J Biol Chem, 1995 Jan 6, 270(1), 99 - 109 A molecular genetic approach for the identification of essential residues in human glutathione S-transferase function in Escherichia coli; Lee HC et al.; The common substrate for glutathione S-transferases (GSTs), 1-chloro-2,4-dinitrobenzene (CDNB), is an inhibitor of Escherichia coli growth . This growth inhibition by CDNB is enhanced when E . coli expresses a functional GST . Cells under growth inhibition have reduced intracellular GSH levels and form filaments when they resume growth . Based on this differential growth inhibition by CDNB we have developed a simple procedure to select for null-mutants of a human GST in E . coli . Null mutations in the human GST gene from hydroxylamine mutagenesis or oligonucleotide-directed mutagenesis can be selected for on agar plates containing CDNB after transformation . The molecular nature of each mutation can be identified by DNA sequence analysis of the mutant GST gene . We have identified three essential amino acid residues in an alpha class human GST at Glu31, Glu96, and Gly97 . Single substitution at each of these residues, E31K, E96K, G97D, resulted in mutant GST proteins with loss of CDNB conjugation activity and failure in binding to the S-hexyl GSH affinity matrix . In contrast, a mutant GST (Y8F) resulting from substitution of the conserved tyrosine near the N terminus has much reduced CDNB conjugation activity but was still capable of binding to the S-hexyl GSH-agarose . Additional mutant GSTs with substitutions at position 96 (E96F, E96Y) and 97 (G97P, G97T, G97S) resulted in changes in both Km and kcat to different extents . The in vitro CDNB conjugation activity of the purified mutant enzymes correlate negatively with the plating efficiencies of strains encoding them in the presence of CDNB . Based on the x-ray structure model of human GST 1-1, two of these residues are involved in salt bridges (Arg19-Glu31, Arg68-Glu96) and the third Gly97 is in the middle of the helix alpha 4 . Our results provide evidence in vivo that Tyr8, Gly97, and the two salt bridges are important for GST structure-function . This molecular genetic approach for the identification of essential amino acids in GSTs should be applicable to any GSTs with CDNB conjugation activity . It should also complement the x-ray crystallographic approach in understanding the structure and function of GSTs. J Biol Chem, 1995 Jan 6, 270(1), 87 - 93 Changing the ion binding specificity of the Escherichia coli H(+)-transporting ATP synthase by directed mutagenesis of subunit c; Zhang Y et al.; Most F1F0 type ATP synthases, including that in Escherichia coli, use H+ as the coupling ion for ATP synthesis . However, the structurally related F1F0 ATP synthase in Propionigenium modestum uses Na+ instead . The binding site for Na+ residues in the F0 sector of the P . modestum enzyme . We postulated that Na+ might interact with subunit c of F0 . Subunit c of P . modestum and E . coli are reasonably homologous (19% identity) but show striking variations around the H(+)-translocating, dicyclohexylcarbodiimide-reactive carboxyl (Asp61 in E . coli) . Several hydrophobic residues around Asp61 were replaced with polar residues according to the P . modestum sequence in the hope that the polar replacements might provide liganding groups for Na+ . One mutant from 31 different mutation combinations did generate an active enzyme that binds Li+, the combination being V60A, D61E, A62S, and I63T . Li+ binding was detected by Li+ inhibition of ATP-driven H+ transport, Li+ inhibition of F1F0-ATPase activity, and Li+ inhibition of F0-mediated H+ transport . The Li+ effects were observed with membrane vesicles prepared from a delta nhaA, delta nhaB mutant background which lacks Na+/H+ antiporters, and with purified, reconstituted preparations of F0 prepared from this background strain . Li+ inhibition was observed at pH 8.5 but not at pH 7.0 . H+ thus appears to compete with Li+ for the binding site . Li+ binding was abolished by replacement of Glu61 by Asp or Ser62 by Ala . The side chains at Ala60 and Thr63 may act in a supporting structural role by providing a more flexible conformation for the Li+ binding cavity . Thr63 does not appear to provide a liganding group since H+ transport in two other mutants, with Gly or Ala in place of Thr63, was also inhibited by Li+ . We suggest that a X-Glu-Ser-Y or X-Glu-Thr-Y sequence may provide a general structural motif for monovalent cation binding, and that the flexibility provided by residues X and Y will prove crucial to this structure. J Biol Chem, 1995 Jan 6, 270(1), 274 - 80 Mechanism of inhibition of human nonpancreatic secreted phospholipase A2 by the anti-inflammatory agent BMS-181162; Burke JR et al.; Many important mediators of inflammation result from the liberation of free arachidonic acid from phospholipid pools which is thought to result from the action of phospholipase A2 (PLA2) . It is believed, therefore, that the inhibition of PLA2 would be an important treatment in many inflammatory disease states . The anti-inflammatory agent BMS-181162 (4-(3'-carboxyphenyl)-3,7-dimethyl-9-(2",6",6"-trimethyl-1"-cyclohexenyl )-2Z,4E , 6E,8E-nonatetraenoic acid) selectively inhibits PLA2 and has been shown to block arachidonic acid release in whole cells . The mechanism of inhibition of human non-pancreatic-secreted PLA2 by BMS-181162 is investigated in this paper . A scooting mode assay in which the enzyme is irreversibly bound to vesicles of 1,2-dimyristoyl-sn-glycero-3-phosphomethanol containing 5 mol % of 1-palmitoyl-2-{1-14C}arachidonoyl-sn-glycero-3-phosphocholine, was used to characterize the inhibition . With this assay system, BMS-181162 inhibited the enzyme in a dose-dependent manner . Compounds which inhibit in the scooting mode have been shown to be competitive inhibitors in the interface (Gelb, M . H., Berg, O., and Jain, M . K . (1991) Curr . Op . Struct . Biol . 1, 836-843) . This was verified by demonstrating that the inhibition was not due to the desorption of the enzyme from the lipid-water interface . Additionally, the compound did not measurably affect the rate of association onto the vesicles . Therefore, the inhibition was not the result of a modulation of the bilayer morphology nor an interaction with the interfacial binding site on the enzyme . The degree of inhibition was dependent on the reaction volume which indicates that the inhibitor is only partially partitioned into the bilayer . After compensating for this partitioning, the dose-dependent inhibition could be defined by kinetic equations describing competitive inhibition at the interface . The equilibrium dissociation constant for the inhibitor bound to the enzyme at the interface (KI*) was determined to be 0.013 mol fraction, thus demonstrating that BMS-181162 represents a novel structural class of tight-binding competitive inhibitors of human nonpancreatic secreted PLA2 . Using Escherichia coli membranes as substrate, to which the enzyme binds to the interface reversibly, the inhibition showed a nonclassical kinetic pattern which is also consistent with a partial partitioning of the inhibitor into the bilayer . This was verified by a direct measurement of the amount of inhibitor remaining in solution . The implications for in vivo efficacy which result from this mechanism are discussed. J Biol Chem, 1995 Jan 6, 270(1), 214 - 7 Inositol hexakisphosphate binds to clathrin assembly protein 3 (AP-3/AP180) and inhibits clathrin cage assembly in vitro; Norris FA et al.; We have isolated an inositol hexakisphosphate binding protein from rat brain by affinity elution chromatography from Mono S cation exchange resin using 0.1 mM inositol hexakisphosphate (InsP6) . The amino acid sequences of six tryptic peptides from the protein were identical to the sequences predicted from the cDNA encoding a previously isolated protein designated as AP-3 or AP180 . This protein is localized in nerve endings and promotes assembly of clathrin into coated vesicles . The isolated protein-bound InsP6 with a dissociation constant of 1.2 microM and a stoichiometry of 0.9 mol of InsP6 bound/mol of AP-3 . Recombinant AP-3 expressed in Escherichia coli also bound InsP6 with a similar affinity . InsP6 inhibited clathrin cage assembly mediated by AP-3, in an in vitro assay, but had little effect AP-3 binding to preformed cages . We speculate that InsP6 and perhaps highly phosphorylated inositol lipids may play a role in coated vesicle formation. J Biol Chem, 1995 Jan 6, 270(1), 170 - 9 A novel mutagenesis strategy identifies distantly spaced amino acid sequences that are required for the phosphorylation of both the oligosaccharides of procathepsin D by N-acetylglucosamine 1-phosphotransferase; Dustin ML et al.; A novel combinatorial mutagenesis strategy (shuffle mutagenesis) was developed to identify sequences in the propiece and amino lobe of cathepsin D which direct oligosaccharide phosphorylation by UDP-GlcNAc:lysosomal enzyme N-acetylglucosamine 1-phosphotransferase . Propiece restriction fragments and oligonucleotide cassettes corresponding to 13 regions of the cathepsin D and glycopepsinogen amino lobes were randomly shuffled together to generate a large library of chimeric molecules . The library was inserted into an expression vector encoding the carboxyl lobe of cathepsin D with a carboxyl-terminal myc epitope and a CD8 transmembrane extension . Transfected COS1 cells expressing the membrane-anchored forms of the cathepsin D/glycopepsinogen chimeras at the cell surface were selected with solid phase mannose 6-phosphate receptor or an antibody to the myc epitope . Plasmids were rescued in Escherichia coli and sequenced by hybridization to the original oligonucleotide cassettes . Two regions of the cathepsin D amino lobe (segments 7 and 12) were found to contribute to proper folding, surface expression, and selective phosphorylation of the carboxyl lobe oligosaccharide . Two different cathepsin D regions (the propiece and segment 5) cooperated with a previously identified recognition element in the carboxyl lobe to allow efficient phosphorylation of both the amino and carboxyl lobe oligosaccharides . Three general models for extending the catalytic reach of N-acetylglucosamine 1-phosphotransferase to widely spaced oligosaccharides are presented. J Biol Chem, 1995 Jan 6, 270(1), 17 - 20 Molecular cloning and expression of rat squalene epoxidase; Sakakibara J et al.; Squalene epoxidase (SE) (EC 1.14.99.7) catalyzes the first oxygenation step in sterol biosynthesis and is suggested to be one of the rate-limiting enzymes in this pathway . Rat SE cDNA was isolated by selecting yeast transformants expressing rat cDNA in the presence of transformants expressing rat cDNA in the presence of terbinafine, an inhibitor specific for fungal SE . The expression of rat SE in the isolated terbinafine-resistant clone was confirmed by its survival in the presence of either terbinafine or an inhibitor specific for mammalian SE, NB-598, but not in the presence of both terbinafine and NB-598 . Rat SE polypeptide deduced from the nucleotide sequence contains 573 amino acids, and its molecular weight is 63,950 Da . The amino acid sequence reveals one potential transmembrane domain, a hydrophobic segment (Leu27 to Tyr43) in the NH2-terminal region . This region also contains a beta 1-alpha A-beta 2 motif, which is the consensus sequence for an FAD binding domain, suggesting that SE is a flavoenzyme . This deduced rat SE sequence is 30.2% identical to the ERG 1 gene, which encodes SE from an allylamine-resistant Saccharomyces cerevisiae mutant . Expression of a full-length rat SE protein in Escherichia coli confirms this polypeptide as a functional SE . This is the first report of the molecular cloning of mammalian SE. J Biol Chem, 1995 Jan 6, 270(1), 163 - 9 Expression and folding of recombinant bovine prethrombin-2 and its activation to thrombin; DiBella EE et al.; Bovine prethrombin-2 has been produced in Escherichia coli using a T7 expression system . The expressed prethrombin-2 formed intracellular inclusion bodies which were solubilized by reversible sulfonation of the cysteines in the presence of 7 M guanidine hydrochloride . Sulfonated prethrombin-2 was refolded in the presence of 4 M guanidine hydrochloride, using oxidized and reduced glutathione as the redox couple . The folded protein was purified by heparin affinity chromatography and activated to thrombin with Echis carinatus snake venom . The resulting thrombin was also purified by heparin affinity chromatography . Kinetic constants were determined for the hydrolysis of H-D-phenylalanyl-L-pipecolyl-L-arginine-p-nitroaniline by recombinant thrombin (kcat = 123 +/- 10 s-1 and Km = 2.91 +/- 0.3 microM) . These values are in good agreement with those determined for wild-type thrombin (kcat = 97 +/- 8 s-1 and Km = 2.71 +- 0.25 microM) . From the thrombin-mediated release of fibrinopeptide A from fibrinogen, kcat/Km was found to be the same for recombinant (17.3 +/- 1.2 microM-1 s-1) and wild-type (16.7 +/- 2.0 microM-1 s-1) thrombin . These results, taken together with circular dichroism spectra and the elution position of prethrombin-2 from a heparin affinity resin, indicate that prethrombin-2 was folded into a conformation similar to that of the wild-type protein . In addition, since E . coli produces deglycosylated enzymes, these findings suggest that the carbohydrate on the B chain of wild-type thrombin does not affect the amidolytic and fibrinolytic activities of thrombin . Finally, this expression system can be used to prepare mutants of prethrombin-2 for future structure-function studies involving thrombin and its substrates; some preliminary results of this type are presented here. Science, 1995 Jan 6, 267(5194), 87 - 90 Experimental tests of the roles of adaptation, chance, and history in evolution; Travisano M et al.; The contributions of adaptation, chance, and history to the evolution of fitness and cell size were measured in two separate experiments using bacteria . In both experiments, populations propagated in identical environments achieved similar fitnesses, regardless of prior history or subsequent chance events . In contrast, the