Proc Natl Acad Sci U S A, 1996 Oct 15, 93(21), 11466 - 70 Cloning of human acetyl-CoA carboxylase-beta and its unique features; Ha J et al.; Acetyl-CoA carboxylase, which has a molecular mass of 265 kDa (ACC-alpha), catalyzes the rate-limiting step in the biosynthesis of long-chain fatty acids . In this study we report the complete amino acid sequence and unique features of an isoform of ACC with a molecular mass of 275 kDa (ACC-beta), which is primarily expressed in heart and skeletal muscles . In these tissues, ACC-beta may be involved in the regulation of fatty acid oxidation, rather than fatty acid biosynthesis . ACC-beta contains an amino acid sequence at the N terminus which is about 200 amino acids long and may be uniquely related to the role of ACC-beta in controlling carnitine palmitoyltransferase I activity and fatty acid oxidation by mitochondria . If we exclude this unique sequence at the N terminus the two forms of ACC show about 75% amino acid identity . All of the known functional domains of ACC are found in the homologous regions . Human ACC-beta cDNA has an open reading frame of 7,343 bases, encoding a protein of 2,458 amino acids, with a calculated molecular mass of 276,638 Da . The mRNA size of human ACC-beta is approximately 10 kb and is primarily expressed in heart and skeletal muscle tissues, whereas ACC-alpha mRNA is detected in all tissues tested . A fragment of ACC-beta cDNA was expressed in Escherichia coli and antibodies against the peptide were generated to establish that the cDNA sequence that we cloned is that for ACC-beta. Virology, 1996 Oct 15, 224(2), 548 - 54 Cloning and expression of a single-chain antibody fragment specific for foot-and-mouth disease virus; Mason P et al.; The gene for a single-chain antibody (VHK) to a conformational epitope on the type A12 foot-and-mouth disease virus (FMDV) particle was assembled and expressed in Escherichia coli . The VHK, purified from periplasmic extracts immunoprecipitated virus as efficiently as its parental monoclonal antibody (MAb) and exhibited the same binding specificity when tested against panel of natural and genetically engineered virus particles . The VHK neutralized type A12 virus in the presence of goat anti-mouse IgG; however, in the absence of the second antibody, only weak neutralizing activity was detected . Preliminary analysis of the mechanism of viral neutralization indicated that both the MAb and the VHK neutralize by the same mechanism . Small amounts of the VHK allowed infection of cells via Fc receptor-mediated adsorption in the presence of the second antibody . These data represent the first report of a single-chain neutralizing antibody for a picornavirus and provide insights into the mechanisms of viral neutralization and virus uptake. Virology, 1996 Oct 15, 224(2), 368 - 79 Nla and Nlb of peanut stripe potyvirus are present in the nucleus of infected cells, but do not form inclusions; Hajimorad MR et al.; We investigated, by immunological and gene-fusion methods, whether the failure of peanut stripe potyvirus (PStV)-encoded nuclear inclusion proteins a (Nla) and b (Nlb) to form nuclear inclusions is due to the lack of their in vivo accumulation or the inability of one or both proteins to be transported into the nucleus Nla domains (Nla-VPg and Nla-proteinase), full-length Nlb, and full-length cylindrical inclusion (CI) protein of PStV were cloned, expressed in Escherichia coli, and used for antisera production . Immunoblot analysis of accumulation of Nla, Nlb, and CI in time course experiments revealed that they accumulated to similar levels in PStV-infected Nicotiana benthamiana . In immunocytochemical studies with electron microscopy, antiserum against Nla-VPg, Nla-Pro, and Nlb specifically labeled Nla and Nlb proteins throughout the nuclei of PStV-infected cells, in the absence of nuclear inclusions . Translational fusions were made between Nla and Nlb to either the green fluorescence protein or the beta-glucuronidase in vectors for transient gene expression or stable expression in transgenic plants respectively . Fusion proteins containing Nla accumulated in the nucleus, whereas fusion proteins containing Nlb accumulated in a punctate pattern in the cytoplasm . These data indicate that at least Nla possesses a nuclear localization signal. Blood, 1996 Oct 15, 88(8), 3208 - 15 Two novel mutations in the reduced nicotinamide adenine dinucleotide (NADH)-cytochrome b5 reductase gene of a patient with generalized type, hereditary methemoglobinemia; Manabe J et al.; Hereditary methemoglobinemia due to reduced nicotinamide adenine dinucleotide (NADH) cytochrome b5 reductase (b5R) deficiency is classified into two types, an erythrocyte (type I) and a generalized (type II) . We investigated the b5R gene of a patient with type II from a white United Kingdom (UK) family and found that the patient was a compound heterozygote for two novel mutations . The first mutation was a C-to-A transversion changing codon 42 (TAC: Tyr) to a stop codon in the one allele . From this mutant allele, the product without the catalytic portion of the enzyme is generated . The second one was a missense mutation at codon 95 (CCC-->CAC) in the other allele with the result that Pro changed to His within the flavin adenine dinucleotide (FAD)-binding domain of the enzyme . To characterize effects of this missense mutation on the enzyme function, we compared glutathione S-transferase (GST)-fused b5R with the GST-fused mutant enzyme with the codon 95 missense mutation (P95H) expressed in Escherichia coll . The mutant enzyme showed less catalytic activity, less thermostability, and a greater susceptibility to trypsin than did the normal counterpart . The absorption spectrum of the mutant enzyme in the visual region differed from that of the wild-type . These results suggest that this amino acid substitution influences both secondary structure and catalytic activity of the enzyme . The compound heterozygosity for the nonsense and the missense mutations apparently caused hereditary methemoglobinemia type II in this patient. Biochemistry, 1996 Oct 15, 35(41), 13485 - 93 A change in the internal aldimine lysine (K42) in O-acetylserine sulfhydrylase to alanine indicates its importance in transimination and as a general base catalyst; Rege VD et al.; O-Acetylserine sulfhydrylase (OASS) is a pyridoxal 5'-phosphate dependent enzyme that catalyzes a beta-replacement reaction forming L-cysteine and acetate from O-acetyl-L-serine (OAS) and sulfide . The pyridoxal 5'-phosphate (PLP) is bound at the active site in Schiff base linkage with a lysine . In the present study, the Schiff base lysine was identified as lysine 42, and its role in the OASS reaction was determined by changing it to alanine using site-directed mutagenesis . K42A-OASS is isolated as an external aldimine with methionine or leucine and shows no reaction with the natural substrates . Apo-K42A-OASS can be reconstituted with PLP, suggesting that K42 is not necessary for cofactor binding and formation of the external Schiff base . The apo-K42A-OASS, reconstituted with PLP, shows slow formation of the external aldimine but does not form the alpha-aminoacrylate intermediate on addition of OAS, suggesting that K42 is involved in the abstraction of the alpha-proton in the beta-elimination reaction . The external aldimines formed upon addition of L-Ala or L-Ser are stable and represent a tautomer that absorbs maximally at 420 nm, while L-Cys gives a tautomeric form of the external aldimine that absorbs at 330 nm, and is also seen in the overall reaction after addition of primary amines to the assay system . The use of a small primary amine such as ethylamine or bromoethylamine in the assay system leads to the initial formation of an internal (gamma-thialysine) or external (ethylamine) aldimine followed by the slow formation of the alpha-aminoacrylate intermediate on addition of OAS . Activity could not be fully recovered, and only a single turnover is observed . Data suggest a significant rate enhancement resulting from the presence of K42 for transimination and general base catalysis. Biochemistry, 1996 Oct 15, 35(41), 13363 - 7 Chemical rescue of Asp237-->Ala and Lys358-->Ala mutants in the lactose permease of Escherichia coli; Frillingos S et al.; Asp237 (helix VII) and Lys358 (helix XI) form a salt bridge in the lactose permease, and neutral replacement of either residue inactivates . Remarkably, noncovalent neutralization of the unpaired Asp or Lys residue, respectively, with n-alkylsulfonates or n-alkylamines of appropriate size restores active transport to high levels in the mutants . Saturation with respect to the concentration of the alkylamines and different size preferences suggest that the alkylamines bind sterically at position 358 . Rescue of Asp237-->Ala by alkylsulfonates is apparently more indiscriminate, since methane-, ethane-, or propane-sulfonate have comparable effects . Sodium and chloride, respectively, are also effective in rescuing the Lys358-->Ala and Asp237-->Ala mutants, while various other compounds are ineffective . In marked contrast to Asp237-->Ala or Lys358-->Ala permease, alkylsulfonates or alkylamines have no effect whatsoever on the activity of mutants with neutral replacements for Asp240, Glu269, Arg302, Lys319, His322, or Glu325 . The results support the conclusion that neutral replacement of one member of the charge pair between Asp237 and Lys358 leads to inactivation because of an unpaired charge in the low dielectric of the membrane . In addition, the findings are consistent with the idea that interactions between Arg302 and Glu325, His 322 and Glu269, and Asp240 and Lys319 play important roles in the mechanism of the permease, which is not the case for either Asp237 or Lys358 or the salt bridge between the two residues. Biochemistry, 1996 Oct 15, 35(41), 13303 - 9 DNA sequence- and structure-selective alkylation of guanine N2 in the DNA minor groove by ecteinascidin 743, a potent antitumor compound from the Caribbean tunicate Ecteinascidia turbinata; Pommier Y et al.; Ecteinascidin 743 is one of several related marine alkaloids isolated from the Caribbean tunicate Ecteinascidia turbinata . It is remarkably active and potent in a variety of in vitro and in vivo systems and has been selected for development as an anticancer agent . The present study investigates the interactions of ecteinascidin 743 with DNA . Ecteinascidin 743 retarded the electrophoretic migration of both supercoiled and relaxed simian virus 40 DNA even in the presence of sodium dodecyl sulfate and after ethanol precipitation, consistent with covalent DNA modifications . Similar results were obtained in a DNA oligonucleotide derived from ribosomal DNA . However, DNA denaturation reversed the DNA modifications . The homopolymeric oligonucleotide dG/dC