responsible for carbon catabolite repression in Aspergillus nidulans represses the transcription of the ethanol regulon . The N-terminal part of the CREA protein encompassing the two zinc fingers (C2H2 class family) and an alanine-rich region was expressed in Escherichia coli as a fusion protein with glutathione-S-transferase . Our results show that CREA is a DNA-binding protein able to bind to the promoters of both the specific trans-acting gene, alcR, and of the structural gene, alcA, encoding the alcohol dehydrogenase I . DNase I protection footprinting experiments revealed several specific binding sites in the alcR and in the alcA promoters having the consensus sequence 5'-G/CPyGGGG-3' . The disruption of one of these CREA-binding sites in the alcR promoter overlapping the induction target for the trans-activator ALCR results in a partially derepressed alc phenotype and derepressed alcR transcription, showing that this binding site is functional in vivo . Our data suggest that CREA represses the ethanol regulon by a double lock mechanism repressing both the trans-acting gene, alcR, and the structural gene, alcA. Curr Eye Res, 1993 Mar, 12(3), 205 - 12 Retinal pigment epithelial cells produce interleukin-1 beta and granulocyte-macrophage colony-stimulating factor in response to interleukin-1 alpha; Planck SR et al.; The retinal pigment epithelium (RPE) is clinically involved in diverse ocular inflammatory diseases . Because perturbed RPE cells produce a variety of inflammatory substances, RPE cells may play an integral part in these diseases . Interleukin-1 (IL-1) and granulocyte-macrophage colony-stimulating factor (GM-CSF) are pleiotropic cytokines with the ability to trigger numerous inflammatory responses . This report shows that cultured human RPE cells synthesize interleukin-1 beta (IL-1 beta) and GM-CSF in response to the potentially inflammatory cytokine, IL-1 alpha, but not to E . coli endotoxin . Control RPE cells made little or no mRNA or protein for either IL-1 beta or GM-CSF . Upon stimulation of the cells by IL-1 alpha, both IL-1 beta and GM-CSF mRNAs were readily apparent by 3 hours, persisted for over 24 hours, and were translated into immunologically detectable proteins . GM-CSF protein was secreted into the culture medium, whereas IL-1 beta protein remained cell associated . The IL-1 alpha-induced mRNA and protein production were inhibited by dexamethasone . These observations provide additional evidence that RPE cells are capable of playing a pivotal role during ocular inflammation. J Inorg Biochem, 1993 Mar, 49(4), 295 - 304 The binding of a complex cobalt ruthenium polyamine by deoxyribonucleic acid and a lipopolysaccharide: a model for a novel class of drugs; Hammershoi A et al.; In this paper we discuss the following: 1 . Synthesis of {Co(H3CsarNHCH2pyRu(NH3)5)} (PF6)5, (CoRu) . 2 . Interaction of CoRu with calf thymus DNA and with lipopolysaccharide from Escherichia coli C (LPS) has been estimated using the absorption of the complex at 242 and 420 nm . 3 . DNA and LPS increase the rate of fall of absorption at 420 nm due to autooxidation of the complex . 4 . The fall in absorption of CoRu(II) at 420 nm can be used to give an approximate measure of binding to DNA and to LPS . 5 . Both macromolecules are aggregated by CoRu at high concentrations and the cation and macromolecule complex can be removed by low speed centrifugation . 6 . The DNA-CoRu complex can also be removed by high speed centrifugation when the cation concentration is too low to cause aggregation (20 microM CoRu/155 microM DNA-P) . Absorption of redissolved complex at 420 nm is restored by reduction with ascorbic acid . 7 . At saturation the ratio of mole CoRu bound/mole DNA-P is 0.16. Acta Physiol Scand, 1993 Mar, 147(3), 251 - 61 Sequential changes in the splanchnic circulation during continuous endotoxin infusion in sedated sheep: evidence for a selective increase of hepatic artery blood flow and loss of the hepatic arterial buffer response; Schiffer ER et al.; On-line recording of the sequential changes in systemic, pulmonary, mesenteric, hepatic and renal circulations during onset of endotoxaemia and at 24 h of established hyperdynamic sepsis were evaluated in seven chronically instrumented and sedated sheep receiving a continuous intravenous infusion of Escherichia coli endotoxin (20 ng min-1 kg-1) . A transient and significant (P < 0.05) pulmonary arterial vaso-constriction was noted after 13 +/- 4 min, and was followed immediately by a simultaneous significant decrease of coeliac trunk, superior mesenteric artery, and portal vein blood flow to below 50% of baseline values . The superior mesenteric artery and portal vein blood flows partially recovered pre-endotoxin levels to 69 and 75% of baseline, respectively, after 70 min of endotoxin infusion . In contrast, the coeliac trunk blood flow remained reduced for a more prolonged period of time, but then completely recovered baseline values at 100 min . The response of the hepatic artery was biphasic, and consisted of a transient (5-10 min) vasoconstriction at 40 min followed by transitory increase of hepatic artery blood flow reaching a maximum of 921% of baseline values at 102 min . Contrasting with the early changes observed in mesenteric vascular resistances mostly unrelated to systemic haemodynamics, the response of the renal vasculature appeared to be more dependent on changes of renal perfusion pressure . A follow-up at 24 h revealed that the continuous intravenous infusion of endotoxin reproduced some of the most characteristic features of human sepsis with increased