evolution of cell size, a trait weakly correlated with fitness, was more strongly influenced by history and chance. Oncogene, 1995 Jan 5, 10(1), 27 - 32 Modulation by copper of p53 conformation and sequence-specific DNA binding: role for Cu(II)/Cu(I) redox mechanism; Hainaut P et al.; The tumor suppressor protein p53 is a metal-binding transcription factor whose conformation and function are altered by mutation in cancers . Using murine p53 translated in vitro, we report here that concentrations of copper within the physiological range (< 30 microM) alter the conformation of wild-type p53 and inhibit sequence-specific DNA-binding . Direct binding of copper to p53 in the form of Cu(I) was demonstrated by Electron Spin Resonance using a purified recombinant protein containing residues 1-343 of murine wild-type p53 fused to E . coli maltose binding protein . Moreover, protection against the effect of Cu(II) sulfate was achieved by the Cu(I)-specific chelator bathocuproinedisulfonic acid but not by scavengers of reactive oxygen species, suggesting that alteration of p53 by copper depends upon a Cu(II)/Cu(I) redox mechanism, but does not require the production of reactive oxygen species . Thus copper at physiological concentrations can interact with wild-type p53 and affect its DNA-binding capacity. Biochem Biophys Res Commun, 1995 Jan 5, 206(1), 414 - 20 Mucosal T cells induce systemic anergy for oral tolerance; Takahashi I et al.; Heat-labile enterotoxin (LT) from enterotoxigenic Escherichia coli is known to possess strong immunoregulatory potential in terms of inhibition of the induction of oral tolerance and adjuvanticity in oral immunization . We found that oral administration of an immunogenic peptide of LT {LT-B(26-45); spanning the residues 26-45 of LT-B} induced systemic unresponsiveness in BALB/c mice resulting in diminished serum IgG responses . It was also shown that the spleen (SP) CD4+ T cells of tolerized mice failed to proliferate, whereas the Peyer's patches (PP) CD4+ T cells responded to the peptide . RT-PCR revealed that the SP CD4+ T cells did not generate IL-2 mRNA, while the PP CD4+ T cells expressed significant levels of IFN-gamma, IL-2, IL-4, and TGF-beta mRNA . Adoptive transfer of LT-B-specific intraepithelial lymphocytes to the tolerant mice abrogated the tolerance . In the reversed mice, LT-B(26-45)-stimulated SP CD4+ T cells expressed significant levels of IFN-gamma, IL-2, IL-4, and IL-6 mRNA . These results indicate that PP CD4+ T cells induce oral tolerance due to systemic T cell anergy. Biochem Biophys Res Commun, 1995 Jan 5, 206(1), 393 - 400 Synthesis of non-translating or translating specialized ribosomes causes feedback regulation of ribosomal RNA synthesis in Escherichia coli; Leipold RJ et al.; Specialized ribosomes carry a mutant anti-Shine-Dalgarno region that disrupts the complementary base pairing that stabilizes the translation initiation complex with E . coli mRNAs . It has been reported that production of specialized ribosomes does not cause the inhibition of chromosomal rRNA synthesis that follows production of wild-type ribosomes . We proposed that enabling translation on specialized ribosomes by providing mRNA with a complementary mutation in the Shine-Dalgarno region would restore feedback regulation and inhibit chromosomal rRNA synthesis . With both our system and the system studied previously, we saw feedback regulation regardless of whether the specialized ribosomes were translating . As reported previously, transcription from plasmid-borne promoters decreased as chromosomal rRNA synthesis was repressed, suggesting that the lambda PL and tac promoters may be sensitive to the effector(s) of feedback regulation. Biochem Biophys Res Commun, 1995 Jan 5, 206(1), 362 - 9 Use of site-directed mutagenesis to identify the galactosyltransferase binding sites for UDP-galactose; Zu H et al.; Site-directed mutagenesis was utilized to identify binding sites for UDP-galactose in galactosyltransferase (EC 2.4.1.22) . Mutant cDNAs were generated by a procedure based on PCR, and the mutated enzymes were expressed in E.coli cells . The mutant enzymes were purified by Ni-NTA Sephadex, and the degree of purification was judged by SDS-PAGE . Purified mutant GTs, F305L, P306V, N307S, N308S, showed dramatic decreases in activities in comparison with the activity of the wild-type GT . Enzyme kinetic analysis revealed that the Km values of F305L, P306V, N307S and N308S for UDP-galactose were, respectively, 9-, 11-, 50- and 20-fold higher than the Km of wild-type GT, but the Km values for manganese were not significantly different from that of the wild-type GT . The quartet mutant F305L/P306V/N307S/N308S showed no activity . From the results of this study it is concluded that amino acids, Phe-305, Pro-306, Asn-307 and Asn-308, in GT are most probably involved in GT catalysis or are located close to the UDP-galactose binding region but are not involved in the binding of manganese. Biochem Biophys Res Commun, 1995 Jan 5, 206(1), 302 - 9 Point mutation in the second phosphatase domain of CD45 abrogates tyrosine phosphatase activity; Ng DH et al.; CD45 is a transmembrane protein tyrosine phosphatase that possesses two phosphatase domains in its cytoplasmic region . Whether both domains function independently as phosphatase enzymes or whether both domains interact to form an active enzyme is unclear . A point mutation of a critical cysteine residue in domain I is known to abolish CD45 activity, implying that the catalytic activity resides in domain I . In this report, mutational analysis of purified, recombinant CD45 cytoplasmic domain protein was performed . It was found that a single amino acid change in domain II (glutamine 1180 to a glycine) resulted in an inactive phosphatase enzyme, whereas two other point mutations in the membrane proximal region of the molecule had no effect on activity . Deletion of the region linking the two phosphatase domains also abolished enzymatic activity . Amino acids crucial for phosphatase activity thus reside in both phosphatase domains of CD45, illustrating that the phosphatase domains of CD45 do not act independently, but are both required for the phosphatase activity of CD45. Biochim Biophys Acta, 1995 Jan 5, 1246(1), 67 - 73 Residues affecting the catalysis and inhibition of rat lens aldose reductase; Carper DA et al.; Aldose reductase (AR), the first enzyme of the polyol pathway, has been implicated in diabetic complications . Results of recent clinical studies have shown that compounds that inhibit aldose reductase (ARIs) and block the flux of glucose through the polyol pathway have provided benefit to diabetic neuropathic patients . Since many ARIs show broad substrate specificity, emphasis on the structure-function properties of the AR enzyme will help in the refinement and design of future inhibitors . To this end, catalysis and inhibition of rat lens aldose reductase was examined following site-directed mutagenesis . Replacement of tyrosine 48 with phenylalanine (Y48F) resulted in an enzyme form with less than 0.25% activity with DL-glyceraldehyde and no detectable activity with p-nitrobenzaldehyde or xylose, although circular dichroism spectra and NADPH binding affinity were similar to wild-type AR . Mutation of histidine 110 to glutamine (H110Q) also resulted in a less active protein with an approximate 3-fold decrease in kcat for the reduction of DL-glyceraldehyde; slight or no activity was measured with other substrates and an increase of 195-fold over wild type was observed in the Km for glyceraldehyde . H110Q was less sensitive to inhibition by aldose reductase inhibitors . The most dramatic change was seen with imeristat, which showed an 1800-fold increase in IC50 . Mutation of cysteine 298 to serine (C298S) affected enzyme function by increasing kcat 2- to 4-fold and increasing Km 15- to 48-fold, with DL-glyceraldehyde, p-nitrobenzaldehyde or xylose as substrates . As a result kcat/Km, catalytic efficiency, dropped to approx . 10% of control . Inhibition of C298S was not noticeably different from wild type . Substitution of histidine 187 or 200 with glutamine (H187Q, H200Q) had little effect on AR catalysis or inhibition . Based on structural and mutagenesis studies of human AR and the conservation of amino acids between human and rat, these data would indicate that Y48, H110, and C298 are important residues in the active site of rat AR and that Y48 is most likely the proton donor during substrate reduction by rat lens aldose reductase . In addition, these studies indicate that mutagenesis of H110 also affects aldose reductase inhibition. EMBO J, 1995 Jan 3, 14(1), 151 - 8 The identity of the base following the stop codon determines the efficiency of in vivo translational termination in Escherichia coli; Poole ES et al.; A statistical analysis of > 2000 Escherichia coli genes suggested that the base following the translational stop codon might be an important feature of the signal for termination . The strengths of each of 12 possible 'four base stop signals' (UAAN, UGAN and UAGN) were tested in an in vivo termination assay that measured termination efficiency by its direct competition with frameshifting . Termination efficiencies varied significantly depending on both the stop codon and the fourth base, ranging from 80 (UAAU) to 7% (UGAC) . For both the UAAN and UGAN series, the fourth base hierarchy was U > G > A approximately C . UAG stop codons, which are used rarely in E . coli, showed efficiencies comparable with UAAN and UGAN, but differed in that the hierarchy of the fourth base was G > U approximately A > C . The rate of release factor selection varied 30-fold at UGAN stop signals, and 10-fold for both the UAAN and UAGN series; it correlated well with the frequency with which the different UAAN and UGAN signals are found at natural termination sites . The results suggest that the identity of the base following the stop codon determines the efficiency of translational termination in E . coli . They also provide a rationale for the use of the strong UAAU signal in highly expressed genes and for the occurrence of the weaker UGAC signal at several recording sites. FEBS Lett, 1995 Jan 3, 357(2), 212 - 6 Expression of an active adenylate-forming domain of peptide synthetases corresponding to acyl-CoA-synthetases; Dieckmann R et al.; Peptide synthetases and acyl-CoA-synthetases form acyl adenylates which are transferred to CoA or enzyme-bound pantetheine . To verify the existence of an adenylate domain in peptide synthetases, a 60.8 kDa fragment of tyrocidine 1-synthetase was constructed by a 1,629 bp deletion, expressed in Escherichia coli, and characterized . The truncated multienzyme activated phenylalanine and substrate analogues with comparable kinetics as the over-expressed synthetase, as judged by ATP-{32P}PP(i) exchange reaction . Thus the N-terminal domain resembling an acyl-CoA-synthetase is an autonomous structural element . This N-terminal domain is followed by a cofactor binding domain, resembling acyl carrier proteins involved in polyketide formation. Carbohydr Res, 1995 Jan 3, 266(1), 95 - 102 A partial reductive-cleavage study of the capsular polysaccharide of Escherichia coli K57; Parolis H et al.; Trideuteriomethylated and methylated derivatives of the capsular polysaccharide of Escherichia coli K57 were partially cleaved by Et3SiH, using Me3SiOSO2 Me and Me3SiOSO2CF3 as catalysts, to produce oligosaccharide-anhydroalditols . The structures of the trideuteriomethylated trisaccharide- and tetrasaccharide-anhydroalditols isolated were established by FABMS and NMR spectroscopy . Although conditions for the selective production of the tetrasaccharide-anhydroalditol could not be established, oligosaccharide-anhydroalditols were isolated in sufficiently high yield to make this an attractive approach for the structural elucidation of the repeating units of bacterial polysaccharides. FEBS Lett, 1995 Jan 2, 357(1), 62 - 4 Crystallization and preliminary crystallographic studies of recombinant dimerization cofactor of transcription factor HNF1/pterin-4 alpha-carbinolamine dehydratase from liver; Ficner R et al.; The bi-functional protein dimerization cofactor of HNF1 (DCoH)/pterin-4 alpha-carbinolamine dehydratase (PCD) is found in liver cell nuclei bound to the transcription factor hepatocyte nuclear factor 1 (HNF1) as well as in the cytoplasm acting as an enzyme involved in the phenylalanine hydroxylation system . Deficiency of DCoH/PCD activity in liver causes an atypical hyperphenylalaninemia and deficiency in human epidermis is related to the depigmentation disorder vitiligo . DCoH/PCD from rat liver, which is identical to the human protein, was expressed in E . coli, purified to homogeneity and crystallized . The crystals belong to the trigonal space group P3(1)21 (or P3(2)21) with unit cell dimensions of a = b = 106.2 A, c = 197.1 A . Native crystals diffract to a resolution of 2.5 A. FEBS Lett, 1995 Jan 2, 357(1), 19 - 22 Two GTPs are consumed on EF-Tu per peptide bond in poly(Phe) synthesis, in spite of switching stoichiometry of the EF-Tu.aminoacyl-tRNA complex with temperature; Dincbas V et al.; Recent observations indicate that the stoichiometry for the complex between EF-Tu.GTP and aminoacyl-tRNA (aa-tRNA) changes with temperature . At 37 degrees C two EF-Tu.GTPs bind one aa-tRNA in an extended ternary complex, but at 0 degrees C the complex has 1:1 stoichiometry . However, the present experiments show that there are two GTPs hydrolyzed on EF-Tu per peptide bond in poly(Phe) synthesis at 37 degrees C as well as at 0 degrees C . This indicates two different pathways for the enzymatic binding of aa-tRNA to the A-site on the ribosome. Biochim Biophys Acta, 1995 Jan 2, 1260(1), 73 - 8 Repression of transient expression by DNA methylation in transcribed regions of reporter genes introduced into cultured human cells; Komura J et al.; We developed a convenient method to methylate all CpG dinucleotides in both strands in a selected region of a plasmid, and investigated the effect of DNA methylation in the transcribed regions of reporter genes on the transient expression in HeLa cells . In a construct containing the chloramphenicol