was modified while neither the dI/dC nor the dA/dT oligonucleotides were, consistent with covalent attachment of ecteinascidin 743 to the exocyclic amino group at position 2 of guanine . Ecteinascidin 743 was then compared to another known DNA minor groove alkylating agent, anthramycin, which has also been shown to alkylate guanine N2 . Footprinting analyses with DNase I and 1,10-phenanthroline-copper and exonuclease III digestions showed that ecteinascidin 743 covers three to five bases of DNA and exhibits a different sequence specificity than anthramycin in the Escherichia coli tyrosine tRNA promoter (tyrT DNA) . The binding of ecteinascidin to DNA was abolished when guanines were substituted with inosines in this promoter . A band shift assay was designed to evaluate the influence of the bases flanking a centrally located guanine in an oligonucleotide containing inosines in place of guanines elsewhere . Ecteinascidin 743 and anthramycin showed similarities as well as differences in sequence selectivity . Ecteinascidin 743-guanine adducts appeared to require at least one flanking guanine and were strongest when the flanking guanine was 3' to the targeted guanine . These data indicate that ecteinascidin 743 is a novel DNA minor groove, guanine-specific alkylating agent. Biochemistry, 1996 Oct 15, 35(41), 13294 - 302 Interaction of pyridine nucleotide substrates with Escherichia coli dihydrodipicolinate reductase: thermodynamic and structural analysis of binary complexes; Reddy SG et al.; E . coli dihydrodipicolinate reductase exhibits unusual nucleotide specificity, with NADH being kinetically twice as effective as NADPH as a reductant as evidenced by their relative V/K values . To investigate the nature of the interactions which determine this specificity, we performed isothermal titration calorimetry to determine the thermodynamic parameters of binding and determined the three-dimensional structures of the corresponding enzyme-nucleotide complexes . The thermodynamic binding parameters for NADPH and NADH were determined to be Kd = 2.12 microM, delta G degree = -7.81 kcal mol-1, delta H degree = -10.98 kcal mol-1, and delta S degree = -10.5 cal mol-1 deg-1 and Kd = 0.46 microM, delta G degree = -8.74 kcal mol-1, delta H degree = -8.93 kcal mol-1, and delta S degree = 0.65 cal mol-1 deg-1, respectively . The structures of DHPR complexed with these nucleotides have been determined at 2.2 A resolution . The 2'-phosphate of NADPH interacts electrostatically with Arg39, while in the NADH complex this interaction is replaced by hydrogen bonds between the 2' and 3' adenosyl ribose hydroxyls and Glu38 . Similar studies were also performed with other pyridine nucleotide substrate analogs to determine the contributions of individual groups on the nucleotide to the binding affinity and enthalpic and entropic components of the free energy of binding, delta G degree . Analogs lacking the 2'-phosphate containing homologs . For all analogs, the total binding free energy can be shown to include compensating enthalpic and entropic contributions to the association constants . The entropy contribution appears to play a more important role in the binding of the nonphosphorylated analogs than in the binding of the phosphorylated analogs. Biochemistry, 1996 Oct 15, 35(41), 13282 - 7 A zinc binding site in viral serine proteinases; De Francesco R et al.; The NS3 protein of hepatitis C virus contains a chymotrypsin-like serine proteinase domain . We built a homology model of this domain which predicts the presence of a tetradentate metal binding site formed by three cysteines and one histidine . These residues are strictly conserved in all known hepatitis C viral genotypes as well as in other recently discovered related hepatitis viruses . We show that the hepatitis C virus enzyme does indeed contain a Zn2+ ion with S3N ligation and that the metal is required for structural integrity and activity of the enzyme . Strikingly, the residues forming the metal binding site are also conserved in the chymotrypsin-like 2A cysteine proteinases of picornaviruses . Remarkably, in these highly variable viral genomes the metal binding site is more conserved than the catalytic residues and thus allows us to define a novel class of zinc binding chymotrypsin-like proteinases and to identify a new attractive target for antiviral therapy. Biochem Biophys Res Commun, 1996 Oct 14, 227(2), 334 - 9 Mutagenic specificity of N-methyl-N'-nitro-N-nitrosoguanidine in the tonB gene on the chromosome of Escherichia coli recA+ and recA- cells; Wang X et al.; DNA base sequence changes induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) mutagenesis has been determined for the endogenous tonB gene of Escherichia coli recA+ strain and its isogenic recA56 strain . In the recA+ strain, base substitutions accounted for 48 mutations among 54 MNNG-induced independent mutations analyzed and consisted of 45 G:C to A:T transitions, two A:T to G:C transitions and one A:T to C:G transversion . In the recA56 strain, 67% (34/51) were base substitutions among which G:C to A:T transition (28/51) predominated, followed by three A:T to T:A transversions, two G:C to T:A transversions, and one A:T to G:C transition . These mutagenic specificities were consistent with the mispairing predicted by the methylation of the O6 position of guanine . In both strains the G:C to A:T mutations were found at guanine residues preceded by either guanine or thymine on the non-transcribed strand of the target gene. Biochim Biophys Acta, 1996 Oct 11, 1313(3), 179 - 86 Human recombinant alpha-parvalbumin and nine mutants with individually inactivated calcium- and magnesium-binding sites: biochemical and immunological properties; Rhyner JA et al.; Human recombinant alpha-parvalbumin (PVwt) and nine mutant proteins, containing inactivating substitutions at positions essential for Ca2+ binding in the CD Ca(2+)-binding site (PVE62V, PVD51A, PVD51A,62V), the EF site (PVE101V, PVD90A, PVD90A,E101V) or in both (PVE62V,E101V, PVD51A,D90A, PVD51A,E62V,D90A,E101V), were expressed and purified . Flow dialysis revealed that PVwt binds 2 Ca2+ with equal K'Ca, of 2.3 x 10(7) M-1 and that Mg2+ competes with a K'Mg.compet . of 4.9 x 10(3) M-1 . The three mutants with an inactivated CD site bind 1 Ca2+ with K'Ca, of 2.0 to 2.3 x 10(7) M-1 and K'Mg.compet . of 3.4 to 4.6 x 10(3) M-1, i.e . very similar to those of PVwt . The mutants with an inactivated EF site bind 1 Ca2+ with K'Ca values of 7.9 x 10(6), 4.5 x 10(6) and 3.6 x 10(6) M-1 for PVD91A, PVE102V and PVE101V,D91A, respectively . The K'Mg.compet values of these mutants are about 4-times lower than in PVwt . The three mutants with both sites inactivated bind neither Ca2+ nor Mg2+ . After excitation at 259 nm, human PV, which contains neither Tyr nor Trp, shows maximal fluorescence emission at 283 nm . Binding of either Ca2+ or Mg2+ to PVwt or to mutants with an inactivated EF site lead to a 1.8-fold decrease in fluorescence intensity, whereas the mutants with an inactivated CD show only a very slight decrease upon binding of Ca2+ or Mg2+ . Specific antibodies against human alpha-parvalbumin were raised in rabbits . Their reactivity was tested against the mutant proteins, and their potential value for location and functional studies was investigated. J Mol Biol, 1996 Oct 11, 262(5), 746 - 55 Glycinamide ribonucleotide transformylase undergoes pH-dependent dimerization; Mullen CA et al.; Glycinamide ribonucleotide transformylase (GART) exhibits closely packed dimers in all crystal forms (pH 6.75), but was demonstrated to be monomeric in solution under conditions of optimal catalytic efficiency (pH 7.5) . We undertook a study of the pH-dependent behavior of GART in solution to determine whether side-chain ionization is responsible for the observed difference in association state . In the pH range 6.8 to 7.5, dimeric GART reversibly dissociates into a monomeric form as demonstrated by dynamic light scattering . The data give a best fit to a cooperative three-proton transfer mechanism: {formula: see text} A comparison of normalized data obtained from difference UV-absorption spectroscopy with the dynamic light scattering data indicates that two or more tyrosine residues per monomer undergo a local conformational change concomitant with dimerization . Fluorescence studies show that the environment of one or both of the tryptophan residues distal to the dimer interface are also perturbed by dimerization . Fitting of the normalized titration curves yields an apparent pKa = 7.16(+/-0.02) and a subnanomolar KD for the transition . Examination of the dimer interface in the crystal structure indicates that there are two histidine residues, H54 and H73, that are likely responsible for the pH-dependent dimerization . There are also two tyrosine residues, Y67 and Y78, which are adjacent to the interface and which may be exposed during dimerization . Our study indicates that under physiological pH conditions, GART exists as a mixture of monomer and dimer in solution . Taken together, the fact that the monomer-dimer transition displays a sharp pH dependence, and the fact that the enzyme activity is maximal under conditions where it is fully monomeric, suggest that enzyme activity may be modulated by subtle pH changes in the cell. J Mol Biol, 1996 Oct 11, 262(5), 706 - 20 The three-dimensional structure of mammalian ribonucleotide reductase protein R2 reveals a more-accessible iron-radical site than Escherichia coli R2; Kauppi B et al.; The three-dimensional structure of mouse ribonucleotide reductase R2 has been determined at 2.3 A resolution using molecular replacement and refined to an R-value of 19.1% (Rfree = 25%) with good stereo-chemistry . The overall tertiary structure architecture of mouse R2 is similar to that from Escherichia coli R2 . However, several important structural differences are observed . Unlike the E . coli protein, the mouse dimer is completely devoid of beta-strands . The sequences differ significantly between the mouse and E . coli R2s, but there is high sequence identity among the eukaryotic R2 proteins, and the identities are localized over the whole sequence . Therefore, the three-dimensional structures of other mammalian ribonucleotide reductase R2 proteins are expected to be very similar to that of the mouse enzyme . In mouse R2 a narrow hydrophobic channel leads to the proposed binding site for molecular oxygen near to the iron-radical site in the interior of the protein . In E . coli R2 this channel is blocked by the phenyl ring of a tyrosine residue, which in mouse R2 is a serine . These structural variations may explain the observed differences in sensitivity to radical scavengers . The structure determination is based on diffraction data from crystals grown at pH 4.7 . Unexpectedly, the protein is