cardiac output and decreased vascular resistances of all vascular beds . We conclude that hepatic artery blood flow is selectively and considerably increased in early endotoxaemia in sheep independently of changes in portal vein blood flow, suggesting a disregulation of the physiologic hepatic arterial buffer response, most probably secondary to an increased liver oxygen demand required for phagocytosis, transport, and digestion of the the sudden overload of bacterial endotoxins. Photochem Photobiol, 1993 Mar, 57(3), 508 - 12 DNA nicking by ultraviolet radiation is enhanced in the presence of iron and of oxygen; Audic A et al.; Double-stranded covalently closed circular supercoiled DNA (ccc DNA) from plasmid pUK 9 was irradiated in vitro at defined wavelengths in the UV region (290, 313 and 365 nm) . The nicking was monitored by electrophoresis on agarose gels, ethidium staining and densitometric quantitation of supercoiled and relaxed moieties . At the explored wavelengths, the dose required for introducing one nick per million phosphodiester bonds diminishes with increased concentration of added ferric iron, whereas the effect of cupric iron is practically negligible . Adding metal chelators or bubbling argon prior to the irradiation results in a dramatic increase in the dose required for introducing one nick per million phosphodiester bonds . Taken together, these results seem to indicate that iron and oxygen play a role as cofactors in the UV-induced nicking of ccc DNA in vitro. J Gen Microbiol, 1993 Mar, 139 ( Pt 3), 639 - 50 Cloning, characterization and sequencing of two haemagglutinin genes from Eikenella corrodens; Rao VK et al.; Eikenella corrodens is emerging as an important human pathogen, in both extra-oral and periodontal infections . From a clone bank of Eikenella corrodens chromosomal DNA produced in Escherichia coli JM109, twenty-two clones expressed Eikenella antigens and of these, two expressed functional haemagglutinins . By virtue of different restriction maps and a lack of homology by Southern hybridization, the two cloned fragments encoding the two haemagglutinins have been shown to be distinct . Maxicell analysis revealed that clone 1, carrying plasmid pVKR201, produces three Eikenella proteins, one of 31.5 kDa and two of approximately 14 kDa each . Expression of each of the proteins appears to be under the control of an Eikenella promoter(s) . Clone 2, carrying plasmid pVKR301, produces two proteins, one of 93 kDa and the second of 17 kDa . Expression of both of these proteins in E . coli requires the lac promoter in the vector . By preparing a series of subclones and testing each by maxicell analysis and for haemagglutination activity, a functional map of the insert of clone 1 was deduced and the 31.5 kDa polypeptide identified as the haemagglutinin . Using similar methods, the 17 kDa protein was found to be the haemagglutinin of clone 2 . The nucleotide sequences of both haemagglutinin genes were determined and are presented . Computer analysis revealed no homology between the two haemagglutinins, and no homology to any previously sequenced proteins . These are the first genes of this genus to be cloned and sequenced. J Gen Microbiol, 1993 Mar, 139 ( Pt 3), 631 - 8 Fusion of the genes encoding Escherichia coli heat-stable enterotoxin b (STb) and the maltose-binding protein to obtain mature STb enterotoxin; Bosse M et al.; The heat-stable enterotoxin b gene (estB) of Escherichia coli was fused to the gene for maltose-binding protein (malE) . The estB gene was cloned into the pMAL-p vector using PCR . The construct consists of the signal sequence of maltose-binding protein, which directs the export of the fusion protein to the periplasm, and the maltose-binding protein fused to the STb polypeptide . A sequence encoding a factor Xa cleavage site is present between malE and estB . The fused genes are controlled by Ptac, a strong inducible promoter . Following IPTG induction, the recombinant strain expressed a 47 kDa protein, which was easily purified from osmotic shock fluid by using preparative electrophoresis and electroelution . Cleavage of the fusion protein with factor Xa generated the maltose-binding protein (42 kDa) and a polypeptide of approximately 5 kDa, corresponding to the molecular mass of mature STb . A monospecific polyclonal rabbit antiserum raised against purified STb reacted in immunoblot with the fusion protein and the cleaved-off peptide . A positive response was observed when testing the osmotic shock fluid containing the fusion protein in a rat intestinal loop assay . On average, 3-4 mg of MBP-STb protein was recovered per litre of induced recombinant strain. J Gen Microbiol, 1993 Mar, 139 ( Pt 3), 575 - 83 Cloning and sequencing of the gene which encodes the highly inducible acetamidase of Mycobacterium smegmatis; Mahenthiralingam E et al.; The acetamidase of Mycobacterium smegmatis NCTC 8159 was purified, and the sequences of its amino-terminus and of two peptides obtained by proteolysis of the protein were obtained . A DNA fragment including the amidase structural gene was cloned in Escherichia coli, using oligonucleotide probes designed on the basis of the peptide sequences and a codon usage table calculated from published sequences of nine protein-antigen-encoding genes of the Mycobacterium tuberculosis complex . Sequence analysis of the cloned DNA revealed that the amidase gene encoded 406 amino acid residues . The nucleotide sequence close to and upstream of the amidase gene contained a probable ribosome-binding site but no identifiable promoter sequences . Three additional