acetyltransferase (CAT) gene linked to the SV40 early promoter, methylation in the CAT structural gene repressed CAT activity . Methylation in the transcribed region of the Escherichia coli lacZ gene driven by the human cytomegalovirus immediate early promoter also inhibited expression of beta-galactosidase activity . These results suggest that methylation in the transcribed region as well as promoter methylation may affect transcription. Biochim Biophys Acta, 1995 Jan 2, 1260(1), 49 - 54 Molecular cloning and functional expression of a cDNA for mouse squalene synthase; Inoue T et al.; Using a probe obtained by PCR amplification, a full-length cDNA encoding squalene synthase was isolated from a mouse liver cDNA library . Its nucleotide sequence had an open reading frame fro a 416 amino acid polypeptide (calculated molecular mass, 48 kDa) . In vitro transcription of the cDNA followed by in vitro translation produced a protein of the expected size . The deduced amino acid sequence was 93%, 88% and 46% identical to those of the rat, human and budding yeast squalene synthases, respectively . Blotting analyses showed that the mRNA is 1.6 kb in size and that less than two copies of the gene are present in the mouse genome . To establish the enzyme activity, the entire coding region was subcloned into an expression plasmid so that it was in frame with the N-terminal region of beta-galactosidase . Escherichia coli, which was transformed with the recombinant plasmid, expressed high activity of converting farnesyl diphosphate into squalene. Biochim Biophys Acta, 1995 Jan 2, 1260(1), 27 - 34 High level expression of human leukemia inhibitory factor (LIF) from a synthetic gene in Escherichia coli and the physical and biological characterization of the protein; Samal BB et al.; LIF is a multi-functional cytokine that elicits effects on a broad range of cell types . In this report, we present the high level expression of human LIF (hLIF) from a chemically synthesized gene template in Escherichia coli where it comprises up to 25% of the cellular protein . The recombinant hLIF, after purification and folding, was examined using CD, FTIR spectroscopy and light scattering . CD and FTIR spectra showed that the hLIF is an alpha-helical protein and has a distinct tertiary structure . The IFTR spectrum resembles that of other four helical bundle proteins including G-CSF and IL-6 . Light scattering analysis indicated that it is a monomeric protein, distinguishing it from M-CSF and interferon gamma, which also belong to the class of four helical bundle proteins but are dimeric . Recombinant hLIF was assayed for its activity on the murine leukemic cell line, M-1 as well as on human leukemic cell line, ML-1 . It inhibited the growth of M-1 cells and differentiated them towards macrophages . However, it did not have any differentiation inducing effect on human leukemic cell lines alone or in combination with other cytokines. Biol Res, 1995, 28(4), 277 - 82 Humoral immune response anti K99 pilus from enterotoxigenic Escherichia coli in experimentally inoculated calves; Bustos C et al.; The bovine model is extremely interesting to study several basic aspects of mucosal local immunity . Many reports have shown that, in young calves, the infectivity of enterotoxigenic Escherichia coli may be inhibited by passively administered antibodies anti K99 pilus . We have measured, by immunoradiometric assays, the IgG response anti K99 pilus in the serum of calves, deprived of colostrum and orally inoculated with enteropathogenic K99+ E . coli . Although variable levels of IgG anti K99 pilus were detected, their protective value could not be ascertained in vivo due to the acute development of the infection . In an effort to correlate the presence of serum antibodies anti K99 pilus with their protective capacity, an ex-vivo assay to monitor the interaction of radiolabeled K99 pilus with the bovine mucosa was standardized . Paradoxically, although K99 pilus, purified by standard procedures, was recognized by polyclonal rabbit and calf antisera, its interaction with the bovine intestinal mucosa, quantitated in the ex-vivo system, was not inhibited by these reagents, indicating that the antibodies did not effectively block those K99 pilus domains involved in the interaction with mucosal receptors. Essays Biochem, 1995, 29, 125 - 36 Molecular chaperones: physical and mechanistic properties; Burston SG et al.; Molecular chaperones can be broadly defined as proteins which interact with non-native states of other protein molecules . This activity is important in the folding of newly synthesized polypeptides and the assembly of multisubunit structures; the maintenance of proteins in unfolded states suitable for translocation across membranes; and the stabilization of inactive forms of proteins which are turned on by cellular signals; and the stabilization of proteins unfolded during cellular stress . The major chaperone classes are hsp60 (including TCP1), hsp70 and hsp90 . All these proteins prevent the aggregation of unfolded proteins and the strength of interaction with their protein substrates is modified by the binding and hydrolysis of ATP . Hsp70 is a dimeric and ubiquitous protein which binds its substrates in an extended conformation through hydrophobic interactions . It binds to newly synthesized proteins and is required for protein transport . In its ATP-bound state it has a low protein affinity but when the nucleotide is hydrolysed to give the ADP state the affinity is increased . Hsp70 in E . coli (DnaK) is regulated by two co-proteins: DnaJ (of which there are homologues in eukaryotes) stimulates hydrolysis of ATP and GrpE promotes the dissociation of ADP to allow rebinding of ATP . Thus DnaJ promotes the association of substrate proteins and GrpE promotes dissociation . Hsp60 is a large, tetradecameric protein with a central cavity in which non-native protein structures are proposed to bind . It is essential for the folding of a huge spectrum of unrelated proteins and is present in all biological compartments except the ER . As in hsp70, the binding of ATP stimulates release of the substrate and its hydrolysis restores high binding affinity . It functions in conjunction with a co-protein, cpn10, which enhances its ability to eject proteins during the ATPase cycle . The enhancement of folding yields arises either from the prevention of irreversible aggregation or the ability to unfold misfolded structures and allow further attempts to arrive at the native state . Proteins of the hsp90 class are found associated with inactive or unstable substrate proteins within the cell, thus preventing their aggregation and/or permitting rapid activation. Essays Biochem, 1995, 29, 113 - 23 The roles of molecular chaperones in vivo; Lund PA; Table 1 summarizes the families of chaperones mentioned in this review, and lists their proposed functions . Many of these proteins are named in the accompanying review of Burston and Clarke . Molecular chaperones are proteins which interact with other proteins and help them to reach their final, active conformation . They appear to do this by binding them in an unfolded or partially folded state and subsequently releasing them in an altered form . This property may endow them with several essential or important roles in addition to helping newly synthesized proteins to fold correctly, such as repairing damaged proteins and assisting proteins in membrane translocation . To confirm that a given protein has molecular chaperone activity in vivo, it is necessary to show that interactions between the chaperone and other proteins do occur in the cell, and that loss of the molecular chaperone leads to the accumulation of inactive or precursor protein . The hsp70 protein family are highly conserved and ubiquitous . Genetic studies confirm that their depletion leads to the accumulation of inactive precursor or other proteins, and immunochemical studies show they associate with nascent polypeptides . They are implicated not only in protein folding, but also in protein transport across membranes and reactivation of heat-damaged proteins . The hsp60 proteins are also ubiquitous and very similar in sequence . Those found in bacteria and organelles, such as mitochondria (the GroEL family), are essential at all temperatures, and particularly after heat shock . Their loss or depletion leads to the formation of protein aggregates and eventual cell death . A co-chaperone protein (GroES) is required for their function . Cytosolic homologues (the TCP1 family) are also essential, though not heat-shock induced; they are believed to have a chaperone role in tubulin assembly and their actual role in the cell may be much broader . Many other proteins may have a chaperone function in vivo . Such a function may be specific to a particular substrate (such as the PapD protein in E . coli); others may be more general (such as hsp90 and SecB) . Evidence is still needed to demonstrate whether all those proteins which show chaperone behaviour in vitro actually have such a role in vivo . It seems likely that different classes of chaperone may overlap in their specificity, and it is certain that the various proteins classed as molecular chaperones fulfil a wide variety of roles in the cell. Nucleic Acids Symp Ser, 1995, (34), 3 - 4 Conversion of GC-->AT recognition and elucidation of AT recognition mechanism in zinc finger transcription factor by permutational approach; Emori T et al.; Zinc finger-DNA recognition has been investigated through GC-->AT conversion of recognition sequence 5'-GGG-GCG-GGG-3' of transcription factor Sp1 containing three zinc fingers . Three critical amino acid residues of the finger 2 in Sp1 were replaced with the corresponding residues of the finger 4 in CF2-II . The finger 2 of the hybrid protein is expected to recognize 5'-ATA-3' sequence . Indeed, the designed protein Sp1QTQ2 specifically bound to ATA and ATAN probes . The present study demonstrates that one zinc finger recognizes total sequence of the target DNA site rather than triplet base sequence. Growth Factors, 1995, 12(4), 251 - 62 Involvement of the fourth alpha-helix of mouse granulocyte-macrophage colony-stimulating factor in binding to the alpha-subunit of the receptor complex; Altmann SW et al.; Mouse granulocyte-macrophage colony-stimulating factor (mGM-CSF) proteins with substitutions for residues located within alpha-helix D were examined for biological activity and receptor binding properties . Alanine substitutions of the surface exposed positions indicated that several residues contribute to the ligand-receptor interface . Position K108 and particularly D102 appeared to dominate the binding epitope recognized by mGM-R alpha . Several amino acid substitutions were made for K108 which reduced binding with concommitant losses in bioactivity . Substitutions for D102 resulted in binding affinities less than 0.1% that of the wild-type mGM-CSF and bioactivity decreased to 1.0% . Comparative analysis using high and low affinity binding conditions indicated that mGM-R beta binding was unaffected by these mutations. Drugs Exp Clin Res, 1995, 21(6), 207 - 10 Is tolerance induction a possible mode of action of OM-89? Willis D, Moore AR, Gowland G, Willoughby DA. Neonatal Wistar rats were injected intraperitoneally with either 20 mu g OM-89 at birth and on day 3 after birth or 2 mg three times weekly until 200 g body weight . At this weight, groups were either implanted subcutaneously with a cotton pellet to induce granuloma, or had avridine polyarthritis or pleurisy induced . Both OM-89 treatment regimes significantly reduced granuloma weight (p < 0.05 and < 0.01), paw volume (p < 0.001) and pleural exudates (p < 0.01) . The results suggest that OM-89 could work via tolerance induction. Mol Biol Rep, 1995-96, 22(2-3), 195 - 200 RNases in ColE1 DNA metabolism; Jung YH et al.; ColE1 DNA replication is initiated by RNA II and inhibited by RNA I . Control of the replication occurs through the interaction between RNA I and RNA II . Therefore, RNases involved in the metabolism of RNA I and RNA II are expected to play a key role in the control of the ColE1 plasmid replication . RNase H, RNase E, RNase III, RNase P, and polynucleotide phosphorylase carry out the many specific reactions of the RNA metabolism. Mol Biol Rep, 1995-96, 22(2-3), 181 - 5 Transfer RNA gene organization and RNase P; Green CJ; Mature tRNAs are remarkably similar in all cells . However, the primary transcripts from tRNA genes can vary considerably due to differences in gene organization . RNase P must be able to recognize the elements that are common to all tRNA precursors to accurately remove the 5'-leader sequences. Mol Biol Rep, 1995-96, 22(2-3), 161 - 9 Recent approaches to probe functional groups in ribonuclease P RNA by modification interference; Hardt WD et al.; Modification interference is a powerful method to identify important functional groups in RNA molecules . We review here recent developments of techniques to screen for chemical modifications that interfere with (i) binding of (pre-)tRNA to bacterial RNase P RNA or (ii) pre-tRNA cleavage by this ribozyme . For example, two studies have analyzed positions at which a substitution of sulfur for the pro-Rp oxygen affects tRNA binding {1} or catalysis {2} . The results emphasize the functional key role of a central core element present in all known RNase P RNA subunits . The four sulfur substitutions identified in one study {2} to inhibit the catalytic step also interfered with binding of tRNA to E . coli RNase P RNA {1} . This suggests that losses in binding energy due to the modification at these positions affect the enzyme-substrate and the enzyme-transition state complex . In addition, the two studies have revealed, for the first time, sites of direct metal ion coordination in RNase P RNA . The potentials, limitations and interpretational ambiguities of modification interference experiments as well as factors influencing their outcome are discussed. Mol Biol Rep, 1995-96, 22(2-3), 151 - 6 Plant mitochondrial RNase P; Marchfelder A; Molecular investigations in mitochondria of higher plants have to take in account the complicated genomic structure