not iron-free, but contains one iron ion bound at one of the dinuclear iron sites . This ferric ion is bound with partial occupancy and is coordinated by three glutamic acids (one bidentate) and one histidine in a bipyramidal coordination that has a free apical coordination position . Soaking of crystals in a solution of ferrous salt at pH 4.7 increased the occupancy on the already occupied site, but without any detectable binding at the second site. J Mol Biol, 1996 Oct 11, 262(5), 615 - 28 Identification of the bases in the ompF regulatory region, which interact with the transcription factor OmpR; Huang KJ et al.; Expression of the outer membrane protein OmpF of Escherichia coli K-12 is influenced by a variety of environmental signals . Most of the signals are thought to regulate OmpF expression at the level of transcription initiation . A key element of this regulation is the interaction between the transcriptional factor OmpR and the cis-acting regulatory region of ompF . In this study, we used a combination of DNase I, dimethyl sulfate and hydroxyl radical footprinting analysis and DNA migration retardation assays to identify the bases within the ompF regulatory region that are in contact with OmpR . Our results indicate that the -107 to -39 region of ompF contains three individual binding sites and that a single OmpR-binding site is capable of interacting with two OmpR molecules . We also establish that a single OmpR-binding site is composed of two half-sites and that both half-sites are required for the formation of stable OmpR/DNA complexes . Comparisons of the sequences protected by OmpR indicate that an OmpR-binding site spans approximately 18 bp and has two highly conserved G/C base-pairs that are separated by three nucleotides . Although the three OmpR-binding sites we identified exhibit limited sequence similarity, this may reflect the fact that two of the sites are incapable of binding OmpR independently and can bind OmpR only if adjacent to another OmpR-binding site . Finally, our DNA migration retardation assays suggest that phosphorylation stimulates the cooperative interactions between OmpR molecules bound at neighboring sites . Therefore, this study provides a detailed understanding of how OmpR interacts with its binding sites immediately upstream of ompF and serves as a foundation for studying how phosphorylation of OmpR results in the regulation of ompF expression in response to environmental signals. J Med Chem, 1996 Oct 11, 39(21), 4162 - 6 Inactivation of S-adenosyl-L-homocysteine hydrolase by amide and ester derivatives of adenosine-5'-carboxylic acid; Wnuk SF et al.; S-Adenosyl-L-homocysteine (AdoHcy) hydrolase has been shown to have (5'/6') hydrolytic activity with vinyl (5') or homovinyl (6') halides derived from adenosine (Ado) . This hydrolytic activity is independent of its 3'-oxidative activity . The vinyl (or homovinyl) halides are converted into 5'(or 6')-carboxaldehydes by the hydrolytic activity of the enzyme, and inactivation occurs via the oxidative activity . Amide and ester derivatives of Ado-5'-carboxylic acid were prepared to further probe the hydrolytic capability of AdoHcy hydrolase . The oxidative activity (but not the hydrolytic activity) is involved in the mechanism of inhibition of the enzyme by the ester and amide derivatives of Ado-5'-carboxylic acid, in contrast to the inactivation of this enzyme by adenosine-derived vinyl or homovinyl halide analogues during which both activities are manifested. J Biol Chem, 1996 Oct 11, 271(41), 25624 - 9 Different inducibility of expression of the two xylanase genes xyn1 and xyn2 in Trichoderma reesei; Zeilinger S et al.; Regulation of formation of the extracellular xylanase system of Trichoderma reesei QM 9414 during growth on xylan, cellulose, and replacement onto a number of soluble inducers was investigated by Northern analysis of xyn1 and xyn2 transcripts and by the use of the Escherichia coli hph (hygromycin B-phosphotransferase-encoding) gene as a reporter . Whereas the xyn1 promoter is active in the presence of xylan and xylose, and virtually silenced in the presence of glucose, the xyn2 promoter enables basal transcription at a low level, but is enhanced in the presence of xylan and xylobiose and also of sophorose or cellobiose . The respective regulatory nucleotide regions were localized on a 221-base pair fragment and a 55-base pair fragment of the xyn1 and xyn2 5'-upstream noncoding sequences, respectively . Electrophoretic mobility shift assays, using cell-free extracts, identified induction-specific protein-DNA complexes: one complex of high mobility was observed under basal, noninduced conditions (glucose) with xyn2, which was in part replaced by a slow-migrating complex upon induction by xylan or sophorose . Both complexes bound to a CCAAT box . With xyn1, the induced complex also binds to a CCAAT box, but this binding is not observed in the presence of the carbon catabolite repressor Cre1, which binds to a nearby located consensus motif. J Biol Chem, 1996 Oct 11, 271(41), 25575 - 81 Stonustoxin is a novel lethal factor from stonefish (Synanceja horrida) venom . cDNA cloning and characterization; Ghadessy FJ et al.; Stonustoxin (SNTX) is a multifunctional lethal protein isolated from venom elaborated by the stonefish, Synanceja horrida . It comprises two subunits, termed alpha and beta, which have respective molecular masses of 71 and 79 kDa . SNTX elicits an array of biological responses both in vitro and in vivo, particularly a potent hypotension that appears to be mediated by the nitric oxide pathway . As a prelude to structure-function studies, we have isolated and sequenced cDNA clones encoding the alpha- and beta-subunits of SNTX from a venom gland cDNA library . The deduced amino acid sequence of neither subunit shows significant homology with any known protein . Protein sequence alignment does, however, show the subunits to be 50% homologous to each other and implies that they may have arisen from a common ancestor . The subunits of this novel toxin lack typical N-terminal signal sequences commonly found in proteins that are secreted via the endoplasmic reticulum-Golgi apparatus pathway, indicating the possibility of its being secreted by a non-classical pathway, which is not clearly understood . The SNTX subunits have been expressed in Escherichia coli as cleavable fusion proteins that cross-react with antibodies raised against the native toxin . To the best of our knowledge, this is the first complete sequence of a fish-derived protein toxin to be reported. J Biol Chem, 1996 Oct 11, 271(41), 25423 - 9 Identification of the cpdA gene encoding cyclic 3',5'-adenosine monophosphate phosphodiesterase in Escherichia coli; Imamura R et al.; We have identified a gene, cpdA, located at 66.2 min of the chromosome of Escherichia coli that encodes cyclic 3',5'-adenosine monophosphate phosphodiesterase (cAMP phosphodiesterase, EC) . The expression of beta-galactosidase, which is a product of the lacZ gene, was repressed in cells that harbored multiple copies of the plasmid carrying the cpdA gene . Northern blotting showed that the transcription of the lacZ gene was inhibited in these cells . Multiple copies of the cpdA gene decreased the intracellular concentration of cAMP, which is a positive regulator for transcription of the lacZ gene . We found that the purified CpdA protein repressed in vitro transcription from the lacP1 promoter by decreasing cAMP . In addition, we showed that the CpdA protein hydrolyzed cAMP to 5'-adenosine monophosphate and that its activity was activated by iron . Our results suggested that regulation of intracellular concentration of cAMP is dependent not only on synthesis of cAMP but also on hydrolysis of cAMP by cAMP phosphodiesterase. J Biol Chem, 1996 Oct 11, 271(41), 25360 - 8 A partially functional DNA helicase II mutant defective in forming stable binary complexes with ATP and DNA . A role for helicase motif III; Brosh RM Jr et al.; To address the functional significance of motif III in Escherichia coli DNA helicase II, the conserved aspartic acid at position 248 was changed to asparagine . UvrDD248N failed to form stable binary complexes with either DNA or ATP . However, UvrDD248N was capable of forming an active ternary complex when both ATP and single-stranded DNA were present . The DNA-stimulated ATPase activity of UvrDD248N was reduced relative to that of wild-type UvrD with no significant change in the apparent Km for ATP . The mutant protein also demonstrated a reduced DNA unwinding activity . The requirement for high concentrations of UvrDD248N to achieve unwinding of long duplex substrates likely reflects the reduced stability of various binary and ternary complexes that must exist in the catalytic cycle of a helicase . The data suggest that motif III may act as an interface between the ATP binding and DNA binding domains of a helicase . The uvrDD248N allele was also characterized in genetic assays . The D248N protein complemented the UV-sensitive phenotype of a uvrD deletion strain to levels nearly equivalent to wild-type helicase II . In contrast, the mutant protein only partially complemented the mutator phenotype . A correlation between the level of genetic complementation and the helicase activity of UvrDD248N is discussed. J Biol Chem, 1996 Oct 11, 271(41), 25247 - 52 Interaction of ATP binding sites in the ArsA ATPase, the catalytic subunit of the Ars pump; Li J et al.; The ArsA ATPase is the catalytic subunit of the Ars pump that catalyzes arsenical extrusion in Escherichia coli, thus providing resistance . The active form of ArsA is a homodimer with four nucleotide binding sites, two from each monomer . The codons for Gly-15 in the N-terminal consensus nucleotide binding sequence and Gly-334 in the C-terminal sequence were individually mutated to cysteine codons . Cells expressing an arsAG334C mutation retained arsenite resistance, while an arsAG15C mutation resulted in substantial reductions in arsenite resistance, transport, and ATPase activity . Selection for suppression of the G15C mutation that restored arsenite resistance yielded an A344V substitution . Ala-344 is located adjacent to the C-terminal nucleotide binding sequence . The second site mutation did not suppress the loss of resistance resulting from G18D, G20S, or T22I substitutions in the N-terminal nucleotide binding site . Cells expressing the G15C/A344V double mutant regained arsenite extrusion . These results suggest a spatial proximity of Gly-15 and Ala-344 and support a model for interaction of the nucleotide binding sites in ArsA. J Biol Chem, 1996 Oct 11, 271(41), 25178 - 83 Molecular design of inhibitors of in vitro oriC DNA replication based on the potential to block the ATP binding of DnaA protein; Mizushima T et al.; DnaA protein, the initiation factor for chromosomal DNA replication in Escherichia coli, is activated by binding to ATP . We earlier reported that 3-acetoxy-2,2'-bi-1H-indol inhibited the ATP binding to DnaA protein (Sasaki, S., Mizushima, T., Hashimoto, T., Maeda, M., and Sekimizu, K . (1994) Bioorg . Med . Chem . Lett . 