potential open-reading frames were found upstream of and very close to the amidase gene, with consensus '-35' and '-10' promoter sites between the first and second of these . It is hoped that the highly inducible expression of the acetamidase gene can be exploited to allow regulated expression of other genes cloned in mycobacteria. Hum Reprod, 1993 Mar, 8(3), 409 - 11 Tumour necrosis factor alpha and interleukin 2 in normal and infected human seminal fluid; Hussenet F et al.; The presence of cytokines such as the tumour necrosis factor alpha (TNF alpha) and interleukin 2 (IL2) in human spermatozoa is still to be defined . The aim of this study was to measure the concentration of both soluble factors in seminal fluid . Data from normal semen samples (n = 24) confirmed the presence of IL2 (258 +/- 84 fmol/ml corresponding to 953 +/- 369 fmol/total volume of ejaculate) and TNF alpha (62.2 +/- 16.4 fmol/ml corresponding to 231.3 +/- 86 fmol/total volume of ejaculate) . A significant positive correlation (r = 0.59; P < 0.01) was observed between the TNF alpha and the IL2 concentrations . The concentrations of these cytokines were not related to sperm parameters . In contrast, IL2 concentrations (196.9 +/- 60.4 fmol/ml; 686.2 +/- 236.7 fmol/total volume of ejaculate) evaluated in 16 seminal fluids with identified bacterial agents were lower than in the control group, whereas TNF alpha concentrations (68.6 +/- 12.3 fmol/ml; 241.3 +/- 78.9 fmol/total volume of ejaculate) were not significantly different from the controls . Further studies are needed to determine the potential role of these cytokines in the physiology of semen and their usefulness as indicators of reproductive pathology. FEMS Microbiol Lett, 1993 Mar 1, 107(2-3), 331 - 6 A stereospecific alignment between the promoter and the cis-acting sequence is required for Lrp-dependent activation of ilvIH transcription in Escherichia coli; Sacco M et al.; The leucine-responsive regulatory protein (Lrp) is a DNA binding protein that affects, either positively or negatively, the expression of several E . coli genes . The ilvIH operon is positively regulated by Lrp and leucine counteracts this effect reducing 5- to 10-fold the efficiency of ilvIH transcription . An investigation of the mechanism of transcription activation of the ilvIH operon by Lrp indicated that: (i) a stereospecific alignment between the ilvIH promoter and the cis-acting sequence upstream of it is required for activation; (ii) a correct distance between the promoter and the adjacent cis-acting sequence is needed for leucine to counteract the positive role of Lrp; (iii) Lrp fails to activate transcription when the cis-acting region is placed several hundred base pairs upstream of the ilvIH promoter. FEMS Microbiol Lett, 1993 Mar 1, 107(2-3), 287 - 92 Cloning and expression of the D-ribulose-1,5-bisphosphate carboxylase/oxygenase form II gene from Thiobacillus intermedius in Escherichia coli; Stoner MT et al.; Both form I and II ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) genes were detected in Thiobacillus intermedius by heterologous hybridization using specific probes from Anacystis nidulans and Rhodobacter sphaeroides, respectively . However, only the previously reported form I enzyme could be demonstrated in cells grown under a number of different conditions . The reason(s) why the form II gene is not expressed in T . intermedius is/are not clear at this time . The form II gene was isolated from a lambda library by screening with the Rb . sphaeroides probe . A SalI fragment from this clone was ligated into pUC8 and transformed into Escherichia coli DH5 alpha . Subclones pTi20IIA and pTi20IIB representing both orientations relative to the lac promoter were isolated . Low levels of RuBisCO activity were detected in both induced and non-induced pTi20IIA indicating the probable expression from a T . intermedius promoter . Induced pTi20IIB produced much higher levels of enzyme activity . Analysis of cell-free extracts using sucrose density gradients confirmed the expression of a form II RuBisCO similar in size to that found in Rhodobacter capsulatus . Other Calvin cycle genes were not clustered with either the form I or form II genes. FEMS Microbiol Lett, 1993 Mar 1, 107(2-3), 231 - 9 Surface films of Escherichia coli colonies; Tetz VV et al.; Escherichia coli colony surfaces were examined using SEM and TEM . The results indicated that bacterial colonies in the course of their development produce surface films which become thicker with increased growth duration . Membrane vesicles contribute to the formation of the surface film . The complex organization of the film suggests that it may perform specific functions. FEMS Microbiol Lett, 1993 Mar 1, 107(2-3), 175 - 8 EnvC, a new lipoprotein of the cytoplasmic membrane of Escherichia coli; Seiffer D et al.; A gene product with an apparent molecular mass of approximately 39,000 Da can be identified in the cytoplasmic membrane of Escherichia coli upon expression of cloned envC . In this communication we report that the product was labelled with {3H}glycerol and {3H}palmitic acid, and a precursor molecule of increased molecular mass was accumulated when cells were treated with globomycin, a specific inhibitor for the prolipoprotein signal peptidase . The same precursor molecule was encoded by an envC mutant gene, in which the cysteine residue in a pentapeptide sequence, Leu-Ile-Ala-Gly-Cys24 within the amino terminal region of EnvC, was replaced by tryptophane (Trp24) . This protein was not labelled with {3H}glycerol . The results demonstrate that the envC gene