of these organelles and their complex mode of gene expression . Recently tRNA processing activities and particularly RNase P-like activities have been described for mitochondria of mono- and dicot plants . The determined biochemical characteristics of these plant mitochondrial tRNA processing enzymes now allow a comparison to the bacterial prototype from which they evolved . The substrate specificity of the plant mitochondrial RNase P in particular has unique selection parameters distinct from the E . coli RNase P. Mol Biol Rep, 1995-96, 22(2-3), 147 - 50 Structure, mechanism and evolution of chloroplast transfer RNA processing systems; Gegenheimer P; Chloroplasts of land plants have an active transfer RNA processing system, consisting of an RNase P-like 5' endonuclease, a 3' endonuclease, and a tRNA:CCA nucleotidyltransferase . The specificity of these enzymes resembles more that of their eukaryotic counterparts than that of their cyanobacterial predecessors . Most strikingly, chloroplast RNase P activity almost certainly resides in a protein, rather than in an RNA.protein complex as in Bacteria, Archaea, and Eukarya . The chloroplast enzyme may have evolved from a preexisting chloroplast NADP-binding protein . Chloroplast RNase P cleaves pre-tRNA by a reaction mechanism in which at least one of the Mg2+ ions utilized by the bacterial ribozyme RNase P is replaced by an amino acid side chain. Mol Biol Rep, 1995-96, 22(2-3), 95 - 7 Early studies of native ribonuclease P, including discovery of its essential RNA component; Stark BC; Early work on E . coli ribonuclease P led to the detailed characterization of the native enzyme, which culminated in the discovery and initial characterization of M1 RNA and the demonstration that E . coli RNase P contains an essential RNA component. Mol Biol Rep, 1995-96, 22(2-3), 81 - 5 Structural and functional similarities between MRP and RNase P; Reddy R et al.; RNase P, the enzyme response for 5'-end processing of tRNAs and 4.5S RNA, has been extensively characterized from E . coli . The RNA component of E . coli RNase P, without the protein, has the enzymatic activity and is the first true RNA enzyme to be characterized . RNase P and MRP are two distinct nuclear ribonucleoprotein (RNP) particles characterized in many eukaryotic cells including human, yeast and plant cells . There are many similarities between RNase P and MRP . These include: (1) sequence specific endonuclease activity; (2) homology at the primary and secondary structure levels; and (3) common proteins in both the RNPs . It is likely that RNase P and MRP originated from a common ancestor. Rom J Physiol, 1995 Jan-Dec, 32(1-4), 71 - 6 Hemostatic balance during the acute inflammatory reaction; with special reference to antithrombin III; Plesca LA et al.; Inflammatory reaction caused by intramuscular injections of turpentine in 5 rabbits led to an obvious increase of not only plasma fibrinogen and factor VIII: C levels, but also of plasma antithrombin III activity, measured by a chromogenic assay as heparin cofactor . This activity rose from 80% +/- 10,8 (mean +/- SEM) before the injection to 123% +/- 6,56 48 hours later . Changes affecting plasma fibrinogen level and antithrombin II activity were much lesser in a group of 5 rabbits given small doses of intravenous endotoxin . It is considered that acute inflammation is accompanied by the setting of the hemostatic balance at a higher level. Intervirology, 1995, 38(6), 346 - 51 Escherichia coli expressed herpes simplex virus gG1 and gG2 proteins in ELISA and immunoblotting assays; Kakkanas A et al.; The type 1 and type 2 glycoprotein G (gG1 and gG2) of herpes simplex virus (HSV) were expressed in Escherichia coli as fusion proteins with the maltose binding protein (MBP) using the pMAL-c2 expression vector . The MBP-gG1 fusion protein contains all but the four amino acids from the amino-terminus of gG1, whereas the MBP-gG2 fusion protein was missing the first 30 amino acids that comprise the signal peptide of the protein . The diagnostic value of these antigens was examined by two methods: (1) immunoblot assay based on MBP-gG1 and MBP-gG2 fusion proteins present in crude E . coli cell extracts and (2) enzyme-linked immunosorbent assay (ELISA) of immunoaffinity-purified recombinant MBP-gG1 and MBP-gG2 fusion proteins . Of 28 serum samples known to have antibody to HSV-1 (10 specimens positive for HSV-1 alone and 18 specimens positive for mixed antibody to HSV-1/HSV-2), 27 were reactive to the MBP-gG1 recombinant protein both in ELISA and in immunoblotting . In addition, of 20 serum samples known to have antibody to HSV-2 (2 specimens positive for HSV-2 alone and 18 samples positive for mixed antibody to HSV-1/HSV-2), 15 were found to be reactive to the MBP-gG2 recombinant protein by ELISA and 16 by immunoblotting . None of the 13 HSV-antibody-negative serum samples showed reactivity to the MBP-gG1 or MBP-gG2 antigens by either assay . Moreover, none of the serum samples known to have antibody to HSV-1 alone showed reactivity to the MBP-gG2 recombinant antigen . This study verified the potential application of the E . coli-expressed recombinant gG1 and gG2 proteins as diagnostic antigens and demonstrated the MBP fusion system to be a simple and effective method of producing adequate amounts of low-cost, easily purified gG antigens. Princess Takamatsu Symp, 1995, 25, 111 - 9 Mechanism of translation initiation on hepatitis C virus RNA; Nomoto A et al.; In vitro translation experiments involving reticulocyte and HeLa cell lysates have demonstrated that translation initiation on hepatitis C virus (HCV) RNA occurs by entry of ribosomes to the internal sequence (internal ribosomal entry site {IRES}) within the 5' noncoding region (5'NCR) . Specific binding factors that bind to the HCV IRES were examined by UV cross-linking tests . A protein p57 (polypyrimidine tract binding protein {PTB}), known to be a binding factor that binds to picornavirus IRES, was found to bind to the HCV IRES . To investigate functions of this PTB in IRES-dependent translation initiation, hyperimmune antibodies to PTB (anti-PTB) were prepared by immunizing rabbits with purified recombinant PTB produced in E . coli . Anti-PTB IgGs were purified and used to deplete PTB in a cell-free protein synthesis system prepared from HeLa cells . In vitro translation experiments using PTB-depleted lysates suggested that host factor dependency of IRES is different in individual IRESs . As we showed previously, function of the IRES typical of group II HCV is more efficient than the IRES typical of group I HCV in a cell-free protein synthesis system from HeLa cells . Recombinant HCV IRESs were constructed and tested for their function in the in vitro system . Most recombinant IRESs showed an initiation activity of the more efficient typical group II IRES level or higher . This observation, together with the fact that group I HCV is the most common isolate of HCV worldwide, suggests that the efficiency of the IRES function of HCV may be generally suppressed in nature, and that the replication ability of HCV that permits its persistent infection in humans may require a delicate balance at a molecular level. Cell Mol Biol Res, 1995, 41(5), 425 - 33 Serine acetyltransferase from Arabidopsis thaliana can functionally complement the cysteine requirement of a cysE mutant strain of Escherichia coli; Murillo M et al.; Serine acetyltransferase, a key enzyme in the L-cysteine biosynthetic pathway of sulfate assimilating organisms, catalyzes the formation of O-acetylserine, the immediate precursor of L-cysteine . In higher plants, it is thought that sulfur assimilation occurs primarily in leaf chloroplasts; however, serine acetyltransferase is not localized exclusively in this tissue and organelle . At least three genes for serine acetyltransferase have been identified in the higher plant Arabidopsis thaliana . Reported here is a cDNA corresponding to one of these genes, SAT1, a 1,079 bp clone with an open reading frame predicted to encode a 34-kDa protein that is able to functionally complement a serine acetyltransferase mutant strain of Escherichia coli . The predicted amino acid sequence of SAT1 shows significant homology with bacterial serine acetyltransferases . SAT1, expressed as a recombinant protein, shows serine acetyltransferase enzyme activity and cross-reacts with an antibody against the homologous E . coli enzyme . The first 40 amino acids of the SAT1 polypeptide resembles a plastid transit peptide, but the polypeptide is probably not plastid localized . Genomic DNA blot analysis of A . thaliana showed that SAT1 is a single copy gene and RNA blot analysis revealed that SAT1 is expressed in both leaves and roots. Acta Biochim Pol, 1995, 42(4), 367 - 80 Structural aspects of the inhibition and catalytic mechanism of thymidylate synthase; Hardy LW; Thymidylate synthase (TS) is a target for anticancer drugs, due to its unique role in the biosynthesis of an essential DNA precursor . The X-ray structures available for several bacterial enzymes have been used to design novel inhibitors of TS, to structurally analyze the binding mode of existing inhibitors, and to propose catalytic roles for amino-acid residues on the protein . The first part of this paper describes some aspects of structure-based drug design, including a recent result from the groups of Montfort and Maley emphasizing the importance of conformational changes in inhibitor binding . The second part of the paper describes the work of the author on the TS mechanism, especially the catalytic roles of active site amino acids Asn177 and Glu58 in TS from Escherichia coli . An important function for Glu58 is proposed to be preventing the excessive stabilization of a covalent intermediate . The use of isotope effects to probe the mechanistic basis for stimulation of E . coli TS by magnesium ions, and to identify differences between the E . coli and human enzymes, is described . The hypothesis that N5 of tetrahydrofolate provides the basicity for deprotonation of the nucleotide is also discussed. Yi Chuan Xue Bao, 1995, 22(3), 178 - 84 {Cloning of the diapause hormone gene of the silkworm (Bombyx mori)}; Xu W et al.; A silkworm genomic library was screened with radiolabelled cDNA of Bombyx mori diapause hormone (Bom DH) . Three positive clones were isolated from approximately 4 x 10(5) plaques, and subjected to further analyses . The DNA inserts excised from positive clones with SalI were ligated into pBluescript KS(+) vector and grown in E . coli XL1 cells . The DNA fragments obtained by EcoRI digest form the insert were subcloned into the same vectors . The nucleotide sequence analysis showed that these clones involved the Bom DH gene . Genomic Southern analysis suggested that the Bom DH is encoded by a single gene . The present study which is the first one on cloning of the Bom DH gene. Folia Microbiol (Praha), 1995, 40(2), 169 - 75 Production and purification of Japanese quail ovalbumin as fusion protein with glutathione S-transferase in Escherichia coli; Visvaderova J et al.; A plasmid encoding a fusion protein interlinked by thrombin recognition sequence between glutathione S-transferase and Japanese quail ovalbumin (without 40 amino acid residues from the 5'-end of the ORF) has been constructed, employing the expression system pGEX-2T . The deglycosylated fusion protein (64 kDa) was purified by affinity chromatography on glutathione agarose beads, analyzed by SDS-polyacrylamide gel electrophoresis, immunochemically detected with antiserum raised against Japanese quail ovalbumin and tested for its stability. Nucleic Acids Symp Ser, 1995, (34), 203 - 4 Codon recognition by tRNA molecules with a modified or unmodified uridine at the first position of the anticodon; Okumura S et al.; Effects of a single nucleoside modification at the first position of the anticodon of a transfer RNA molecule on its codon reading properties were investigated by use of a cell-free protein synthesis . We prepared two artificial tRNA molecules that differ only in the nucleotide at the first position of the anticodon . One has an unmodified uridine and the other has a 5-methoxyuridine (mo5U) . These molecules were charged with labeled serine and introduced into a cell-free protein synthesis directed by a designed mRNA, and the relative codon reading efficiencies were calculated . The results showed that the modification of U into mo5U elevates the reading efficiencies of the UCU and UCG codons but reduces that of the UCA codon. Nucleic Acids Symp Ser, 1995, (34), 181 - 2 Synthesis of oligonucleotide having a bent structure by incorporation of an interresidually cyclized uridylyl (3'-5')uridine unit; Seio K et al.; The chemical synthesis and conformational properties of interresidually cyclized uridylyl(3'-5')uridine derivatives were studied in order to introduce a stable turn structure into oligonucleotides . These cyclized molecules were analogs of uridylyl(3'-5')5-{methylamino(methyl)}-uridine which is the component of the U turn structure of tRNAarg E . coli . The conformational properties of these cyclic dinucleoside monophosphates were studied using NMR and CD spectroscopy with the aid of molecular mechanics and molecular dynamics simulations . These experiments indicated that the turn conformation could be stabilized by introducing a cyclic structure as expected . On the basis of these results, the chemical synthesis of phosphoramidite units of these cyclic dinucleoside monophosphate derivatives were studied to construct oligonucleotides having a stable bent structure. Nucleic Acids Symp Ser, 1995, (34), 153 - 4 Structural studies of tRNAs by capillary high performance liquid chromatography/electrospray mass spectrometry; Hayashi N et al.; Capillary high performance liquid chromatography/ electrospray mass spectrometry (LC/ ESI/MS) was applied to studies of nucleic acids . Since the presence of salt adducts obscures the molecular peaks and hinders the precise determination of molecular mass, conditions were sought in which effective online desalting and efficient ionization could be achieved simultaneously . In the presence of triethylamine, which is routinely used for the reversed-phase