4, 1771-1774) . In the present study, derivatives of 3-acetoxy-2,2'-bi-1H-indol with different lengths of aliphatic chains at the 3-O position were synthesized, and their potential to inhibit the ATP binding to DnaA protein was examined . Elongation of the aliphatic chain resulted in inhibition of the ATP binding to DnaA protein at lower concentrations . Among the derivatives, 3-{N-(11-carboxyundecyl)}carbamoylmethoxy-2,2'-bi-1H-indol (structure 7 (3-CUCM-BI)) exhibited the most potent inhibition with an IC50 value of 7 microM . The mode of the inhibition was competitive . We further demonstrated that structure 7 (3-CUCM-BI) inhibited DNA replication of the oriC plasmid in a system reconstituted from purified proteins . This inhibition was specific for the initiation of DNA replication rather than for the elongation . The inhibition was overcome by preincubation of DnaA protein with ATP . Furthermore, structure 7 (3-CUCM-BI) showed little inhibition on DNA synthesis in the ABC primosome system . We propose that structure 7 (3-CUCM-BI) functions in the in vitro oriC DNA replication by inhibiting the ATP binding to DnaA protein. J Biol Chem, 1996 Oct 11, 271(41), 25126 - 30 Identification of histone H2A.X as a growth factor secreted by an androgen-independent subline of mouse mammary carcinoma cells; Watabe Y et al.; Shionogi carcinoma 115 (SC 115) cells and Chiba subline 2 (CS 2) cells are clones of an androgen-responsive mouse tumor cell line and its autonomous subline, respectively . We have shown previously that CS 2 cells produce a heparin-binding growth factor that stimulates the growth of SC 115 cells as well as the growth of themselves . In this study, a growth factor was purified from serum-free conditioned media of CS 2 cells cultured without testosterone . A heparin-binding fraction showed growth- promoting activity on SC 115 cells and BALB/3T3 cells . The amino acid sequence analysis revealed that the components were identical to histones H2A.1 and H2A.X . Since histone H2A purified from bovine thymus had almost no growth-promoting activity on SC115 cells, histone H2A.X was assumed to be a growth factor . cDNA of histone H2A.X was cloned from a library of CS 2 cells, and its sequence was confirmed . The expressed product of histone H2A.X cDNA in Escherichia coli showed remarkable stimulatory effects on growth of SC 115 cells cultured in the absence of testosterone . These results indicate that histone H2A.X is secreted from CS 2 cells cultured without testosterone and plays a role as a growth factor. J Biol Chem, 1996 Oct 11, 271(41), 25083 - 8 Heterologous expression of three plant serpins with distinct inhibitory specificities; Dahl SW et al.; For the first time, inhibitory plant serpins, including WSZ1 from wheat, BSZ4, and the previously unknown protein BSZx from barley, have been expressed in Escherichia coli, and a procedure for fast purification of native plant serpins has been developed . BSZx, BSZ4, and WSZ1 were assayed for inhibitory activity against trypsin, chymotrypsin, and cathepsin G, and cleavage sites in the reactive center loop were identified by sequencing . BSZx proved to be a potent inhibitor with specific, overlapping reactive centers either at P1 Arg for trypsin or at P2 Leu for chymotrypsin . At 22 ;C, the apparent rate constant for chymotrypsin inhibition at P2 (ka = 9.4 x 10(5) M-1 s-1) was only four times lower than for trypsin at P1 (ka = 3.9 x 10(6) M-1 s-1), and the apparent inhibition stoichiometries were close to 1 . Furthermore, our data suggest that cathepsin G was inhibited by BSZx (ka = 3.9 x 10(6) M-1 s-1) at both the P1 Arg and P2 Leu . These results indicate a unique adaptability of the reactive center loop of BSZx . WSZ1 inhibited chymotrypsin (ka = 1.1 x 10(5) M-1 s-1) and cathepsin G (ka = 7.6 x 10(3) M-1 s-1) at P1 Gln and not, as for BSZx, at the more favorable P2 Leu . BSZ4 inhibited cathepsin G (ka = 2.7 x 10(4) M-1 s-1) at P1 Met but was hydrolyzed by trypsin and chymotrypsin . The three plant serpins formed stable SDS-resistant complexes with the proteinases in accordance with the kinetic data. J Biol Chem, 1996 Oct 11, 271(41), 25079 - 82 Selectivity of the renal collecting duct water channel aquaporin-3; Echevarria M et al.; Aquaporin-3 (AQP3) is a water channel found in the basolateral cell membrane of principal cells of the renal collecting tubule as well as in other epithelia . To examine the selectivity of AQP3, the permeability to water (Pf), urea (Pur), and glycerol (Pgly) of Xenopus oocytes injected with cRNA encoding AQP3 was measured . Oocytes injected with cRNA encoding either human or rat aquaporin-1 (AQP1) were used as controls . Although both aquaporins permit water flow across the cell membrane, only AQP3 was permeable to glycerol and urea (Pgly > Pur) . The uptake of glycerol into oocytes expressing AQP3 was linear up to 165 mM . For AQP3 the Arrhenius energy of activation for Pf was 3 kcal/mol, whereas for Pgly and Pur it was >12 kcal/mol . The sulfhydryl reagent p-chloromercuriphenylsulfonate (1 mM) abolished Pf of AQP3, whereas it did not affect Pgly . In addition, phloretin (0.1 mM) inhibited Pf of AQP3 by 35%, whereas it did not alter Pgly or Pur . We conclude that water does not share the same pathway with glycerol or urea in AQP3 and that this aquaporin, therefore, forms a water-selective channel. J Biol Chem, 1996 Oct 11, 271(41), 25063 - 6 Nucleotides reveal polynucleotide phosphorylase activity from conventionally purified GroEL; Ybarra J et al.; GroEL, as conventionally purified, can be incubated with nucleotides to produce high molecular weight material with an absorption maximum at 260 nm . This material is most clearly demonstrated when samples are subjected to gel filtration under conditions where GroEL is monomeric . There is a time-dependent increase in the high molecular weight material that occurs on incubation with ADP or, more slowly, with ATP . This material is generated during incubation, and none is present in the initial samples . Experiments with nucleases, proteases, radiolabeled nucleotides, and chemical cleavage reagents demonstrate that the high molecular weight material is polyadenylic acid whose formation is inhibited by phosphate . These results are consistent with the GroEL samples containing polynucleotide phosphorylase activity . Nondenaturing gels stained with acridine orange, after incubation in ADP, reveal that the activity producing the poly(A) coelectrophoreses with authentic polynucleotide phosphorylase . Conditions that remove the tryptophan-like fluorescence from preparations of GroEL also remove the PNPase activity . Thus, this activity is not associated with GroEL itself . The results are consistent with reports that GroEL can associate with RNase E and with other studies showing that RNase E and PNPase can form complexes . Thus, the present experiments support suggestions that GroEL can participate in multiprotein complexes that are involved in mRNA processing and degradation. Gene, 1996 Oct 10, 175(1-2), 223 - 31 Molecular cloning of higher plant homologues of the high-affinity nitrate transporters of Chlamydomonas reinhardtii and Aspergillus nidulans; Trueman LJ et al.; The crnA nitrate transporter from Aspergillus nidulans was identified as belonging to the major facilitator superfamily (MFS) of membrane transporters . Degenerate oligonucleotides corresponding to the crnA sequences at the locations of two conserved sequence motifs were designed and used in the polymerase chain reaction (PCR) to amplify related sequences from barley root poly(A)+ RNA . A 130 bp cDNA fragment with sequence similarities to crnA was amplified and used as a probe to screen a barley root cDNA library . Two full-length clones (pBCH1 and pBCH2) were isolated . The nt sequences of pBHC1 and pBCH2 are closely related (80% identical) and potentially encode hydrophobic polypeptides of 54.7 and 55.0 kDa respectively, with twelve predicted transmembrane domains . The encoded polypeptides are 41-43% identical to the A . nidulans CRNA protein and 56-57% identical to NAR-3, a high-affinity nitrate transporter from the eukaryotic alga Chlamydomonas reinhardtii . Phylogenetic analysis indicated that crnA, nar-3 and the barley homologues belong to a new family within the MFS, a family that also includes narK, the gene for a nitrite efflux pump in Escherichia coli . In northern blots, BCH1 hybridised to a mRNA species of 1.9 kb which is rapidly induced in barley roots by NO3-, but not by NH4+, and genomic Southern blots indicated that there may be seven to ten BCH1-related genes in the barley genome. Gene, 1996 Oct 10, 175(1-2), 199 - 201 The 5' regulatory region from the Drosophila pseudoobscura hsp82 gene results in a high level of reporter gene expression in Lucilia cuprina embryos; Coates CJ et al.; We have previously examined the efficiency of two Drosophila melanogaster promoters to enable reporter gene expression in embryos of the Australian sheep blowfly, Lucilia cuprina . Both the hsp70 heat-shock promoter and the actin5C promoter resulted in low levels of expression of a reporter gene in these embryos . In this study, the D . pseudoobscura hsp82 promoter (phsp82) was tested for its ability to direct the expression of the Escherichia coli chloramphenicol acetyltransferase-encoding gene (cat) . We report that the level of CAT activity in L . cuprina embryos was comparable to that obtained with the same construct in D . melanogaster, indicating that phsp82 functions efficiently in this non-drosophilid insect . The results suggest that phsp82 may be utilised in other non-drosophilid insects in which poor expression levels are obtained from constructs containing the hsp70 or actin5C promoters. Gene, 1996 Oct 10, 175(1-2), 83 - 7 Cloning of a gene from Escherichia coli that confers resistance to fosmidomycin as a consequence of amplification; Fujisaki S et al.; A gene conferring resistance to fosmidomycin (Fs) was cloned from the gene pool of a wild-type strain of Escherichia coli . The cloned DNA fragment was sequenced and shown to encode a putative polypeptide of 406 amino acids (aa) with a molecular weight of 43303 . The gene mapped at 10.9 min on the E . coli chromosome and was designated fsr (fosmidomycin resistance) . Maxicell analysis revealed that the Fsr protein migrated in sodium dodecyl sulfate-polyacrylamide-gel electrophoresis as a broad band of 35 kDa . A comparison between the aa sequence of Fsr and sequences in a protein database revealed 18% homology to the bacterial drug-export