product represents a new lipoprotein of the cytoplasmic membrane of E . coli. DNA Cell Biol, 1993 Mar, 12(2), 191 - 7 Immobilization and recovery of fusion proteins and B-lymphocyte cells using magnetic separation; Ljungquist C et al.; A new approach to facilitate immobilization and affinity purification of recombinant proteins and selected human B lymphocytes has been developed . Using magnetic beads with attached DNA containing the Escherichia coli lac operator, fusion proteins comprising the DNA-binding lac repressor could be affinity-purified and recovered by gentle elution conditions, such as with a lactose analogue or by enzymatic means using either deoxyribonuclease (DNase) or restriction endonucleases . The results show for the first time that a DNA-binding protein can be used for affinity purification of fusion proteins as exemplified by the specific and gentle recovery of beta-galactosidase and alkaline phosphatase from bacterial lysates using immunomagnetic separation . The approach was further extended to cell separation by the efficient recovery and elution of human CD37 B lymphocytes from peripheral blood. Anal Biochem, 1993 Mar, 209(2), 238 - 46 On the activities of Escherichia coli exonuclease III; Hoheisel JD; Exonuclease III (Exo III) of Escherichia coli is a DNA-modifying enzyme very frequently used in molecular biology . The experiments described here were carried out with the aims of reliably controlling exonuclease activity and of learning more about the enzyme's specificities . The dependence of Exo III activity on factors such as temperature (including heat inactivation), the concentrations of Exo III and NaCl, and the concentration and shape of 3' termini was investigated . Double stranded DNA was found to be a competitive inhibitor of the enzyme activity . Some four nucleotide 3' protrusions were shown to be sensitive to Exo III digestion . The synchronism of deletion was also examined . Implications for the proposed mechanism of activity are discussed. Plasmid, 1993 Mar, 29(2), 94 - 116 Control of mini-R1 plasmid replication: a computer simulation; Rosenfeld R et al.; A molecular model for the control of plasmid R1 replication has been proposed by Nordstrom, Molin and Light (Plasmid 12, 71-90, 1984), involving three genes: repA, copA, copB . RepA codes for a polypeptide whose synthesis is required for initiation; replication is controlled by regulating this synthesis . CopA encodes a small, unstable, untranslated RNA molecule that inhibits translation of the repA message whereas copB produces a protein that inhibits transcription from the repA promoter . We have recast this model into precise mathematical terms and tested it by computer simulation of a synchronous culture in steady-state balanced growth, composed of individual Escherichia coli cells harboring the small, unstable derivative, mini-R1 . All single-cell steady-state distributions obtained are independent of initial conditions, and the average values of various plasmid-related variables are similar to those measured experimentally . The relationship between the number of replication events per cell and the copy number at birth, as predicted by the model, mitigates against a sensitive correction mechanism for cells born with other than average copy number and is much closer quantitatively to a nonresponse system, although there is a weak dependence on copy number . The effect of the convergent transcription initiated at the repA and copB promoters on the expression of the copA gene is found to contribute little to the stability of mini-R1 replicons under steady-state growth conditions or to their potential for survival following infection . In fact, the role of the entire CopB control loop is shown to be quite minor, both in steady state and after infection . It is pointed out that genetic manipulations are far more easily performed in silico than in vivo but that results of the kind presented here are very often possible only when simulating individual cells. Plasmid, 1993 Mar, 29(2), 142 - 6 Conjugative transfer and in vitro/in vivo stability of the broad-host-range IncP R751 plasmid in Brucella spp; Verger JM et al.; Escherichia coli strain K12 BM14, carrying plasmid R751 (51.4 kb; Tpr, Tra+, IncP), was used as donor in matings with reference strains of the six Brucella nomenspecies, not known so far to harbor naturally occurring plasmids . R751 was easily transferred to and between Brucella spp., at frequencies ranging between 10(-1) and 10(-4) . All Brucella transconjugants stably maintained plasmid R751 both in vitro and in vivo in our experimental conditions . No genetic modification of the plasmid during and after the conjugal transfer process could be deduced from the comparative restriction analysis (BamHI, HindIII, and EcoRI) in each Brucella transconjugant and in the E . coli donor. Plasmid, 1993 Mar, 29(2), 117 - 24 Partition of plasmid R1: a computer simulation; Rosenfeld R et al.; A computer-simulated population of individual Escherichia coli cells harboring plasmid R1 parA+/parB- has been used to analyze three possible modes of plasmid segregation: equi-partition, in which plasmids are partitioned equally to daughter cells at cell division; single-site inheritance, in which the products of the most recent replication event are partitioned equally and the remaining plasmids are distributed randomly; and pair-site partition, in which a single, randomly chosen plasmid is actively partitioned to each daughter cell at division and the rest are distributed randomly . Comparison between predicted and experimental plasmid