separation of nucleic acid, small oligo-nucleotides could be measured satisfactorily, while RNA tended to show more pronounced adduct peaks . When tributylamine was used instead of the shorter analogue, the adduct peak formation was well suppressed, and the hydrophobic nature of the counter ion made the nucleotides binding to the column tighter, resulting in the better chromatographic separation . The efficient ionization and suppression of the ion adducts make it possible to determine precisely the molecular mass of large nucleotides such as tRNA. Nucleic Acids Symp Ser, 1995, (34), 105 - 6 Study of JA300 as agent having high transglycosylation activity; Izumi M et al.; Escherichia coli JA300 as the strain catalyzing transglycosylation of nucleic acids has so strong activity . We scrutinized where the high faculty of this strain come from . Using PCR methods with our primer pair MI-1 (up; GTT GAA TTC GCC TTT GTT ATG TCA C) and MI-2 (down; GTT CCG CTA ACG GAA CAC CTA GGC CT), we isolated deoD (the coding region of PNPase) of JA300 . Nucleotide sequence analysis indicated that deoD of JA300 is definitely same as that of parent strain K12 . The fact that the transcription of deoD of JA300 is more active than that of K12 was also revealed after the analysis by Northrn blot hybridization . These data may suggest that the varieties of gene concerning regulation contribute to the difference of PNPase activity between them. Nucleic Acids Symp Ser, 1995, (34), 91 - 2 Characterization and transcriptional regulation of the modABCD genes for molybdenum transport in Escherichia coli; Miyake H et al.; The mod genes coding for molybdenum transport system were isolated from Escherichia coli by means of complementation of the tor mutation that causes no synthesis of some molybdenum-containing enzymes . The nucleotide sequence of 3048-base pair showed four open reading frames including the previously reported modC gene . The genes were named modA, modB, modC, and modD . Promoter analysis by lacZ assay and primer extension showed the genes modA and modB individually have their own promoters . No transcriptional regulation of the two genes were observed in the presence of molybdate, nitrate, and oxygen in E . coli strain DH5 alpha. Nucleic Acids Symp Ser, 1995, (34), 63 - 4 Conformational changes of purine repressor DNA-binding domain upon complexation with DNA; Nagadoi A et al.; The purine repressor (PurR) consists of two functional domains: an N-terminal DNA-binding domain and a C-terminal corepressor-binding domain . Recently, the structure of PurR-corepressor-operator ternary complex was determined by X-ray crystallography . In the complex the DNA-binding domain, consisting of 56 amino acids, was composed of four helices . Here, we have determined the solution structure of the DNA-binding domain in its DNA free state by NMR . It consists of three helices and the fourth helix (the hinge helix) region is diordered . The architecture of the first three helices of its DNA free state is very similar to that of its DNA-bound form . The hinge helix is induced by the specific DNA binding and by the dimerization of PurR which is provided by the corepressor-binding domain. Nucleic Acids Symp Ser, 1995, (34), 7 - 8 Model building of DNA repair enzyme 3-methyladenine DNA glycosylase-DNA complex; Yamagata Y et al.; We report the model structure of enzyme-DNA complex based on X-ray structure of 3-methyladenine-DNA glycosylase II and mutagenic studies . The enzyme structure does not have any recognizable DNA binding motifs . The DNA was located in the large cleft between the two all alpha domains with the damaged adenine base moiety (3-methyladenine) of DNA duplex positioned in the entrance region of the cleft including the tryptophan and aspartic acid residues . For the target 3-methyladenine to interact with these residues, the 3-methyladenine has to flipped out from the DNA helix . A number of the positively charged amino acid residues adopt at the negatively charged phosphate moieties . It is necessary to kink the DNA helix slightly to form the good interactions between both molecules . The damaged base must be held in place by a number of interactions including the weak van der Waals and electrostatic attractions. Nucleic Acids Symp Ser, 1995, (34), 5 - 6 Structural requirement of four-way junction DNA for binding to and cleavage by RuvC; Shida T et al.; Sixteen bimobile four-way junction DNAs were designed to clarify the consensus sequence at the resolution site by the E . coli RuvC protein . The consensus sequence is 5'-T decreases X-3' (X: A, G, C, and T) without exception, but the sequences at the cleaved sites are not necessarily symmetric . The presence of symmetrically located two AT base-pairs at the junction is the minimal requirement for resolution. Biochimie, 1995, 77(12), 925 - 30 The hinge region of Escherichia coli ribosomal protein L7/L12 is required for factor binding and GTP hydrolysis; Dey D et al.; A variant form of Escherichia coli ribosomal protein L7/L12 that lacked residues 42 to 52 (L7/L12: delta 42-52) in the hinge region was shown previously to be completely inactive in supporting polyphenylalanine synthesis although it bound to L7/L12 deficient core particles with the normal stoichiometry of four copies per particle (Oleinikov AV, Perroud B, Wang B, Traut RR (1993) J Biol Chem, 268, 917-922) . The result suggested that the hinge confers flexibility that is required for activity because the resulting bent conformation allows the distal C-terminal domain to occupy a location on the body of the large ribosomal subunit proximal to the base of the L7/L12 stalk where elongation factors bind . Factor binding to the hinge-truncated variant was tested . As an alternative strategy to deleting residues from the hinge, seven amino acid residues within the putative hinge region were replaced by seven consecutive proline residues in an attempt to confer increased rigidity that might reduce or eliminate the bending of the molecule inferred to be functionally important . This variant, L7/L12:(Pro)7, remained fully active in protein synthesis . Whereas the binding of both factors in ribosomes containing L7/L12:delta 42-52 was decreased by about 50%, there was no loss of factor binding in ribosomes containing L7/L12:(Pro)7, as predicted from the retention of protein synthesis activity . The factor:ribosome complexes that contained L7/L12:delta 42-52 had the same low level of GTP hydrolysis as the core particles completely lacking L7/L12 and EF-G did not support translocation measured by the reaction of phe-tRNA bound in the A site with puromycin . It is concluded that the hinge region is required for the functionally productive binding of elongation factors, and the defect in protein synthesis reported previously is due to this defect . The variant produced by the introduction of the putative rigid Pro7 sequence retains sufficient flexibility for full activity. Rocz Akad Med Bialymst, 1995, 40(2), 408 - 13 Stimulated monocytes release factor XIII subunit A and fibronectin in vitro; Kloczko J et al.; Factor XIII catalyses the formation of covalent bonds between several proteins involved in wound healing . Subunit A of factor XIII has been found inside and on the surface of monocytes and macrophages . In our study we investigated the ability of cultured monocytes to release factor XIII A subunit and its substrate-fibronectin . Human monocytes were cultured for up to 36 hours with lipopolysaccharide (LPS) from Escherichia coli . In supernatants of LPS stimulated monocytes we found 22 +/- 4 nM/ml and 40 +/- 6 nM/ml of factor XIII subunit A after 24 and 36 hours of incubation respectively . We also observed an increase of fibronectin concentration in supernatants of stimulated monocytes . The obtained results suggest that activated monocytes might be a source of factor XIII and fibronectin, which are critical for tissue repair processes. Rocz Akad Med Bialymst, 1995, 40(2), 267 - 75 Study of lithogenicity of bile among patients suffering from cholecystolithiasis and among patients not suffering from this disease; Kamocki Z et al.; On the basis of biochemical and morphological research, the authors decided to attempt to determine the degree of lithogenicity of the bile among patients suffering from cholecystolithiasis (100 patients) and those not suffering from this disease (31 patients) . Having analyzed the obtained data, it can be concluded that the degree of lithogenicity of bile is influenced by the concentration of cholesterol, bile acids and phospholipids as well as the damage of the liver and of the wall of the gallbladder as well as its kinetics . Lithogenic bile was noticed in 84% of the patients of group I and in 54% in group II . However, the degree of lithogenicity was significantly higher in patients suffering from cholecystolithiasis . According to the authors, the degree of lithogenicity is the deciding factor in the creation of bile stones. Adv Exp Med Biol, 1995, 380, 437 - 42 Zinc-binding of the cysteine-rich domain encoded in the open reading frame of 1B of the RNA polymerase gene of coronavirus; Yoo D et al.; We cloned and sequenced the second open reading frame of the RNA polymerase gene, ORF1b, of bovine coronavirus . In the region representing nucleotide positions 4919-5677 upstream from the initiation codon of the 32K non-structural protein gene, we identified two putative functional domains . One of these domains contained four leucine residues repeated exactly in every seventh position, and the other domain represented a cluster of cysteine and histidine residues . The DNA sequence representing these domains was cloned and expressed in Escherichia coli as fusion proteins with glutathione S-transferase from Schistosoma japonicum . A high level expression of the cysteine-rich domain was achieved as a fusion protein when the bacterial culture was induced with IPTG . In a solid phase zinc binding assay using the recombinant fusion protein, we found that the protein containing the cysteine-rich domain was able to bind to radioactive zinc in vitro, demonstrating that the polypeptide encoded by the ORF1b of coronavirus is a zinc-binding protein. Adv Exp Med Biol, 1995, 380, 423 - 30 Characterization of the leader papain-like protease of MHV-A59; Bonilla PJ et al.; Sequence analysis of the mouse hepatitis virus, strain A59 (MHV-A59) genome predicts the presence of two papain-like proteases encoded within the first open reading frame of the replicase gene . The more 5' of these domains, the leader papain-like protease, is responsible for the cleavage of the amino terminal protein, p28 . We have defined the core of this protease to between amino acids 1075 and 1344 from the beginning of ORF 1a . Deletion analysis coupled with in vitro expression, was used to study p28 cleavage by this leader protease . Expression of a series of deletion mutants showed processing of p28, albeit at lower levels in some of them . Reduced p28 production resulting from a 0.4 kb deletion positioned between p28 and the protease domain suggests an involvement of this region in catalytic processing . Some mutants display cleavage patterns indicative of a second cleavage site . Interestingly, this newly identified cleavage site maps to a position similar to the expected cleavage site of a p65 polypeptide detected in MHV-A59 infected cells . Mutagenesis of the catalytic H1272 residue demonstrates that both cleavages observed are mediated by the leader papain-like protease encoded in ORF 1a. Adv Exp Med Biol, 1995, 380, 317 - 20 Identification and characterization of the porcine reproductive and respiratory virus ORFs 7, 5 and 4 products; Mounir S et al.; Porcine reproductive and respiratory syndrome virus (PRRSV) causes abortions and respiratory diseases in pigs . The PRRSV genome is a positive-sense polyadenylated RNA molecule of about 15 kb . Along with the genes for structural proteins (envelope, matrix and nucleocapsid proteins) PRRSV genome contains a number of ORFs potentially encoding nonstructural proteins (ns) . To investigate the nature of the PRRSV ORFs 7, 5 and 4 products, we have cloned the envelope (ORF5) nucleocapsid (ORF7) and ns4 (ORF4) protein genes in the bacterial expression vector pMAL(TM)-c2 under control of the "tac" promoter and expressed the proteins in E . coli . The recombinant proteins were recognized by porcine and rabbit hyperimmune serums to PRRSV, suggesting their structural nature. Virus Genes, 1995, 11(2-3), 239 - 57 The evolution of small DNA viruses of eukaryotes: past and present considerations; Shadan FF et al.; Historically, viral evolution has often been considered from the perspective of the ability of the virus to maintain viral pathogenic fitness by causing disease . A predator-prey model has been successfully applied to explain genetically variable quasi-species of viruses, such as influenza virus and human immunodeficiency virus (HIV), which evolve much faster rates than the host . In contrast, small DNA viruses (polyomaviruses, papillomaviruses, and parvoviruses) are species specific but are stable genetically, and appear to have co-evolved with their host species . Genetic stability is attributable primarily to the ability to establish and maintain a benign persistent state in vivo and not to the host DNA proofreading mechanisms . The persistent state often involves a cell cycle-regulated episomal state and a tight linkage of DNA amplification mechanisms to cellular differentiation . This linkage requires conserved features among viral regulatory proteins, with characteristic host-interactive domains needed to recruit and utilize host machinery, thus imposing mechanistic constrains on possible evolutionary options . Sequence similarities within these domains are seen amongst all small mammalian DNA viruses and most of the parvo-like viruses, including those that span the entire spectrum of evolution of organisms from E . coli to humans that replicate via a rolling circle-like mechanism among the entire spectrum of organisms throughout evolution from E . coli to humans . To achieve benign inapparent viral persistence, small DNA viruses are proposed to circumvent the host acute phase reaction (characterized by minimal inflammation) by mechanisms that are evolutionarily adapted to the immune system and the related cytokine communication networks . A striking example of this is the relationship of hymenoptera to polydnaviruses, in which the crucial to the recognition of self, development, and maintenance of genetic identity of both the host and virus . These observations in aggregate suggest that viral replicons are not recent "escapies" of host replication, but rather provide relentless pressure in driving the evolution of the host through cospeciation. Biochimie, 1995, 77(11), 875 - 9 Importance of the replication origin sequestration in cell division of Escherichia coli; Meury J et al.; The DNA adenine methyltransferase of Escherichia coli methylates adenines at GATC sequences . The mutant deficient in this methylase has no apparent deficiency in the cell division process in spite of the absence of both synchrony in initiations of chromosomal DNA replication and sequestration of replication origin (oriC) at hemimethylated state . However, the dam mutant cannot resume cell division after hyperosmotic shock differing from the wild-type strain . This inhibition is not provoked by induction of the cell division inhibitor, SfiA protein . Although the FtsZ protein is present in the dam mutant in a reduced amount compared to wild-type, the quantitative difference of this protein is not the main reason of division arrest provoked by hyperosmotic shock . This observation supports the idea of oriC-membrane interaction playing a role both in chromosome partitioning and cell division as predicted by replicon theory. Biochimie, 1995, 77(11), 848 - 53 In vitro inhibition of RecA-mediated homologous pairing by UmuD'C proteins; Szpilewska H et al.; The process of SOS mutagenesis in Escherichia coli requires: i) the replisome enzymes; ii) RecA protein; and iii) the formation of the UmuD'C protein complex which appears to help the replisome to resume DNA synthesis across a lesion . It has recently been shown that the UmuD'C complex, if overproduced, inhibits recombinational repair of a UV-damaged plasmid DNA as well as homologous recombination in an Hfr x F- cross . Since UmuD'C proteins might inhibit an early recombination step by interacting with a RecA nucleo-protein filament, we checked whether UmuD'C proteins will inhibit RecA promoted homologous pairing in vitro . We tested the inhibitory action of UmuD'C proteins in a crude bacterial extract containing possible cofactors such as chaperone proteins that ensure the proper folding of UmuC and the assembly of the UmuD'C complex in vivo . We used a novel recombination assay in which RecA protein promotes the formation of a stable plectonemic joint between a circular single-stranded DNA immobilized onto a membrane and an incoming homologous linear duplex DNA . Under these conditions we show that UmuD'C proteins inhibit the formation of joint molecules. Drug Metabol Drug Interact, 1995, 12(3-4), 285 - 97 Characterization of cytochrome P450TYR, a multifunctional haem-thiolate N-hydroxylase involved in the biosynthesis of the cyanogenic glucoside dhurrin; Halkier BA et al.; The haem-thiolate N-hydroxylase cytochrome P450TYR involved in the biosynthesis of the tyrosine-derived cyanogenic glucoside dhurrin in Sorghum bicolor had recently been isolated . Reconstitution of enzyme activity by insertion of cytochrome P450TYR and NADPH-cytochrome P450-reductase into L-alpha-dilauroylphosphatidylcholine micelles and using tyrosine as substrate results in the formation of p-hydroxyphenylacetaldehyde oxime . Quantitative substrate binding spectra demonstrate that tyrosine and N-hydroxytyrosine are mutually exclusive substrates that bind to the same active site of cytochrome P450TYR . The multifunctionality of cytochrome P450TYR has been confirmed in reconstitution experiments using recombinant cytochrome P450TYR expressed in Escherichia coli . It was earlier reported that an in vitro microsomal system catalyzing all but the last step in the biosynthetic pathway for cyanogenic glucosides exhibits catalytic facilitation (channelling) . This observation is explained by the multifunctionality of cytochrome P450TYR . The cytochrome P450TYR sequence represents the first amino acid sequence of a functionally characterized cytochrome P-450 enzyme from a monocotyledonous plant and the first sequence of an N-hydroxylase with high substrate specificity . Multifunctional N-hydroxylases of the cytochrome P-450 type have not previously been reported in living organisms. Folia Microbiol (Praha), 1995, 40(6), 588 - 94 In vivo cloning of a carboxy-terminal rpoB allele which confers altered transcriptional properties; Rowland GC et al.; A 3'-terminal mutation of the gene encoding the beta subunit of Escherichia coli RNA polymerase was isolated using an in vivo polA(Ts) technique . Cloning of the allele was monitored by virtue of the fact that the deletion delta(rpoB)1570-1 resulted in an altered-size restriction fragment . DNA sequencing confirmed the predicted nature and location of the mutation: delta(rpoB)1570-1 involved an in-frame deletion of 186 bp (62 codons) encoding amino acid residues 967-1028 . The phenotype conferred by delta(rpoB)1570-1 is discussed with respect to conserved domains within the beta polypeptide. Bioseparation, 1995, 5(6), 359 - 67 Cloning, expression and purification of Ppl-1, a kappa-chain binding protein, based upon protein L from Peptostreptococcus magnus; Bottomley SP et al.; Protein L is a multi-domain, cell wall constituent of certain strains of Peptostreptococcus magnus, which binds to the variable domain of the L-chains of Ig . A gene fragment which codes for a single Ig-binding domain of protein L (Ppl-1) was cloned into a modified pKK223-3 vector and over-expressed in E . coli JM103 . A rapid protein purification protocol is described . In these studies, purified Ppl-1 was immobilised on to an agarose gel and tested against an array of Igs and Ig fragments . It was found that Ppl-1-bound Igs from a number of different sources via interactions with the L kappa-chain . An enzyme linked immunosorbant assay has been developed to assay the binding of Ppl-1 to IgG . The incubation of Ppl-1 with human serum does not produce an immunoprecipitate, thus suggesting one unique interaction per binding domain which has been confirmed by ELISA . These experiments demonstrate the potential value of Ppl-1 as an immunological tool and as an affinity chromatography ligand for the purification of Igs. Bioseparation, 1995, 5(6), 351 - 8 Partitioning of proteins in dextran/hydrophobically modified dextran aqueous two-phase systems; Lu M et al.; Partitioning of proteins was studied in aqueous two-phase systems composed of the polymers dextran and hydrophobically modified dextran . The modified dextrans were benzoyl dextran with a degree of substitution of 0.17 and valeryl dextran with a degree of substitution of 0.20 . Phase diagrams for the systems of dextran/benzoyl dextran and dextran/valeryl dextran were determined at room temperature . The proteins studied were beta-galactosidase, bovine serum albumin, beta-lactoglobulin, lysozyme, myoglobin and cytochrome C . The partition coefficients of a series of salts were determined in dextran/benzoyl dextran two-phase systems . The addition of salts had strong effect on the partitioning of proteins . This effect was related to protein net charge and the position of the ions in the Hofmeister series . Cross partitioning of bovine serum albumin was studied in a dextran/benzoyl dextran aqueous two-phase system. Bioseparation, 1995, 5(6), 329 - 37 Isolation of a recombinant intracellular beta-galactosidase by ammonium sulfate fractionation of cell homogenates; Zhang Z et al.; The Escherichia coli beta-galactosidase (EC 3.2.1.23) expressed intracellularly as soluble, biologically active enzyme in the yeast Saccharomyces cerevisiae was recovered from clarified homogenates of the yeast cells by precipitation with crystalline ammonium sulfate . Effects of salt saturation (0-80%) of the homogenate, the initial total protein level (2-20 g.L-1) and the processing pH (6-8) on the enzyme and protein recovery were investigated . As the ammonium sulfate concentration increased, the enzyme was precipitated preferentially and at 30% salt saturation nearly all had been recovered in the precipitate . In contrast, at this salt concentration only 25% of the total protein had precipitated . Preferential precipitation of beta-galactosidase was associated with the hydrophobic nature of this large protein . Complete precipitation of the total protein required salt concentrations exceeding 70% of saturation . The salt concentration needed for complete recovery of the enzyme was not sensitive to the processing pH . Over the salt saturation level of 20-50%, the enzyme precipitation followed the Cohn equation . The salting-out constant was strongly affected by the initial protein level in the homogenate; higher values were observed at lower protein concentrations . The salting-out constant was unaffected by the processing pH; however, the Cohn parameter B was pH dependent in addition to being affected by the initial protein concentration . Within the beta-galactosidase stability range of pH 6-8, pH variations alone (no added salt) proved ineffective in precipitating the enzyme. Micron, 1995, 26(6), 511 - 20 Direct structure determination by electron crystallography: protein data sets; Dorset DL; Applications of the Sayre equation to the electron crystallographic analysis of protein structures are demonstrated . Starting with a lower-resolution basis phase set obtained from the Fourier-transform of an electron micrograph, it is possible, for example, to extend directly to the higher resolution of the electron diffraction pattern . Examples of such analyses include bacteriorhodopsin, halorhodopsin and the Omp F porin from the outer membrane of Escherichia coli . If a multisolution approach is taken, it may also be possible to carry out ab initio phase determinations, as demonstrated with low-resolution diffraction amplitudes from a negatively-stained membrane protein crystal. Folia Microbiol (Praha), 1995, 40(5), 541 - 6 Purification and partial immunochemical characterization of proteins of fimbriae F107 from Escherichia coli isolated from edema disease of pigs; Rosocha J et al.; The paper describes the isolation, purification and characterization of F107-fimbrial proteins, obtained by thermoelution from Escherichia coli 107/86 . Isolation of the pure F107 protein was done by FPLC chromatography, employing Superose 12, Mono Q, and Phenyl-Superose columns . The highest purity of the F107 protein was achieved with Superose 12 HR 10/30 . Purity checking by a HPLC system Waters 625 LC (Millipore) proved the absence of protein admixtures in a fraction from Superose 12 . Analysis of the molar mass of F107 proteins by SDS PAGE revealed that F107 fimbriae consist of two proteins, one of M = 43 kDa (minor), and other of M = 18.9 kDa (major) . Western blot analysis with rabbit polyclonal antiserum confirmed that the 18.9 kDa protein was the major characteristic unit of F107 fimbriae. Zhen Ci Yan Jiu, 1995, 20(3), 36 - 9 {Histochemical observation of the effect of electroacupuncture on the livers of rats with endotoxic shock}; Huang W et al.; The experimental rats were randomly divided into three groups: i.e . control group; endotoxic shock group (the model of endotoxic shock was induced by intravenous administration of E . Coli endotoxin, 16 mg/kg); and electroacupuncture (EA) group ("Renzhong" or "Zusanli" acupoint was stimulated for 15 minutes at 1 hr after injection of endotoxin) . The experimental rats were decapitated at 75 minutes after injecting endotoxin . Their livers were taken out for cryostat section, and histochemical observation . The results were as follows: 1) The glycogen in the hepatic cells of endotoxic shocked rats were almost completely depleted . The activities of SDH Mg(++)-ATPase and G-6-Pase and 5'-Nase were decreased; especially the activities of 5'-Nase in the biliary canaliculi and sinusoids were apparently reduced . 