proteins that mediate resistance to tetracycline and chloramphenicol . Hydropathy analysis of the Fsr protein revealed twelve putative transmembrane segments . The degree of FsR of transformants depended on the number of copies of the plasmid that contained fsr . The levels of ubiquinone-8 and undecaprenyl phosphate in cells that harbored a high-copy-number plasmid that included fsr were almost the same as those in the cells without the plasmid . These results suggest that Fsr does not have any direct effect on the biosynthesis of isoprenoid in E . coli, and that the mechanism for FsR involves the efflux of the drug by a process that is facilitated by Fsr. Nature, 1996 Oct 10, 383(6600), 550 - 3 Assembly of microtubule-associated protein tau into Alzheimer-like filaments induced by sulphated glycosaminoglycans; Goedert M et al.; The paired helical filament (PHF) is the major component of the neurofibrillary deposits that form a defining neuropathological characteristic of Alzheimer's disease . PHFs are composed of microtubule-associated protein tau, in a hyperphosphorylated state . Hyperphosphorylation of tau results in its inability to bind to microtubules and is believed to precede PHF assembly . However, it is unclear whether hyperphosphorylation of tau is either necessary or sufficient for PHF formation . Here we show that non-phosphorylated recombinant tau isoforms with three microtubule-binding repeats form paired helical-like filaments under physiological conditions in vitro, when incubated with sulphated glycosaminoglycans such as heparin or heparan sulphate . Furthermore, heparin prevents tau from binding to microtubules and promotes microtubule disassembly . Finally, we show that heparan sulphate and hyperphosphorylated tau coexist in nerve cells of the Alzheimer's disease brain at the earliest known stages of neurofibrillary pathology . These findings, with previous studies which show that heparin stimulates tau phosphorylation by a number of protein kinases, indicate that sulphated glycosaminoglycans may be a key factor in the formation of the neurofibrillary lesions of Alzheimer's disease. Biochemistry, 1996 Oct 8, 35(40), 13222 - 30 Activation of a Ca(2+)-dependent protein kinase involves intramolecular binding of a calmodulin-like regulatory domain; Huang JF et al.; Ca(2+)-dependent protein kinases (CDPKs) are regulated by a C-terminal calmodulin-like domain (CaM-LD) . The CaM-LD is connected to the kinase by a short junction sequence which contains a pseudosubstrate autoinhibitor . To understand how the CaM-LD regulates a CDPK, a recombinant CDPK (isoform CPK-1 from Arabidopsis, accession no . L14771) was made as a fusion protein in Escherichia coli . We show here that a truncated CDPK lacking a CaM-LD (e.g . mutant delta NC-26H) can be activated by exogenous calmodulin or an isolated CaM-LD (Kact approximately 2 microM) . We propose that Ca2+ activation of a CDPK normally occurs through intramolecular binding of the CaM-LD to the junction . When the junction and CaM-LD are made as two separate polypeptides, the CaM-LD can bind the junction in a Ca(2+)-dependent fashion with a dissociation constant (KD) of 6 x 10(-6) M, as determined by kinetic binding analyses . When the junction and CaM-LD are tethered in a single polypeptide (e.g . in protein JC-1), their ability to engage in bimolecular binding is suppressed (e.g . the tethered CaM-LD cannot bind a separate junction) . A mutation which disrupts the putative CaM-LD binding sequence (e.g . substitution LRV-1444 to DLPG) appears to block intramolecular binding, as indicated by the restored ability of a tethered CaM-LD to engage in bimolecular binding . This mutation, in the context of a full-length enzyme (mutant KJM46H), appears to block Ca2+ activation . Thus, a disruption of intramolecular binding correlates with a disruption of the Ca2+ activation mechanism . CDPKs provide the first example of a member of the calmodulin superfamily where a target binding sequence is located within the same polypeptide. Biochemistry, 1996 Oct 8, 35(40), 13180 - 5 Different sensitivities to acid denaturation within a family of proteins: implications for acid unfolding and membrane translocation; Evans LJ et al.; Colicins A, B, and N form a family of membrane pore-forming toxins with > 50% sequence identity in their toxic C-terminal domains . The colicin A C-terminal domain has been shown to insert into model membranes via an acidic molten-globule insertion intermediate, and thus this family provides a means to compare acid unfolding of related proteins . Unlike the domains of colicins A and B which are acidic, that of colicin N is very basic with fewer Asp and Glu residues . If surface positive charge density is the crucial factor in acidic molten globule formation, colicin N should begin to unfold at higher pH values than colicins A or B . However, comparison of their CD spectra reveals that colicins A and B both form acidic molten globules but colicin N does not . None of the proteins forms a denaturant-induced molten globule at neutral pH where the proteins exhibit very similar stabilities . The acidic unfolding cannot therefore be due to excess positive surface charge and may be caused by a subset of acidic residues as has been predicted for myoglobin . The difference between the colicins is confirmed by their in vivo membrane insertion, with colicins A and B inserting much faster than colicin N . Stopped-flow circular dichroism measurements of colicin A insertion into vesicles confirmed that a molten globule insertion intermediate occurs at the membrane surface. Biochemistry, 1996 Oct 8, 35(40), 13147 - 56 Substrate specificity of Escherichia coli MutY protein; Bulychev NV et al.; The MutY protein of Escherichia coli removes mismatched deoxyadenine residues from DNA . In this study, duplex oligodeoxynucleotides containing modified bases are used as model substrates for this enzyme . In contrast to a recent report {Lu, A.-L., et al . (1995) J . Biol . Chem . 270, 23582}, dA:8-oxo-dG appears to be the preferred natural substrate for MutY, as evidenced by the specificity constants (kcat/Km) for dA:8-oxo-dG and dA:dG of 39 600 x 10(-6) and 383 x 10(-6) (min-1 nM-1), respectively . kcat for the duplex containing dA:dG was highest at lower pH; the rate of cleavage for the duplex containing dA:8-oxo-dG was unaffected over a pH range of 5.5-8.0 . The presence of an 8-oxo function in dG increased significantly the rate of removal of dA from all substrates tested . Replacement of dA by rA reduced the specificity constant of dA:8-oxo-dG to 294 x 10(-6) (min-1 nM-1), whereas replacement of dA by 2'-O-methyladenosine virtually abolished enzymatic activity . Modifications of the dG moiety generally were better tolerated than those of dA; however, introduction of a methyl ether at the 6 position of dG produced a noncleavable substrate and replacement of dG by 2'-O-methylguanosine generated a substrate with a low specificity constant . Rates of cleavage of duplexes containing dA:dC and dA:tetrahydrofuran were three orders of magnitude lower than the reference substrate . Duplexes containing a carbocyclic analog of dA were not cleaved . A model is proposed to explain the recognition of DNA substrates by MutY and the catalytic properties of this enzyme. Biochemistry, 1996 Oct 8, 35(40), 12993 - 3000 Folding intermediates of a beta-barrel membrane protein . Kinetic evidence for a multi-step membrane insertion mechanism; Kleinschmidt JH et al.; The mechanism of folding and membrane insertion of integral membrane proteins, including helix bundle and beta-barrel proteins is not well understood . A key question is whether folding and insertion are coupled or separable processes . We have used the beta-barrel outer membrane protein A (OmpA) of Escherichia coli as a model to study the kinetics of folding and insertion into dioleoylphosphatidylcholine (DOPC) bilayers, as a function of temperature by gel electrophoresis, protease digestion, and fluorescence spectroscopy . OmpA was unfolded in 8 M urea solution (without detergent), and refolding and membrane insertion was initiated by rapid dilution of the urea concentration in the presence of phospholipid vesicles . In addition to the kinetically unresolved hydrophobic collapse in water, the time course of refolding of OmpA into DOPC bilayers exhibited three kinetic phases over a large temperature range . The first step was fast (k1 = 0.16 min-1) and not very dependent on temperature . The second step was up to two orders of magnitude slower at low temperatures (2 degrees C), but approached the rate of the first step at higher temperatures (40 degrees C) . The activation energy for this process was 46 +/- 4 kJ/mol . A third slow process (k3 = 0.9 x 10(-2) min-1 at 40 degrees C) was observed at the higher temperatures . These results suggest that at least two membrane-bound intermediates exist when OmpA folds and inserts into lipid bilayers . We also show that both membrane-bound intermediates can be stabilized in fluid lipid bilayers at low temperatures . These intermediates share many properties with the adsorbed/partially inserted form of OmpA that was previously characterized in gel phase lipid bilayers {Rodionova et al . (1995) Biochemistry 34, 1921-1929} . Temperature jump experiments demonstrate, that the low-temperature intermediates can be rapidly converted to fully inserted native OmpA . On the basis of these and previous results, we present a simple folding model for beta-barrel membrane proteins, in which folding and membrane insertion are coupled processes which involve at least four kinetically distinguishable steps. FEBS Lett, 1996 Oct 7, 394(3), 316 - 20 Quaternary structure of human nucleoside diphosphate kinase isoforms HA and HB in solution; Schaertl S; Human isoforms of nucleoside diphosphate kinase, NDPK-HA and NDPK-HB, have been expressed in E . coli and purified . Their apparent molecular masses have been determined by FPLC gel filtration . Absolute molecular masses were measured by equilibrium ultracentrifugation and sedimentation coefficients determined from the sedimentation velocity . Under near-physiological conditions, NDPK-HA has a mass of 101 +/- 3 kDa, close to that calculated for a hexamer (102.11 kDa), whilst NDPK-HB has a mass of 71 +/- 3 kDa, close to a tetramer (68.67 kDa) . The sedimentation coefficients, 5.15 +/- 0.2 and 3.41 +/- 0.1 x 10(-13) s, for HA and HB also indicate a hexamer and a tetramer respectively . This suggests, although the crystal structure shows a hexameric quaternary arrangement {Webb et al . (1995) J . Mol . Biol . 251, 574-587}, that NDPK-HB forms tetramers in solution like bacterial NDPK {Williams et al . (1993) J . Mol . Biol . 