loss-frequency enabled us to rule out the first of these models as a likely mode of action in R1 but was inconclusive regarding the other two . The parA region would therefore seem to partition actively only one pair of plasmids to each daughter cell, the precise selection rule involved remaining unresolved . This question is not easily decided with current technology, as we show, but our simulation results also predict that the isolation of rep(Ts) mutants will provide an experimental system in which a clear distinction is possible between two plasmids that are the products of the most recent replication event and two that are chosen strictly at random. Prostaglandins Leukot Essent Fatty Acids, 1993 Mar, 48(3), 229 - 32 Indomethacin-induced gastrointestinal ulcers in rats: effects of dietary fatty acids and endotoxin; Turek JJ et al.; The purpose of this study was to determine how dietary n-3 and n-6 polyunsaturated fatty acids (PUFA) affected the gastrointestinal response to lipopolysaccharide (LPS) and indomethacin (INDO) in the rat . Rats were fed diets containing 12.5% linseed oil (LO-enriched in short-chain n-3 PUFA) or corn oil (CO-enriched in n-6 PUFA) . After 30 days on the diets, rats were given one of three treatments 1:10 mg/kg O55:B5 Escherichia coli LPS intraperitoneally (i.p.), 2:25 mg/kg INDO subcutaneously (s.c.), 3: a combination of 10 mg/kg i.p . LPS and 25 mg/kg (s.c.) INDO given 30 min before LPS . 20 h after challenge, rats were given an intravenous injection of Monastral Blue B to stain ulcer areas in the gastrointestinal tract . Lipopolysaccharide did not result in any Monastral Blue B vascular leakage in the gastrointestinal tract . Rats on the LO diet had significantly increased stomach and intestinal ulcers compared to CO fed rats . When rats were challenged with LPS and INDO, the LPS almost completely eliminated small intestinal ulcers, but enhanced ulcer development in the stomach. Mol Microbiol, 1993 Mar, 7(5), 795 - 803 TraM of plasmid R1 regulates its own expression; Schwab M et al.; Regulation of the traM gene, which encodes a factor essential for conjugation of resistance plasmid R1, was studied in vivo using translational gene fusion . traM"lacZ fusion constructs were transferred to the chromosome via the recombinant phage lambda RZ5 . The level of beta-galactosidase expressed by the lysogens indicates that the traM promoters are very active . Expression of traM was diminished five- to sixfold when the single-copy plasmids R1 or R1-19 were present in trans . When recombinant plasmids carrying traM were present at higher copy numbers, traM expression was reduced as much as 45-fold . The negative effect of R1 plasmids on traM expression in trans, which we interpret as autoregulation, was observed regardless of whether the plasmids were conjugatively repressed or derepressed . Site-specific mutagenesis of the region encoding the N-terminus of the TraM protein eliminated the autoregulative effect indicating that the N-terminal amino acids of the protein are important to its DNA-binding function . The autoregulatory behaviour of TraM is allele specific . R1- or P307-encoded TraM molecules were found to recognize only the cognate DNA. Mol Microbiol, 1993 Mar, 7(5), 693 - 704 Functional analysis of the cya promoter of Bordetella pertussis; Goyard S et al.; The cyaA gene of Bordetella pertussis and of Bordetella bronchiseptica encodes a toxin which is a bifunctional protein exhibiting adenylate cyclase and haemolytic activities . In Bordetella, virulence factors are synthesized under the control of the bvg regulatory locus, in response to environmental signals . In Escherichia coli the cyaA gene is not expressed, nor is it activated by bvg indicating that the activation of cya by bvg is indirect . To characterize cis-acting regulatory regions required for the activation of the cyaA gene we constructed cyaA-lacZY fusions containing progressive deletions in the promoter upstream region and isolated promoter mutations by chemical and site-directed mutagenesis . Deletion analysis shows that a region extending from -569 to -136 bp upstream from the start site of transcription is required for transactivation by bvg, suggesting that multiple binding sites are involved in the activation of the cyaA promoter . No single or double mutations in the promoter upstream region were found which conferred inactive or bvg-independent Cya phenotype . A double mutation in positions +10 and +13, relative to the transcription start site, rendered the promoter bvg-independent and functional in E . coli . The constitutive mutations create a new transcription start site, 20 bp downstream from the wild-type site, by providing new -10 and -35 elements recognized by RNA polymerase alone. J Pediatr Surg, 1993 Mar, 28(3), 334 - 6; discussion 336-7 The effect of endotoxin on the neonatal erythrocyte; Todd JC 3rd et al.; Intestinal ischemia is considered a major factor in the development of necrotizing enterocolitis (NEC) . Despite this, the majority of affected infants lack documentation of clinical events associated with obvious gut hypoperfusion . Recent evidence in adults suggests that endotoxin may impair flow in the microcirculation through alterations in erythrocyte deformability . As the gut serves as a semipermeable reservoir of endotoxin in the stressed neonate, such localized activity may result in intestinal ischemia at the microcirculatory level through alterations in the red cell membrane . This study evaluates the role of endotoxin on neonatal erythrocyte membrane viscosity . Paired anticoagulated whole blood specimens