2) The content of hepatic glycogen in EA group was increased, but some of them was still depleted . The activities of SDH, Mg(++)-ATPase and G-6-Pase were slightly increased as compared with that of the endotoxic shock group . The activity of 5'-Nase was obviously increased after EA . The preliminary results indicated that EA at "Renzhong" or "Zusanli" acupoint of rats with endotoxic shock might play certain role on improving the hepatic metabolism and promoting the membrane transport action. Semin Thromb Hemost, 1995, 21(4), 390 - 401 Hemostasis in childhood acute lymphoblastic leukemia: coagulopathy induced by disease and treatment; Mitchell LG et al.; Thromboembolic events (TE) are serious complications of treatment for childhood acute lymphoblastic leukemia (ALL) that result in significant morbidity and occasionally mortality . These events are strongly associated with the administration of L'asparaginase (ASP) . There have been many studies reporting TE and assessing the coagulopathy associated with treatment . The intention of these studies was to determine a potential mechanism for thrombosis . This article reviews the current literature in this area . First, data on thrombotic complications in terms of incidence, location, diagnosis, and timing of events are summarized . The second section discusses the coagulopathy associated with the disease and treatment . To minimize the effects of confounding treatments, the data are divided into sections covering pretreatment, after ASP only, after combination chemotherapy without ASP, and after combination chemotherapy with ASP . In addition, the effects of glucocorticoid steroids on the hemostatic system are discussed . As thrombin regulation is critically important to hemostasis, the next section of the review discusses the regulation of thrombin in children with ALL, both in vitro and in vivo, and the link between impaired thrombin regulation and TE in this population . Finally current hypothesis on mechanisms for TE and proposed preventative strategies are examined. Adv Neuroimmunol, 1995, 5(4), 463 - 78 Nitric oxide: an ancestral immunocyte effector molecule; Franchini A et al.; The presence and the role of nitric oxide synthase (NOS) were investigated in the molluscan hemocytes by immunocytochemical, biochemical and functional approaches . Using an anti-NOS polyclonal antibody, immunoreactivity was observed in the hemocytes, and this reactivity increased after stimulation of the animals with Escherichia coli, indicating that this enzyme is inducible . The NOS inducibility was also histochemically demonstrated by detection of NADPH-diaphorase activity . Biochemical studies show that the enzyme is 70% cytoplasmatic and 30% membrane bound and that the inducible form is mainly cytoplasmatic . The nitrite + nitrate and citrulline formation, the inhibition by N omega-nitro-L-arginine, the Km value for arginine, the calcium and co-enzyme dependence show that the molluscan NOS shares the same properties as the NOS isoenzymes so far studied . However, it cannot be identified with any of these enzymes . It appears to be in some way similar to an inducible form of human hepatocyte NOS . Also cytokines are able to induce NOS . In vitro studies have shown that hemocytes produce nitric oxide (NO), a bactericide substance, and that there is a relationship between the NO system and phagocytosis . The presence of NO in the invertebrate hemocyte demonstrates that critical molecules have been conserved over the course of evolution. J Mol Neurosci, 1995, 6(2), 121 - 30 Decreased incorporation of D-glucosamine into glycosphingolipids of intact Familial Dysautonomia lymphoblasts; Strasberg PM et al.; Familial Dysautonomia (FD) is an autosomal recessive Ashkenazi Jewish genetic disease, of unknown etiology, involving deficits in both autonomic and sensory functions . Previously, we found statistically significant increases in globotriaosylceramide (Gb3) in FD fibroblasts and lymphoblasts, and a decrease in ganglioside levels . FD fibroblasts exhibited pleiomorphic changes at the light microscopy level, suggestive of changes in the plasma membrane . We described an increase in Gb3 on the surface of synchronized cells at the G1/S boundary of the cell cycle, based on Gb3-verotoxin (derived from E . coli) interactions . Using D-glucosamine-1-14C as an in vitro precursor, we herein report a marked decrease in the rate of incorporation of D-glucosamine into the sialic acid and the N-acetylgalacto/glucosamine moieties of gangliosides and neutral glycosphingolipids in intact FD compared to control lymphoblasts . The total ganglioside content of FD cells (primarily GM3, measured as incorporation of 3H from NaB3H4) was also decreased . These data indicate differences in the turnover of sialic acid and N-acetylated sugar constituents in FD vs normal cells. Ann Fr Anesth Reanim, 1995, 14(6), 514 - 6 {Reactive hemophagocytic syndrome: a probably underestimated cause of thrombocytopenia in intensive acre patients}; Francois B et al.; Thrombocytopenia is a common feature in ICU patients which occurs usually in case of infection or septic shock . Its mechanisms, which are often unclear, include the haemophagocytic syndrome initially linked with histiocytic proliferation but probably also associated with infectious diseases . This syndrome is characterized by a phagocytosis of medullar blood cells . Reactive haemophagocytic syndrome can probably lead to thrombocytopenia in ICU patients as in this case report of a E . Coli infection. Chin J Biotechnol, 1995, 11(4), 275 - 80 Studies on high salt tolerance of transgenic tobacco; Liu J et al.; With E . coli mtlD gene (encoding mannitol 1-phosphate dehydrogenase) and gutD gene (encoding glucitol 6-phosphate dehydrogenase) cloned, plant expression vector pBIGM had been obtained by inserting mtlD and gutD genes into binary vector pBin438 . Tobacco was transformed with A . tumefaciens LBA4404 containing pBIGM . Results of molecular hybridization of transformed plants indicated that mtlD and gutD genes had integrated into the genomic DNA of tobacco plants . Experiments of salt tolerance and analysis of sugar alcohols showed that the accumulation of different sugar alcohols in transgenic tobacco plants had increased salt tolerance of tobacco. Chin J Biotechnol, 1995, 11(4), 213 - 9 Effect of 3'-untranslated region on expression of TNF alpha cDNA in Escherichia coli; Chang J et al.; A construct pRL-rhTNF alpha 2, in which the 110 bp 3'-untranslated region of rhTNF alpha cDNA was deleted, was transformed in E.coli . The expression level of several positive transformants was determined . The results showed that the expression of pRL-TNF alpha 2 was stable and the biological characteristics of the protein product were the same as that of pRL-rhTNF alpha . Furthermore, the expression level of pRL-rhTNF alpha 2 was increased . It is suggested that the 3'-untranslated region may have some effects on gene expression . A TA-rich sequence, TTTATTA, contained in the 3'-untranslated region of pRL-rhTNF alpha may be involved in the inhibitory effect on gene expression. Ciba Found Symp, 1995, 193, 41 - 58; discussion 59-70 The cell lineage of neuronal subtypes in the mammalian cerebral cortex; Parnavelas JG et al.; We have studied the lineage relationships of pyramidal and nonpyramidal neurons, the principal neuronal types in the cerebral cortex, using a recombinant retrovirus that carries the gene encoding Escherichia coli beta-galactosidase as a lineage marker . The phenotype of every cell of clones of beta-galactosidase-labelled neurons generated by intraventricular injection of recombinant retrovirus in rat embryos at different stages of cortical neurogenesis was identified using light and electron microscopy as well as immunohistochemistry for known markers of neuronal subtypes . We found that clonally related neurons in adult rats showed the same morphological and neurotransmitter phenotypes, suggesting that lineages of pyramidal and nonpyramidal neurons are specified as early as E14, the time of onset of neurogenesis . However, when we followed the development of cortical cell lineages, we noted that a significant number of neuronal clones showed a mixed pyramidal/nonpyramidal cell composition during the first three weeks of life . We suggest that the change in the composition of neuronal clones between the third week of postnatal life and adulthood may either be due to changes in the phenotype of some developing neurons or, more likely, to selective cell death. J Cardiovasc Pharmacol, 1995, 25 Suppl 2, S23 - 9 Neutrophil activation, tumor necrosis factor, and survival after endotoxic and hemorrhagic shock; Barroso-Aranda J et al.; Polymorphonuclear neutrophils (PMNs) may contribute to organ injury in both hemorrhagic and endotoxic shock . Both models of shock exhibit a "flight of the leukocytes," but the mechanisms for entrapment of leukocytes in the microcirculation differ . The objective of this study was to investigate lipopolysaccharide (LPS)-induced shock and hemorrhagic shock with similar survival rates, in terms of circulating PMNs, activated circulating PMNs, plasma tumor necrosis factor (TNF) activity, and PMN adhesion . In the LPS protocol, rats received 6.5 mg/kg E . coli LPS i.v., which resulted in 50% survival . In the hemorrhagic shock protocol, rats were maintained for 3 h at 40 mm Hg mean arterial pressure, and survival during a 24-h observation period was 40% . LPS injection and hemorrhage caused rapid neutropenia in survivors and nonsurvivors . Low circulating PMN counts persisted during hypotension in the hemorrhagic protocol and among nonsurvivors in the LPS protocol, but in both protocols a tendency toward significantly higher circulating PMN counts in survivors compared with nonsurvivors was found . In both protocols, survivors had significantly lower fractions of circulating activated PMNs and lower adhesion of circulating PMNs to nylon fibers . In the LPS protocol, higher plasma TNF activity was found in nonsurvivors than in survivors, but no TNF activity in plasma could be found throughout the hemorrhagic protocol . These results indicate that nonsurvivors in both shock models exhibit higher levels of PMN activation . No correlation was detected between PMN activation and plasma TNF activity to suggest that TNF serves as the primary mediator of circulating PMN activation. Hum Antibodies Hybridomas, 1995, 6(4), 129 - 36 A genetically engineered fusion protein M4/TNF with increased bifunctional activity refolded in the presence of protein disulfide isomerase; Yang J et al.; Recombinant DNA techniques were used to clone, to construct and to express a fusion protein M4/TNF in Escherichia coli . The fusion protein includes the chimeric F(ab')2 fragment (M4) recognizing the human tumor-associated TAG72 antigen and the tumor necrosis factor alpha (TNF) moiety . The M4/TNF purified from inclusion bodies of the bacteria homogenates was further solubilized in a denaturing buffer containing 6 mol l-1 guanidine and refolded in a refolding buffer . Our results showed that the M4/TNF refolded in a buffer containing 6 mmol l-1 oxidized glutathione (GSSG), 0.2 mmol l-1 dithioerythione (DTE) and 0.5 mumol l-1 protein disulfide isomerase (PDI) displayed a 4-fold higher anti-TAG72 immunoreactivity and a 5-fold higher TNF activity than that refolded in the same refolding buffer but without PDI . Our data thus indicates that the protein disulfide isomerase not only facilitates the correct formation of disulfide-bonds of the antibody molecule, but also the correct refolding of the TNF moiety in vitro. Skin Pharmacol, 1995, 8(6), 300 - 8 Cellular and topical in vivo inflammatory murine models in the evaluation of inhibitors of phospholipase A2; Glaser KB et al.; Several novel inhibitors of human synovial fluid phospholipase A2 (HSF-PLA2) were evaluated in cellular models of inflammatory mediator release (murine macrophage and human neutrophil) and topical in vivo inflammatory skin models in mice to ascertain the scope of effects which might be observed for PLA2 inhibitors . Potent inhibition of HSF-PLA2 in vitro can be observed with compounds such as scalaradial and ellagic acid, which both have IC50 values of 0.02 microM (using autoclaved {3H}-arachidonic-acid (AA)-labelled Escherichia coli membranes as substrate) . Luffariellolide, a manoalide analog, and aristolochic acid are less potent (IC50 = 5 and 46 microM, respectively) in this assay . An interesting observation is that ellagic acid in cellular assays does not inhibit macrophage eicosanoid production and only 30% inhibition of PAF biosynthesis can be obtained at 50 microM in the human neutrophil . Possibly due to its irreversible mechanism of action, scalaradial retained its potent activity in both the macrophage (IC50 for PGE2 production = 0.05 microM) and neutrophil assays (IC50 for PAF biosynthesis = 1 microM) . Aristolochic acid is active in these cellular assays (macrophage IC50 = 2.5 microM and neutrophil IC50 = 100 microM), but is consistently less active than either scalaradial or luffariellolide . The relative potencies of these compounds were determined in several murine in vivo inflammatory models such as oxazolone contact hypersensitivity, AA-induced ear edema and phorbol ester (PMA)-induced ear edema . In the mouse model of oxazolone contact hypersensitivity, these PLA2 inhibitors have little effect (< or = 30% inhibition at 400 micrograms/ear) with scalaradial and luffariellolide being less effective than either aristolochic or ellagic acid . PMA-induced ear edema was effectively inhibited by scalaradial, luffariellolide and aristolochic acid (ED50 = 70, 50 and 50 micrograms/ear, respectively) whereas ellagic acid was less effective (ED50 = 230 micrograms/ear) . In AA-induced ear edema, these PLA2 inhibitors had minimal effects, as would be expected for compounds which inhibit PLA2 . These results, especially those of ellagic acid, suggest that caution should be taken in the extrapolation of potency against a purified human extracellular type PLA2 to the scope of activities these compounds might have in the cellular and in vivo models . The consistency of scalaradial and luffariellolide may be inherent to their irreversible mechanism of action, which is a factor to be accounted for in the extrapolation of enzyme data to cellular and in vivo models. Intervirology, 1995, 38(3-4), 187 - 91 Occurrence of the antibody against human papillomavirus type 16 virion protein L2 in patients with cervical cancer and dysplasia; Kanda T et al.; Infection with HPV 16 is believed to be a major risk factor for cervical cancer . To correlate HPV 16 infection and carcinogenesis in the cervix, we examined by ELISA 326 sera from healthy females and patients with cervical cancer, cervical intraepithelial neoplasia, or dysplasia, for the presence of IgG antibodies against HPV 16 virion protein L2 expressed in Escherichia coli . Whereas 2 of 208 were positive in the healthy females, 4 of 23 and 6 of 90 were positive in the patients with cervical cancer and dysplasia, respectively . The findings indicate that infection with HPV 16 is related to cancer and dysplasia of the cervix . The anti-L2 antibody did not occur coincidentally with the antibodies against the HPV 16 early proteins E4 and E7, which are specifically but independently associated with patients with cervical cancer. Chin J Biotechnol, 1995, 11(3), 185 - 91 Cloning and overexpression of thermostable DNA polymerase gene in Escherichia coli; Jin C et al.; Thermostable DNA polymerase genes had been amplified from Thermus aquaticus YT-1 using the PCR technique . The amplified 2.5-kb DNA fragment was inserted into pUC18 and confirmed to be a thermostable DNA polymerase gene by restriction mapping and DNA sequencing . The insert fragment was then combined into an expression vector, pBV221 . This recombinant plasmid overexpressed a 94-kDa of recombinant protein in E . coli . A 100-mL E . coli . culture could yield 1.5 x 10(5) units of Taq DNA polymerase, which could be applied in the PCR. Chin J Biotechnol, 1995, 11(3), 157 - 62 Purification of recombinant human granulocyte-macrophage colony stimulating factor expressed in Escherichia coli; Ling M et al.; Recombinant human granulocyte-macrophage