234, 1230-1247}. FEBS Lett, 1996 Oct 7, 394(3), 311 - 5 Loop mutations affect ferritin solubility causing non-native aggregation of subunits or precipitation of fully assembled polymers; Jappelli R et al.; As a consequence of elevated expression rates, the intracellular aggregation of polypeptide chains is commonly observed in E . coli . Although wild-type human ferritin, a polymeric iron storage protein, accumulates in the soluble form at high level in the bacterial cytoplasmic fraction, some amino acid substitutions in an exposed loop direct the synthesis of a highly insoluble product . We found that two mechanisms can lead to the aggregation of ferritin . While some mutations prevent ferritin polymerisation, others cause the precipitation of molecules in the assembled state. J Mol Biol, 1996 Oct 4, 262(4), 437 - 58 Mechanism, specificity and general properties of the yeast enzyme catalysing the formation of inosine 34 in the anticodon of transfer RNA; Auxilien S et al.; In yeast, inosine is found at the first position of the anticodon (position 34) of seven different isoacceptor tRNA species, while in Escherichia coli it is present only in tRNAArg . The corresponding tRNA genes all have adenosine at position 34 . Using as substrates in vitro T7-runoff transcripts of 31 plasmids carrying each natural of synthetic tRNA gene harbouring an anticodon with adenosine 34, we have characterised a yeast enzyme that catalyses the conversion of adenosine 34 to inosine 34 . The homologous E . coli enzyme modifies adenosine 34 only in tRNAs with an arginine anticodon ACG . The base conversion occurs by a hydrolytic deamination-type reaction . This was determined by reversed phase high-pressure liquid chromatography/electrospray mass spectrometry analysis of the reaction product after in vitro modification in {18O}water . This newly characterised tRNA:adenosine 34 deaminase was partially purified from yeast . It has a molecular mass of approximately 75 kDa, and it does not require any cofactor, except magnesium ions, to deaminate adenosine 34 efficiently in tRNA . The observed dependence of the enzymatic reaction on magnesium ions probably reflects the need for a correct tRNA architecture . Enzymatic recognition of tRNA does not depend on the presence of any "identify" nucleoside other than adenosine 34 . Likewise, the presence of pseudouridine 32 or 1-methyl-guanosine 37 in the anticodon loop does not interfere with inosine 34 biosynthesis . However, the efficacy of adenosine 34 to inosine 34 conversion depends on the nucleotide sequence of the anticodon loop and its proximal stem, the best tRNA substrates being those with a purine at position 35 . Mutations that affect the size of the anticodon loop or one of several three-dimensional base-pairs abolish the capacity of the tRNA to be substrate for the yeast tRNA:adenosine 34 deaminase . Evidently, the activity of yeast tRNA:adenosine 34 deaminase depends more on the global structural feature (conformational stability/flexibility) of the L-shaped tRNA substrates than on the identity of any particular nucleotide other than adenosine 34 . An apparent K(m) of 2.3 nM for its natural substrate tRNASer (anticodon AGA) was measured . Altogether, these results suggest that a single enzyme can account for the presence of inosine 34 in all seven cytoplasmic A34-containing precursor tRNAs in yeast. J Mol Biol, 1996 Oct 4, 262(4), 407 - 12 High-level misincorporation of lysine for arginine at AGA codons in a fusion protein expressed in Escherichia coli; Calderone TL et al.; The expression of eukaryotic genes in Escherichia coli is one of the most frequently used tools of modern science . The arginine codon AGA is a common codon in eukaryotic genes but is particularly rare in E . coli . We report here 36 to 42% misincorporation of lysine at three AGA codons in a well-expressed protein . This misincorporation yields a protein whose electrospray mass spectrum (ESMS) shows peaks at the expected mass (M), M-28, M-56 and M-84 with intensities representing 34.5(+/-0.7), 37.5(+/-1.1), 21.2(+/-1.7) and 6.6(+/-0.5) % of the total intensity, respectively . Replacement of either all three AGA codons or the two closest to the 3' end of the gene by the more common CGC arginine codon gave a protein with a single ESMS peak . Misincorporation could also be eliminated by the co-expression of the tRNA(UCL)Arg gene, argU . These studies demonstrate that misincorporation of amino acids at rare codons of recombinant proteins can be far higher than previously thought. J Mol Biol, 1996 Oct 4, 262(4), 389 - 95 The preprotein translocase of the inner mitochondrial membrane: evolutionary conservation of targeting and assembly of Tim17; Bomer U et al.; The preprotein translocase of the inner mitochondrial membrane has only been described in Saccharomyces cerevisiae to date . We report that the essential subunit Tim17 is highly conserved in evolution . The targeting and assembly of yeast Tim17 as well as that of human and Drosophila melanogaster Tim17 were characterized with isolated yeast mitochondria . Targeting signals in the mature protein direct the Tim17 precursors to the receptor Tom70 on the mitochondrial surface . In a membrane potential-dependent step the precursors insert into the inner membrane, adopt a characteristic topology and assemble with Tim23 . The mechanisms of targeting and assembly were indistinguishable between the Tim17s from distinct organisms, indicating a high evolutionary conservation. J Chromatogr A, 1996 Oct 4, 746(1), 17 - 24 Two-step chromatographic procedure for purification of basic fibroblast growth factor from recombinant Escherichia coli and characterization of the equilibrium parameters of adsorption; Seeger A et al.; A two-step chromatographic procedure for purification of basic fibroblast growth factor (bFGF) from high-cell-density cultures of recombinant E . coli is described . Heparin-Sepharose as a material which shows a high affinity to endothelial growth factors was used as sorbent for purification of bFGF from the soluble cell fraction . A one-step affinity chromatographic procedure resulted in very pure bFGF . However, this one-step affinity isolation of bFGF caused the loss of around 60% of the recombinant protein . A combination of ion-exchange chromatography with heparin-Sepharose affinity chromatography was favored for bFGF purification . A first cation-exchange chromatographic step resulted in a solution of bFGF with a purity of around 70% . The weak cation exchanger CM Sepharose C50 was preferred in comparison to the strong cation exchanger S-Sepharose because of the higher recovery of bFGF . With the ion-exchange chromatographic step prior to the heparin-Sepharose affinity chromatography, the total yield of recovery of bFGF increased to 56% compared to 40% using the one-step purification procedure with heparin-Sepharose . To characterize the equilibrium parameters of adsorption, batch experiments for the calculation of maximum capacities and dissociation constants for CM-Sepharose C50 and heparin-Sepharose were carried out . The equilibrium experiments revealed that adsorption of bFGF to the ion-exchange sorbent followed single-site interaction according to the Langmuir model of adsorption . The adsorption of bFGF to heparin-Sepharose was described by a double Langmuir approach of two independent binding sites with different maximum capacities and dissociation constants . The purified bFGF showed a high biological activity and circular dichroic spectra of a proper folded molecule . The analysis of the N-terminal amino acid sequence revealed a mixture of two fractions of bFGF, which both are characterized by the cleavage of the first amino acid methionine . In addition, half of the bFGF molecules lacked the second amino acid alanine. Cell, 1996 Oct 4, 87(1), 115 - 25 A structural model for the HIV-1 Rev-RRE complex deduced from altered-specificity rev variants isolated by a rapid genetic strategy; Jain C et al.; A broadly applicable genetic strategy was developed for investigating RNA-protein interactions and applied to the HIV-1 Rev protein . By rapidly screening thousands of Rev-RNA interactions in Escherichia coli, we isolated Rev suppressor mutations that alleviated the deleterious effect of mutations in RRE stem-loop IIB, the high affinity RNA-binding site for Rev . All of these suppressor mutations map to a single arginine-deficient face of a Rev alpha-helix, and some alter the binding specificity of the protein, providing genetic evidence for direct contacts between specific Rev amino acids and RNA nucleotides in the RNA complex of Rev . The spatial constraints suggested by these data have enabled us to model the structure of this complex. J Biol Chem, 1996 Oct 4, 271(40), 25027 - 34 Characterization of truncated forms of the KdpD protein, the sensor kinase of the K+-translocating Kdp system of Escherichia coli; Puppe W et al.; The expression of the kdpFABC operon, coding for the K+-translocating Kdp system, is controlled by the two regulatory proteins, KdpD and KdpE, which belong to the group of sensor kinase/response regulator systems . This study describes the construction and analysis of KdpD sensor kinases, in which different deletions in the N-terminal part of the protein were introduced . Truncated KdpD proteins, in which the membrane-spanning segments were deleted, had lost their phosphorylation capacity . Truncated KdpD proteins, in which the four membrane-spanning helices were untouched, were still phosphorylated, and the phosphoryl group could be transferred to the response regulator KdpE in vitro . Furthermore, these truncated KdpD proteins cause dephosphorylation of KdpE(P), which is comparable with that of the wild-type protein . To investigate the effect of the deletions on signal transduction in vivo the corresponding kdp genes were transferred to the chromosome . Growth studies with the mutant strains are in accord with the data obtained from the in vitro studies . Furthermore, kdp expression was investigated using a KdpA-LacZ fusion . The data obtained support the notion that the extent of kdp expression is modulated by the N-terminal part of KdpD. J Biol Chem, 1996 Oct 4, 271(40), 24954 - 61 A thumb subdomain mutant of the large fragment of Escherichia coli DNA polymerase I with reduced DNA binding affinity, processivity, and frameshift fidelity; Minnick DT et al.; In Klenow fragment DNA polymerase, a flexible 50-amino acid subdomain at the tip of the thumb which includes two alpha helices has been suggested to interact with the duplex template-primer (Beese, L.S., Derbyshire, V . and Steitz, T.A . (1993) Science 260, 352-355) . The present study investigates the properties of Klenow polymerase containing a 24-amino acid deletion (residues 590-613) that removes a portion of the tip of the thumb . The mutant polymerase has relatively normal dNTP binding and catalytic rate . However, its DNA