were obtained from the umbilical cord of 10 neonates at delivery . Samples were incubated with either 2 micrograms/mL of E coli endotoxin (LPS) or an equal volume of saline (control) . Following incubation, erythrocytes were isolated, washed, and incorporated with the fluorescent membrane probe TMA-DPH . Membrane viscosity was assessed by spectroscopic analysis of the fluorescent emissions induced by excitation of the probe at 365 nm . Results were calculated as anisotropy and analyzed for differences by ANOVA . Endotoxin resulted in a significant increase in red cell membrane viscosity as compared to control (LPS 291.2 +/- 5.1 v Control 271.7 +/- 3.3, P < .01) . As the effects of endotoxin are known to be primarily the result of white blood cell (WBC) activation, this study was repeated in an additional 10 neonates in whom WBCs were removed prior to endotoxin/saline incubation.(ABSTRACT TRUNCATED AT 250 WORDS) Plant Mol Biol, 1993 Mar, 21(5), 929 - 35 Tissue-specific expression of a plant turgor-responsive gene with amino acid sequence homology to transport-facilitating proteins; Guerrero FD et al.; We report the isolation of a turgor-responsive gene of pea, trg-31, whose transcription induced within 30 min after the loss of leaf turgor . Structure of the coding region and 1.4 kb of 5' untranslated region was determined by DNA sequencing . A 3 kb promoter fragment from trg-31 was fused to a beta-glucuronidase (GUS) reporter gene in pBI101, tobacco leaf disks and mature plants analyzed for turgor-responsive induction of GUS mRNA . Significant amino acid sequence homology exists between trg-31 and putative transport proteins of bovine, Phaseolus, soybean and Escherichia coli membranes. Plant Mol Biol, 1993 Mar, 21(5), 913 - 8 Structure of a cyanobacterial gene encoding the 50S ribosomal protein L9; Malakhov MP et al.; The rplI gene encoding the ribosomal protein L9 was found 4 kbp downstream from the desA gene, but on the opposite strand, in the genome of the cyanobacterium Synechocystis PCC6803 . The deduced amino acid sequence is homologous to the sequences of the L9 proteins from Escherichia coli and chloroplasts of Arabidopsis and pea . The gene is present as a single copy in the chromosome and is transcribed as a mRNA of 0.64 kb . An open reading frame of unknown function (ORF291) was found in the upstream region of the rplI gene. Plant Mol Biol, 1993 Mar, 21(5), 835 - 45 Isolation and characterization of the genes encoding allophycocyanin subunits and two linker proteins from Synechocystis 6714; DiMagno L et al.; Genes encoding the phycobilisome core subunits allophycocyanin alpha and beta and a small core linker protein in Synechocystis sp . strain PCC 6714 were cloned and sequenced . These genes form an operon, apcABC, with a single transcription start site and two possible termination sites, one following apcB and the other following apcC . The promoter region, like those of the apcABC operons of other cyanobacteria, does not resemble the consensus promoter sequences of Escherichia coli . However, the apcABC promoters identified in four strains of cyanobacteria have conserved sequences centered at -50 and -10 with respect to the start of transcription . The apcE gene, encoding the protein that links the phycobilisome core to the thylakoid membrane, was also cloned from Synechocystis 6714 and sequenced . It is unlinked to the apcABC operon . As in other Synechocystis strains, the LCM polypeptide encoded by the apcE gene contains three repeats of the basic phycobiliprotein linker domain . The apcE gene promoter sequence bears little resemblance to either the E . coli consensus or the apcABC promoter region, but it is similar to the corresponding regions of other cyanobacterial apcE genes . In these cases, there are conserved sequences centered at -40 and -10 with respect to the transcription start site . These conserved promoter elements from the apcABC and apcE genes were also identified in the corresponding 5'-flanking regions of eleven transcript starts for cpc genes encoding phycocyanin subunits in cyanobacteria and algal chloroplasts . These results suggest that a factor yet to be described participates in transcription of phycobiliprotein genes. Plant Mol Biol, 1993 Mar, 21(5), 753 - 64 The Rubisco activase (rca) gene is located downstream from rbcS in Anabaena sp . strain CA and is detected in other Anabaena/Nostoc strains; Li LA et al.; A gene encoding ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) activase (rca) was found downstream from the rbcLrbcS operon in the heterocystous cyanobacterium Anabaena sp . strain CA . Two unknown open reading frames were shown to be located between rbcS and rca in strain CA and all the genes, rbcLrbcS, ORF1, ORF2, and rca were in the same transcriptional orientation . The deduced amino acid sequence of the Anabaena Rubisco activase showed both similarities and differences to the plant enzyme with considerable differences at the carboxy and amino termini . Proposed ATP-binding sites were conserved in the cyanobacterial protein . Recombinant cyanobacterial Rubisco activase, however, reacted with antisera to spinach Rubisco activase . Hybridization studies, using the Anabaena sp . strain CA rca gene as a heterologous probe, detected homologous sequences in heterocystous Anabaena/Nostoc strains but not in unicellular or nonheterocystous filamentous cyanobacteria, suggestive of a close evolutionary relationship of chloroplasts and heterocystous cyanobacteria. Plant Mol Biol, 1993 Mar, 21(5), 739 - 52 Isolation and characterization of a cDNA encoding NADP(+)-specific isocitrate