colony stimulating factor (rhGM-CSF) was expressed as inclusion bodies (IB) in E . coli . A simple and effective protocol has been worked out for the purification . IB collected after the breakage of bacteria through sonication were subjected to repeated washing followed by solubilization in TE buffer (50 mmol/L of Tris.HCl, 1 mmol/L of EDTA, pH 8.3) containing 8 mol/L urea and 10 mmol/L DL-dithiothreitol . By means of Sephacryl-200 HR, refolding, and Q Sepharose Fast Flow, rhGM-CSF was obtained with a purity of 99% . The total protein recovery was 10% and specific activity of rhGM-CSF was 1 x 10(7) u/mg . The sequence of N-terminal 16 amino acid residues of purified rhGM-CSF was determined and found to be identical to the native protein . This study provided useful parameters for mass production of rhGM-CSF. Acta Vet Scand, 1995, 36(4), 499 - 508 Hematological and blood biochemical effects of fasting and subsequent oral administration of endotoxin in prepubertal gifts; Holst H et al.; The main objective of the present study was to investigate the effects of short-term fasting in gilts on endocrinological and blood biochemical parameters and, further, the effects of subsequent oral endotoxin (ET) administration . Group 1 was fasted for 30 h and then received feed with ET added . Group 2 was fasted for 30 h but received standard feed at refeeding . In group 3, gilts were fed every 6 h for 30 h . The major effects of fasting were: gradually increased concentration of plasma prostaglandin F2 alpha metabolite, serum total bilirubin, serum free fatty acids, and decreased serum glucose . The values were normalized within 1-4 h of refeeding . Twelve hours after refeeding, the ET-refed gilts showed higher levels of serum total bile acids and polymorphonuclear leukocytes than those in group 2 . It is possible that the observed changes during fasting reflect either an increased intestinal uptake of naturally present endotoxin or a reduced endotoxin detoxifying capacity of the liver . The increased bile acid concentration and polymorphonuclear leukocyte count following refeeding with ET-feed may indicate that orally administered ET is to some extent absorbed from the gut. Scand J Infect Dis, 1995, 27(4), 369 - 73 Phagocytosis and oxidative burst of blood phagocytes in chronic obstructive airway disease; Muns G et al.; Acute diseases and chronic conditions might be associated with altered blood neutrophil functions . The aim of this study was to investigate teh effects of stable chronic obstructive pulmonary disease (COPD) and superimposed acute bacterial bronchopneumonia (ABP) on the phagocytosis and oxidative metabolism of blood phagocytes . The 129 participants were assigned to 6 groups: group 1, healthy controls; group 2, previously healthy controls with ABP; group 3, patients with stable COPD on inhaled corticosteroids (IC); group 4, individuals with stable COPD on IC and ABP; group 5, patients with stable COPD requiring oral corticosteroids (OC); group 6, individuals with COPD on OC and ABP . Phagocytosis and oxidative burst was measured . Both tests showed a significant difference between the groups without acute infection when compared to the patients with ABP . No differences were observed between patients with stable COPD without ABP (groups 3 and 5) and group 1 . The number of phagocytized E . coli/cell was 9.1 +/- 0.37, 7.9 +/- 0.27 and 8 .2 +/- 0.28 (groups 2, 4, and 6, respectively) reversed the observed changes . We conclude, that acute bacterial bronchopneumonia is associated with an impairment of phagocytosis and oxidative metabolism in blood . We suggest, that stable COPD has no influence on phagocytosis and oxidative burst. J Cell Sci Suppl, 1995, 19, 73 - 7 Recognition and processing of damaged DNA; Lindahl T; Base excision-repair, which is required for correction of spontaneous hydrolytic and oxidative damage to DNA as well as lesions inflicted by alkylating agents, is a relatively well understood repair pathway . Mammalian factors involved in this pathway are reviewed, with emphasis on current uncertainties . Most DNA replication and repair enzymes in mammalian cell nuclei, e.g . DNA polymerases alpha, beta, delta, and epsilon, have direct counterparts in yeast . In contrast, the abundant enzymes in mammalian cell nuclei that bind and are activated specifically by DNA strand interruptions, poly(ADP-ribose) polymerase and DNA-dependent protein kinase, have not been detected in yeast; nor has p53, which is elevated in response to DNA strand breaks . We have found a family of four distinct DNA ligases in human cell nuclei, whereas only a single DNA ligase has been detected in yeast . It would appear that the cellular responses to DNA strand breaks may differ markedly between higher and lower eukaryotes. J Cell Sci Suppl, 1995, 19, 51 - 8 Analysis of the temporal program of replication initiation in yeast chromosomes; Friedman KL et al.; The multiple origins of eukaryotic chromosomes vary in the time of their initiation during S phase.In the chromosomes of Saccharomyces cerevisiae the presence of a functional telomere causes nearby origins to delay initiation until the second half of S phase . The key feature of telomeres that causes the replication delay is the telomeric sequence (C(1-3)A/G(1-3)T) itself and not the proximity of the origin to a DNA end . A second group of late replicating origins has been found at an internal position on chromosome XIV . Four origins, spanning approximately 140 kb, initiate replication in the second half of S phase . At least two of these internal origins maintain their late replication time on circular plasmids . Each of these origins can be separated into two functional elements: those sequences that provide origin function and those that impose late activation . Because the assay for determining replication time is costly and laborious, it has not been possible to analyze in detail these 'late' elements . We report here the development of two new assays for determining replication time . The first exploits the expression of the Escherichia coli dam methylase in yeast and the characteristic period of hemimethylation that transiently follows the passage of a replication fork . The second uses quantitative hybridization to detect two-fold differences in the amount of specific restriction fragments as a function of progress through S phase . The novel aspect of this assay is the creation in vivo of a non-replicating DNA sequence by site-specific pop-out recombination . This non-replicating fragment acts as an internal control for copy number within and between samples . Both of these techniques are rapid and much less costly than the more conventional density transfer experiments that require CsCl gradients to detect replicated DNA . With these techniques it should be possible to identify the sequences responsible for late initiation, to search for other late replicating regions in the genome, and to begin to analyze the effect that altering the temporal program has on chromosome function. Nucleic Acids Symp Ser, 1995, (33), 95 - 8 Spinach chloroplast RNase P: a putative protein enzyme; Thomas BC et al.; Ribonuclease P (RNase P) is the enzyme responsible for endonucleolytically separating the 5'-leader sequence from precursor tRNA molecules . In bacteria, and in the nuclei and mitochondria of all eukaryotes studied so far, RNase P contains an RNA subunit which is necessary for activity in vitro and in vivo . In contrast, we showed earlier that partially-purified RNase P from spinach chloroplasts had physical properties inconsistent with the presence of any RNA . We now report that the properties of the chloroplast enzyme, after 500 to 1500-fold purification, are consistent with enzymatic activity residing in a approximately 70 kDa polypeptide . Gel filtration chromatography on Sephacryl S-200 and S-300 provides a mass for chloroplast RNase P of approximately 70 +/- 5 kDa . A single polypeptide of approximately 70-80 kDa can be crosslinked to iodoUMP-substituted pre-tRNA . The labeling intensity of this polypeptide corresponds closely to the peak of RNase P activity on Sephacryl S-200 chromatography . Unlike the bacterial ribozyme-type RNase P, chloroplast RNase P is not a metalloenzyme . We showed previously that phosphodiester bond cleavage by the E . coli RNA enzyme absolutely requires Mg2+ or Mn2+ coordinated to the pro-Rp oxygen of the scissile phosphodiester phosphate . In contrast, we now find that chloroplast RNase P has no such requirement, and can accurately and efficiently cleave pre-tRNA containing an Rp-thio-substitution at the scissile bond . These data are entirely consistent with the hypothesis that RNase P in plant chloroplasts is not a ribozyme, but a conventional protein enzyme. Nucleic Acids Symp Ser, 1995, (33), 85 - 8 Autoregulation of RNase E synthesis in Escherichia coli; Jain C et al.; RNase E plays a central role in controlling mRNA degradation in E . coli . We have investigated the mechanism of RNase E autoregulation . Our data indicate that RNase E autoregulates its synthesis by controlling the decay rate of its own transcript (rne mRNA), which is unusually sensitive to the level of cellular RNase E activity . Feedback regulation is mediated in cis by the rne 5' untranslated region (5' UTR), which can confer this property onto heterologous mRNAs to which it is fused . The marked sensitivity of rne mRNA to regulation by RNase E is also due in part to the susceptibility of nascent rne transcripts to RNase E-mediated degradation. Nucleic Acids Symp Ser, 1995, (33), 76 - 8 Three dimensional model for the 16S ribosomal RNA that incorporates information for the mRNA track; Wollenzien P et al.; A three dimensional model for the 16S rRNA in the ribosome is described that accommodates information for mRNA.16S rRNA interactions as well as accommodating information that has been used as constraints for determining the internal 16S rRNA structure . mRNA.16S rRNA interactions have been summarized from experiments that employ photoaffinity crosslinking to identify sites at which the mRNA comes into close contact with the 16S rRNA . The mRNA track that has been constructed follows a path completely around the middle of the 30S subunit, occupying a space above 16S rRNA domains I and II and below 16S rRNA domain III . The mRNA track contains regions associated with contacts with the 16S rRNA in, and just upstream of, the P tRNA site, associated with contacts around the A site and associated with neighborhoods upstream of the Shine-Dalgarno region and downstream of the decoding region . Structural constraints that come from UV-induced RNA-RNA crosslinks, suggest that the mRNA decoding region lies in a groove between three 16S rRNA duplex regions facing the 50S subunit. Nucleic Acids Symp Ser, 1995, (33), 70 - 2 Mutations at three sites in the Escherichia coli 23S ribosomal RNA binding region for protein L11 cause UGA-specific suppression and conditional lethality; Murgola EJ et al.; A single nucleotide change, G to A, at nucleotide position 1093 of E . coli 23S ribosomal RNA was found to cause UGA-specific suppression (D.K . Jemiolo, F.T . Pagel and E.J . Murgola, Proc . Natl . Acad . Sci . USA, in press) . To obtain new kinds of UGA-specific suppressors in 23S rRNA, we used segment-directed mutagenic PCR, and targeted first the 1405 nucleotide SnaBI/I-CeuI segment, which includes position 1093, of the rrnB operon cloned into a multicopy plasmid . The mutagenized fragments were subcloned into the plasmid vector and used to transform to ampicillin resistance (Ampr) a recipient strain containing a UGA mutation in trpA . The Ampr transformants were then screened for suppression of UGA . After purification, Trp+ transformants were tested for association of the suppressor phenotype first with the plasmid and then specifically with the SnaBI/I-CeuI fragment . In one screening, four different kinds of mutational change were found, all at three sites within a highly conserved hexanucleotide loop in domain II of 23S rRNA . This region is part of the site for binding of the large subunit protein L11, which has been shown to be involved in peptide chain termination in a specific way . All of the mutants (G1093A, G1093 delta, A1095 delta, and U1097 delta) suppress UGA mutations, but not UAA or UAG mutations, and all four types exhibit high-temperature conditional lethality when highly expressed . Several mechanisms can be suggested for the UGA-specific suppression exhibited by these mutants, including altered interaction with protein L11, Second-site mutations that overcome the conditional lethality of G1093A indicate that intramolecular interactions within 23S rRNA may play a role in peptide chain termination at the UGA stop codon. Nucleic Acids Symp Ser, 1995, (33), 68 - 9 Three dimensional model for the 16S ribosomal RNA in the Escherichia coli ribosome; Minchew P et al.; Molecular modeling has been performed to help correct and refine an approximate three dimensional structure for Eschericia coli 16S rRNA that originated as hand-built wire model . The wire model was built to accommodate the base pairs in 16S rRNA (1), mRNA.16S rRNA interactions (2,3), rRNA.ribosomal protein interactions (see 4), ribosomal protein-ribosomal protein distances (5), intramolecular RNA-RNA UV-crosslinks (6,7) and site-directed mutagenesis experiments (8) . In the computer model, individual base-paired regions were constructed separately and then were installed into the expected locations to produce the entire structure . We describe here the considerations, steps and results of this refinement process. Nucleic Acids Symp Ser, 1995, (33), 59 - 62 Photolabile oligoDNA probes of internal Escherichia coli ribosomal structure; Cooperman BS et al.; We are examining the spatial arrangement of ribosomal components in the vicinity of rRNA sequences of particular significance for ribosome structure and function through the use of radioactive, photolabile derivatives of oligoDNAs complementary to such sequences . The oligoDNA probes bind to their target sequences in intact ribosomal subunits, and, on photolysis, incorporate into neighboring ribosomal components . Labeled ribosomal proteins are subse