binding affinity is reduced by more than 100-fold relative to the intact polymerase and its ability to conduct processive synthesis is also reduced . Although the mutant polymerase has relatively normal base substitution fidelity, it has strongly reduced frameshift fidelity, being especially error-prone for single nucleotide addition errors in homopolymeric runs . The addition error rate increases as the length of the reiterated sequence increases, indicative of errors initiated by template-primer strand slippage . These observations suggest a role for the tip of the thumb of Klenow polymerase in determining DNA binding, processivity and frameshift fidelity, perhaps by tracking the minor groove of the duplex DNA . The results are discussed in light of remarkably similar observations with T7 DNA polymerase in the presence or absence of thioredoxin, an accessory subunit that affects these same properties. J Biol Chem, 1996 Oct 4, 271(40), 24862 - 8 DNA binding by cut homeodomain proteins is down-modulated by protein kinase C; Coqueret O et al.; The Drosophila and mammalian Cut homeodomain proteins contain, in addition to the homeodomain, three other DNA binding regions called Cut repeats . Cut-related proteins thus belong to a distinct class of homeodomain proteins with multiple DNA binding domains . Using nuclear extracts from mammalian cells, Cut-specific DNA binding was increased following phosphatase treatment, suggesting that endogenous Cut proteins are phosphorylated in vivo . Sequence analysis of Cut repeats revealed the presence of sequences that match the consensus phosphorylation site for protein kinase C (PKC) . Therefore, we investigated whether PKC can modulate the activity of mammalian Cut proteins . In vitro, a purified preparation of PKC efficiently phosphorylated Cut repeats, which inhibited DNA binding . In vivo, a brief treatment of cells with calphostin C, a specific inhibitor of PKC, led to an increase in Cut-specific DNA binding, whereas phorbol 12-myristate 13-acetate, a specific activator of PKC, caused a decrease in DNA binding . The PKC phosphorylation sites within the murine Cut (mCut) protein were identified by in vitro mutagenesis as residues Thr415, Thr804, and Ser987 within Cut repeats 1-3, respectively . Cut homeodomain proteins were previously shown to function as transcriptional repressors . Activation of PKC by phorbol 12-myristate 13-acetate reduced transcriptional repression by mCut, whereas a mutant mCut protein containing alanine substitutions at these sites was not affected . Altogether, our results indicate that the transcriptional activity of Cut proteins is modulated by PKC. J Biol Chem, 1996 Oct 4, 271(40), 24806 - 10 Heat shock-induced DNA relaxation in vitro by DNA gyrase of Escherichia coli in the presence of ATP; Kataoka K et al.; Genetic studies revealed that DNA gyrase seems to catalyze immediate and transient DNA relaxation after Escherichia coli cells are exposed to heat shock (Ogata, Y., Mizushima, T., Kataoka, K., Miki, T., and Sekimizu, K . (1994) Mol . Gen . Genet . 244, 451-455) . We have now obtained biochemical evidence to support this hypothesis . DNA gyrase catalyzed an increase in the linking number of DNA and relaxation of negatively supercoiled DNA, under physiological concentrations of ATP . Analyses by gel filtration chromatography of each subunit revealed that DNA relaxation activity co-migrated with each subunit . The linking number of DNA increased as the temperature increased . Further, the reaction was inhibited by nalidixic acid or by oxolinic acid . Based on these results, we propose that DNA gyrase participates in a concerted reaction with DNA topoisomerases in the immediate relaxation of DNA in cells exposed to heat shock. J Biol Chem, 1996 Oct 4, 271(40), 24736 - 40 Identification of residues of spinach thioredoxin f that influence interactions with target enzymes; Geck MK et al.; The necessity for two types of thioredoxins (Trx f and m) within chloroplasts of higher plants that mediate the same redox chemistry with various target enzymes is not well understood . To approach this complex issue, we have applied site-directed mutagenesis to the identification of residues of Trx f that affect its binding to and selectivity for target enzymes . Based upon amino acid sequence alignments and the three-dimensional structure of Escherichia coli thioredoxin, putative key residues of Trx f were replaced with residues found at corresponding positions of Trx m to generate the mutants K58E, Q75D, N74D, and deletion mutants DeltaAsn-74 and DeltaAsn-77 . Kinetics of activation of oxidized recombinant sorghum leaf NADP-dependent malate dehydrogenase and oxidized spinach chloroplastic fructose-1,6-bisphosphatase by wild-type Trx f, wild-type Trx m, and Trx f mutants were compared . All of the mutants are less efficient than wild-type Trx f in the activation of fructose-1,6-bisphosphatase and are altered in both S0.5 and Vmax . In contrast to literature reports, the activation of NADP-dependent malate dehydrogenase does not display rate saturation kinetics with respect to the concentration of Trx f, thereby signifying very weak interactions between the two proteins . The mutants of Trx f likewise interact only weakly with NADP-dependent malate dehydrogenase, but the apparent second-order rate constants for activation are increased compared to that with wild-type Trx f . Thus, Lys-58, Asn-74, Gln-75, and Asn-77 of Trx f contribute to its interaction with target enzymes and influence target protein selectivity. J Biol Chem, 1996 Oct 4, 271(40), 24720 - 7 Localization of the effector-specifying regions of Gi2alpha and Gqalpha; Medina R et al.; Heterotrimeric G proteins transmit hormonal and sensory signals received by cell surface receptors to effector proteins that regulate cellular processes . Members of the highly conserved family of alpha subunits specifically modulate the activities of a diverse array of effector proteins . To investigate the determinants of alpha subunit-effector specificity, we localized the effector-specifying regions of alphai2, which inhibits adenylyl cyclase, and alphaq, which stimulates phosphoinositide phospholipase C using chimeric alpha subunits . The chimeras were generated using an in vivo recombination method in Escherichia coli . The effector-specifying regions of both alphai2 and alphaq were localized within the GTPase domain . An alphaq/alphai2/alphaq chimera containing only 78 alphai2 residues within the GTPase domain robustly inhibited adenylyl cyclase . This alphai2 segment includes regions corresponding to two of the three regions of alphas that activate adenylyl cyclase, but does not include any of the alpha subunit regions that switch conformation upon binding GTP . Replacement of the alphaq residues that comprise the helical domain with the homologous alphai2 residues resulted in a chimeric alpha subunit that activated phospholipase C . Combined with previous studies of the effector-specifying residues of alphas and alphat, our results suggest that the effector specificity of alpha subunits is generally determined by the GTPase and not the helical domain. J Biol Chem, 1996 Oct 4, 271(40), 24662 - 9 Mechanism of translesion DNA synthesis by DNA polymerase II . Comparison to DNA polymerases I and III core; Paz-Elizur T et al.; Bypass synthesis by DNA polymerase II was studied using a synthetic 40-nucleotide-long gapped duplex DNA containing a site-specific abasic site analog, as a model system for mutagenesis associated with DNA lesions . Bypass synthesis involved a rapid polymerization step terminating opposite the nucleotide preceding the lesion, followed by a slow bypass step . Bypass was found to be dependent on polymerase and dNTP concentrations, on the DNA sequence context, and on the size of the gap . A side-by-side comparison of DNA polymerases I, II, and III core revealed the following . 1) Each of the three DNA polymerases bypassed the abasic site analog unassisted by other proteins . 2) In the presence of physiological-like salt conditions, only DNA polymerase II bypassed the lesion . 3) Bypass by each of the three DNA polymerases increased dramatically in the absence of proofreading . These results support a model (Tomer, G., Cohen-Fix, O . , O'Donnell, M., Goodman, M . and Livneh, Z . (1996) Proc . Natl . Acad . Sci . U . S . A . 93, 1376-1380) by which the RecA, UmuD, and UmuC proteins are accessory factors rather than being absolutely required for the core mutagenic bypass reaction in induced mutagenesis in Escherichia coli. J Biol Chem, 1996 Oct 4, 271(40), 24649 - 54 Escherichia coli orf17 codes for a nucleoside triphosphate pyrophosphohydrolase member of the MutT family of proteins . Cloning, purification, and characterization of the enzyme; O'Handley SF et al.; The product of the Escherichia coli orf17 gene is a novel nucleoside triphosphate pyrophosphohydrolase with a preference for dATP over the other canonical (deoxy)nucleoside triphosphates, and it catalyzes the hydrolysis of dATP through a nucleophilic attack at the beta-phosphorus to produce dAMP and inorganic pyrophosphate . It has a pH optimum between 8.5 and 9.0, a divalent metal ion requirement with optimal activity at 5 mM Mg2+, a Km of 0.8 mM and a kcat of 5.2 s-1 at 37 degrees C for dATP . dAMP is a weak competitive inhibitor with a Ki of approximately 4 mM, while PPi is a much stronger inhibitor with an apparent Ki of approximately 20 microM . The enzyme contains the highly conserved signature sequence GXVEX2ETX6REVXEEX2I designating the MutT family of proteins . However, unlike the other nucleoside triphosphate pyrophosphohydrolases with this conserved sequence, the Orf17 protein does not complement the mutT- mutator phenotype, and thus must serve a different biological role in the cell. J Biol Chem, 1996 Oct 4, 271(40), 24604 - 9 Modeling of a mutation responsible for human 3-hydroxy-3-methylglutaryl-CoA lyase deficiency implicates histidine 233 as an active site residue; Roberts JR et al.; 3-Hydroxy-3-methylglutaryl-CoA (HMG-CoA) lyase is inactivated by diethyl pyrocarbonate (DEPC); activity can be fully restored by incubation with hydroxylamine . Protection against DEPC inactivation is afforded by a substrate analogue, suggesting an active site location for a DEPC target . Included in the inherited defects that map within the HMG-CoA lyase gene is a point mutation that results in an arginine substitution for histidine 233, one of only two invariant histidines . These observations prompted a functional test of the importance of His-233 . The mutant lyases H233R, H233A, and H233D were overexpressed in Escherichia coli, isolated, and kinetically characterized . In H233D, DEPC targets one less histidine than was measured using wild-type lyase, supporting the assignment of wild-type lyase His-233 