dehydrogenase from soybean (Glycine max); Udvardi MK et al.; A cDNA that encodes an NADP-specific isocitrate dehydrogenase (IDH) was cloned from a soybean nodule cDNA library by complementation of an Escherichia coli mutant that lacked IDH . DNA sequence analysis showed that the 1583 bp soybean cDNA could encode a protein that shares 63.9% amino acid sequence identity with the Saccharomyces cerevisiae NADP-IDH and long sequences of identity to an IDH from pig . Southern blot analysis suggests that this gene corresponds to a gene family made up of no more than two loci . The IDH cDNA hybridized to a 1.7 kb soybean mRNA and the relative amount of this transcript in soybean leaves, nodules and roots was 1:3.4:7.7 . In alfalfa, a 1.7 kb mRNA was also found but the ratios for the corresponding tissues were 1:7.4:7.7 . IDH activity was detected in the complemented E . coli strain and the electrophoretic mobility of this activity in nondenaturing polyacrylamide gels was identical to that of an IDH in extracts from soybean cotyledons or nodule cytosol . NADP-IDH specific activity in the E . coli host strain varied with growth phase; the highest rates (ca . 180 nmol/min per mg protein) were observed in late-stationary-phase cells . The enzyme had a broad pH optimum of 8.0 to 9.5 and had an absolute metal cofactor requirement, preferring Mn2+ below pH 8.0 and Mg2+ above pH 8.0 . The Km for isocitrate and NADP was 21 microM and 11 microM respectively with Mn2+ as cofactor and 13 microM and 12 microM with Mg2+ as cofactor. Mutagenesis, 1993 Mar, 8(2), 149 - 54 Determination of target nucleotides involved in 7-methoxy-2-nitro-naphtho{2,1-b}furan (R7000)-DNA adduct formation; Touati E et al.; The characterization of target nucleotides involved in the binding to DNA of 7-methoxy-2-nitro-naphtho{2,1-b}furan (R7000), a very potent genotoxic nitrofuran derivative, was investigated . Since R7000 undergoes metabolic activation prior to interacting with DNA, plasmids containing AT-rich and GC-rich sequences were devised and treated by R7000 in bacterial cells presenting nitroreductase activity . The nucleotide modifications to these homogeneous fragments that resulted from R7000 treatment were analyzed using the 'postlabeling' method . A preferential binding to the GC segment was demonstrated . Using a modification of the Maxam-Gilbert sequencing technique, it was demonstrated that activated R7000 creates alkali-labile phosphodiester bonds at the positions of guanines . In addition, the analysis of DNA replication-blocking properties of R7000 lesions was performed using avian myeloblastosis virus (AMV) reverse transcriptase as DNA polymerase . The termination of DNA replication occurred preferentially at the sites of guanine residues in the template strand, indicating that one nucleotide was inserted opposite a lesion . All these results indicate that guanine residues are the preferential sites of formation of R7000-DNA adducts. Mutagenesis, 1993 Mar, 8(2), 133 - 9 Spontaneous mutagenesis in Escherichia coli harbouring plasmid pKM101: DNA sequence analysis of forward lacI- mutations; Gordon AJ et al.; To investigate the influence of plasmid pKM101 on spontaneous mutagenesis, 198 lacI- mutations generated in Escherichia coli harbouring pKM101 were characterized at the DNA sequence level . pKM101 by itself did not enhance the lacI- forward mutation frequency . In general, the resultant distribution of mutation highlights particular sequences at which a variety of mutational events repeatedly occur, including 5'-(G/C)TGG-3', 5'-CCAGG-3', 5'-GATC-3' and 5'-TCGCG-3' sequences . Specifically, the distribution of mutation within base substitution and deletion classes distinguishes the pKM101 spectrum from the wild-type distribution (i.e . absence of pKM101) . An even distribution amongst base substitutions was observed which corresponds to a 2.9- to 6.3-fold increase in occurrence of low frequency events (A-->G,T,C; G-->C); high frequency events in the wild-type distribution (G-->A,T) were not influenced by the presence of pKM101 . One complex event was recovered which was comprised of two base substitutions separated by 4 bp . An 11-fold increase in small deletion events (3-6 bp) was also observed . The observed pKM101 spectrum does not closely resemble the mutational consequences of SOS induction in the absence of mutagenic treatment (recA441 spectrum) but does return a distribution like that obtained in E . coli deficient in polymerase I activity (polA1 spectrum). J Nutr, 1993 Mar, 123(3), 481 - 8 Oral arginine supplementation does not affect lymphocyte proliferation during endotoxin-induced inflammation in rats; Torre PM et al.; Earlier studies from this laboratory were unable to confirm reported immunostimulatory effects of supplemental dietary arginine on healthy, unstressed young or aged rats . The present study was undertaken to determine effects of oral arginine supplementation on in vitro measures of immune function using a stressed rat model . The stressor used was intraperitoneal injection of bacterial lipopolysaccharide (1 mg/kg body wt) . Four-month-old male Sprague-Dawley rats were placed in either a control or an arginine-supplemented (7.5 g/L arginine-HCl in drinking water) group for 7 d, after which control and supplemented rats received injections of endotoxin or phosphate-buffered saline . Rats were killed 3 d following injections . Endotoxin treatment resulted in lower food intake, less thymic cellularity and greater splenic weight . Endotoxin injections also enhanced proliferative response of rat splenocytes to pokeweed mitogen (1 mg/L) and lipopolysaccharide (25 and 100 mg/L) and enhanced response of thymocytes to concanavalin A (10 mg/L), phytohemagglutinin (25 and 100 mg/L) and pokeweed mitogen (1 mg/L) . Supplemental arginine did not reduce thymic weight loss or influence mononuclear cell proliferation or interleukin-2 production in the presence or absence of endotoxin stress . These data indicate no benefit of arginine supplementation during endotoxin stress in rats. J Dairy Sci, 1993 Mar, 76(3), 722 - 7 Effects of intramammary endotoxin infusion on milking-induced oxytocin release; Gorewit RC; One overt sign of clinical coliform mastitis in dairy cows is the failure to eject milk normally or to "milk out" the udder . The effect, if any, of coliform mastitis on oxytocin release is unknown . Therefore, the objective of this study was to determine the effect of endotoxin mastitis on milking-induced release of oxytocin in lactating cows . Fifteen multiparous pregnant lactating Holstein cows were divided into three groups of 5 cows each . Cows in group 1 served as controls and received an intramammary infusion of sterile physiological saline . Cows in groups 2 and 3 received intramammary infusions of 12.5 and 25 micrograms of Escherichia coli endotoxin, respectively . Serum concentrations of oxytocin were measured by radioimmunoassay before, during, and after milkings commencing at 6 and 12 h after treatment . Rectal temperatures and milk SCC were monitored to follow the course of inflammation and to verify the biological activity of infused endotoxin . Endotoxin resulted in a 1.5- to 2-fold increase in milking-induced oxytocin release compared with that of control treatments . The effect was most prominent during the first 6 h after infusion and coincided with the peak pyretic response . This study shows that endotoxin-induced mastitis potentiates, rather than inhibits, milking-induced oxytocin release. Int J Biochem, 1993 Mar, 25(3), 427 - 32 Comparative analysis of the antibodies against capsular polysaccharides of Escherichia coli K-92 and K-235: an immunochemical method for the identification of polysialic acids; Rodriguez-Aparicio LB et al.; 1 . The antibodies produced against the capsular poly-N-acetylneuraminic acid (poly-Neu5Ac) of E . coli K-92 (alpha 2-8-, alpha 2-9-linked) were 100-fold less sensitive than those obtained against E . coli K-235 capsular polysaccharide (CP) (alpha 2-8-linked) and recognized both kinds of polymers to a similar extent . 2 . The partial hydrolysis of each purified polysaccharide revealed that E . coli K-92 CP is more labile at acidic pH than the polymer alpha 2-8-linked of E . coli K-235 . 3 . The antisera against CP from E . coli K-92 bound its own oligomers in which the number of Neu5Ac units was higher than three, whereas they only cross-reacted with the oligomers derived from E . coli K-235 containing a number of residues higher than 12 . 4 . The antisera against E . coli K-235 CP that recognized alpha 2-8 oligomers with a number of Neu5Ac residues higher than 5, also reacted, although very weakly, with those containing alpha 2-8 and alpha 2-9 linkages in which the carbon length was higher than (Neu5Ac)3 . 5 . Both types of antibodies were also able to recognize the native antigens in living bacteria and could be employed for the recognition of the type of linkage presents in different sialylpolymers. Int J Biochem, 1993 Mar, 25(3), 359 - 66 Expression of a putative catalytic domain of the human APEX nuclease (a major apurinic/apyrimidinic endonuclease) in Escherichia coli; Ono Y et al.; 1 . Sequence analyses of APEX nuclease, a mammalian major apurinic/apyrimidinic (AP) endonuclease homologous to Escherichia coli exonuclease III, suggested that APEX nuclease is organized into two domains, a Mr 6000 N-terminal domain containing nuclear location signals and a Mr 29,000 C-terminal catalytic domain . 2 . In order to study the enzyme structure further, vectors expressing APEX nuclease (pTAPXH1) and the Mr 29,000 C-terminal region (pTAPXH61) were constructed using cDNA (APX cDNA) for the human APEX nuclease and pTrc99A plasmid . The constructs were introduced into BW2001 strain (xth-11, nfo-2) cells of E . coli to produce transformants designated as BW2001/pTAPXH1 and BW2001/pTAPXH61, respectively . Both the APEX nuclease expressed in BW2001/pTAPXH1 and the Mr 29,000 C-terminal peptide expressed in BW2001/pTAPXH61 were partially purified by column chromatography and highly purified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis . 3 . The purified APEX nuclease and the Mr 29,000 C-terminal peptide both showed equally high AP endonuclease activity which indicates that the Mr 29,000 C-terminal region of the APEX nuclease is (or contains) the AP endonuclease domain. Cell Immunol, 1993 Mar, 147(1), 148 - 57 The influence of DNA structure on the in vitro stimulation of murine lymphocytes by natural and synthetic polynucleotide antigens; Messina JP et al.; Although DNA is generally considered to be a poor immunogen, recent evidence suggests that DNA from various species differ in their immunological activity and that bacterial DNA can induce the in vitro proliferation of normal murine B cells . To delineate structural features of DNA associated with mitogenic activity, the response of murine lymphocytes to various natural and synthetic polynucleotides was determined . Both ss and dsDNA from two different bacterial strains were equally effective in inducing proliferation . This response was independent of adenosine methylation, since DNA from dam- Escherichia coli stimulated proliferation . Among the synth