as one of the DEPC targets . Substitution of His-233 results in diminution of activity by approximately 4 orders of magnitude . Km values of the mutant lyases for both substrate HMG-CoA and activator divalent cation (Mg2+ or Mn2+) are comparable to the values measured for wild-type enzyme, indicating that these enzymes retain substantial structural integrity . This conclusion is reinforced by the observation that the affinity label, 2-butynoyl-CoA, stoichiometrically modifies the mutant lyases, indicating that they contain a full complement of active sites . In view of these data suggesting that the structures of these mutant lyases closely approximate that of the wild-type enzyme, their observed 10(4)-fold diminution in catalytic efficiency supports assignment to His-233 of a role in the chemistry of HMG-CoA cleavage. J Biol Chem, 1996 Oct 4, 271(40), 24449 - 57 A steady-state and pre-steady-state kinetic analysis of the NTPase activity associated with the hepatitis C virus NS3 helicase domain; Preugschat F et al.; The helicase domain of hepatitis C virus NS3 (genotype 1b) was expressed in Escherichia coli and purified to homogeneity . The purified protein catalyzed the hydrolysis of nucleoside triphosphates (NTP) and the unwinding of duplex RNA in the presence of divalent metal ion . The enzyme was not selective for the NTP substrate . For example, UTP and acyclovir triphosphate were hydrolyzed efficiently by the enzyme . The rate of NTP hydrolysis was stimulated up to 27-fold by oligomeric nucleic acids (NA) . Furthermore, NA bound to the enzyme with concomitant quenching of the intrinsic protein fluorescence . The dissociation constants of the enzyme for selected NA in the absence of NTP were between 10 and 35 microM at pH 7.0 and 25 degrees C . The enzyme had maximal affinity for NA with 12 or more nucleotides . A detailed steady-state and pre-steady-state kinetic analysis of ATP hydrolysis was made with (dU)18 as the effector . The kcat values for ATP hydrolysis in the presence and absence of (dU)18 were 80 s-1 and 2.7 s-1, respectively . The association (dissociation) rate constants for the enzyme and (dU)18 in the presence and absence of ATP were 5.7 microM-1 s-1 (3.9 s-1) and 290 microM-1 s-1 (2.27 s-1), respectively . The association (dissociation) rate constants for the enzyme and ATP in the presence and absence of (dU)18 were 0.4 microM-1 s-1 (<0.5 s-1) and 0.9 microM-1 s-1 (<10(-1) s-1), respectively . These data were consistent with a random kinetic mechanism. J Biol Chem, 1996 Oct 4, 271(40), 24349 - 52 Cloning and functional analysis of the beta-carotene hydroxylase of Arabidopsis thaliana; Sun Z et al.; An Arabidopsis thaliana cDNA encoding the enzyme beta-carotene hydroxylase was identified by functional complementation in Escherichia coli . The product of this cDNA adds hydroxyl groups to both beta rings of the symmetrical beta-carotene (beta,beta-carotene) to form zeaxanthin (beta,beta-carotene-3,3'-diol) and converts the monocyclic beta-zeacarotene (7',8'-dihydro-beta,psi-carotene) to hydroxy-beta-zeacarotene (7',8'-dihydro-beta,psi-carotene-3-ol) . The epsilon rings of delta-carotene (epsilon,psi-carotene) and alpha-zeacarotene (7',8'-dihydro-epsilon,psi-carotene) are poor substrates for the enzyme . The predicted amino acid sequence of the A . thaliana enzyme resembles the four known bacterial beta-carotene hydroxylase enzymes (31-37% identity) but is much longer, with an N-terminal extension of more than 130 amino acids . Truncation of the cDNA to produce a polypeptide lacking the first 69 amino acids does not impair enzyme activity in E . coli . Truncation to yield a polypeptide of a length comparable with the bacterial enzymes (lacking 129 N-terminal amino acids) resulted in the accumulation of the monohydroxy intermediate beta-cryptoxanthin (beta,beta-carotene-3-ol), predominantly, when beta-carotene was provided as the substrate . It is suggested that amino acid residues 70-129 of the A . thaliana enzyme may play a role in formation of a functional homodimer. Gene, 1996 Oct 3, 174(2), 293 - 7 Sequence analysis of the 2,4-dichlorophenol hydroxylase gene tfdB and 3,5-dichlorocatechol 1,2-dioxygenase gene tfdC of 2,4-dichlorophenoxyacetic acid degrading plasmid pEST4011; Koiv V et al.; Chlorocatechol 1,2-dioxygenase (CC12O) and 1,2-dichlorophenol hydroxylase (DCPH) encoding genes tfdC and tfdB are located on a 4.2-kb DNA fragment cloned from the 2,4-dichlorophenoxyacetic acid (2,4D) degrading plasmid pEST4011 . The nucleotide sequences of tfdC and tfdB were determined . The DCPH is coded by a 1758-bp gene and CC12O is coded by a 762-bp gene . The deduced M(r) of these proteins are 64.09 kDa and 28.2 kDa, respectively . Expression analysis of tfdB and tfdC in Escherichia coli suggested that these genes form one operon, tfdCB. Biochem Biophys Res Commun, 1996 Oct 3, 227(1), 211 - 5 Electron paramagnetic resonance (EPR) studies on hydrogenase-1 (HYD1) purified from a mutant strain (AP6) of Escherichia coli enhanced in HYD1; DerVartanian ME et al.; Hydrogenase-1 (HYD1), overexpressed by twofold, has been purified to homogeneity and to a high specific activity from a mutant strain (AP6) of Escherichia coli which lacks hydrogenase-2 . Plasma emission spectroscopy indicated that 0.93 atom of nickel and 11.4 iron atoms were present in HYD1 . EPR studies on the as isolated HYD1 detected a complex 3Fe-4S signal and a Ni(III) species . Reduction with hydrogen gas caused disappearance of both the 3Fe-4S cluster and initial Ni(III) signals . At the same time the EPR signature (small g = 2.19 signal) of the activated hydrogenase appeared . The detection of a 4Fe-4S cluster signal was noted . Reduction of HYD1 with sodium dithionite caused all nickel signals to disappear . The 4Fe-4S complex intensity was slightly increased . The EPR responses in the three oxidation-reduction states are consistent with other known (NiFe)-hydrogenases. Parasite Immunol, 1996 Oct, 18(10), 507 - 13 Host-protective fragments and antibody binding epitopes of the Taenia ovis 45W recombinant antigen; Lightowlers MW et al.; The protective efficacy of a recombinant Taenia ovis vaccine antigen, 45W, was compared in sheep vaccine trials with antigen expressed by the full length 45W cDNA and by incomplete copies of the cDNA . Vaccine, trials were also carried out using antigen expressed by a cDNA (45S) having a sequence similar, but not identical, to 45W . Stability of the 45W antigen expressed in Escherichia coli was found to be increased after deletion of cDNA sequence encoding 19 COOH-terminal amino acids . This truncated form of the antigen was designated 45WB/X . Vaccination of sheep with antigen expressed by 45W, 45WB/X, as well as full length 45W and 45S cDNAs, induced high levels of protection . Vaccination with antigen expressed by an incomplete copy of the 45S cDNA clone did not induce protection . Comparison of deduced amino acid sequences for these clones suggests that the host-protective epitope(s) of the 45W antigen occur on either or both of the 23 and 9 amino acid peptides at the amino and carboxyl termini of 45W, respectively . Antibody binding epitopes of 45W were investigated in ELISA using overlapping 9 amino acid peptides . Protection was found to correlate with the induction of antibody to two 9 amino acid peptides. Braz J Med Biol Res, 1996 Oct, 29(10), 1317 - 20 Distinct antigenic subtypes of human beta interferon can be distinguished by neutralization; Golgher RR et al.; Different molecular configurations of human beta interferon were titrated with the standard reference antiserum of the National Institutes of Health (NIH) which had been prepared with natural beta fibroblast interferon in order to determine to what extent differences in these configurations would influence the neutralization of the antiviral action of interferon . Neutralization tests were carried out in Vero cells by diluting both interferon and antiserum . Encephalomyocarditis virus was employed as challenge virus . The neutralization titer was considered to have been reached when the effect of eight units of interferon was reduced to one . Two natural beta interferons prepared from fibroblasts and from amniotic membranes gave similar high titers . However, titers were reduced five-fold with recombinant interferons expressed in Escherichia coli, which do not contain carbohydrate, one with the natural sequence and a mutant with a single amino acid substitution (cysteine for serine) . The NIH antiserum did not neutralize the effect of a protein fraction from amniotic membranes antigenically different from the human alpha, beta or gamma interferons but having the biological activity of interferon . We conclude that the carbohydrate moieties of human beta interferons are essential for their recognition by the NIH antiserum and that antibodies specific for human recombinant beta interferon, which does not contain carbohydrate, are needed. Res Microbiol, 1996 Oct, 147(8), 609 - 13 The qmeA (ts) mutation of Escherichia coli is localized in the fabI gene, which encodes enoyl-ACP reductase; Kleerebezem M et al.; The phenotypes of temperature-sensitive qmeA and fabI mutants of Escherichia coli appear to be very similar . Furthermore, the qmeA mutation could be complemented by the fabI gene on a plasmid, and the fabI allele derived from the qmeA mutant strain harbours a nucleotide substitution identical to that from a previously characterized fabI mutant . These results show that the qmeA gene is, in fact, identical to the fabI gene, which encodes enoyl-ACP reductase, involved in fatty acid elongation. Res Microbiol, 1996 Oct, 147(8), 597 - 608 In vivo positive effects of exogenous pyrophosphate on Escherichia coli cell growth and stationary phase survival; Biville F et al.; We have studied the effect of exogenous pyrophosphate on growing cells of Escherichia coli . In the presence of 10 mM of pyrophosphate, the entry into the stationary phase was delayed and thus a significant increase in the growth yield was observed (25 to 35%) when the bacteria were grown in glucose minimal medium . Furthermore, the synthesis of 52 polypeptides was affected, as demonstrated by two-dimensional electrophoresis . Among the 22 proteins identified by comparison with the E . coli gene-protein index and/or by microsequencing procedures, 15 were involved either in catabolic or anabolic pathways of the intermediary metabolism or in stress responses . Subsequent physiological experiments enabled us to conclude that pyrophosphate exerted a direct or indirect effect on bacterial growth by (1) conferring upon cells a better capacity to use carbon sources and (2) inducing biosynthetic processes . Finally, we show that exogeneous pyrophosphate enhanced the stationary phase survival of E . coli cells. Appl Biochem Biotechnol, 1996 Oct-Nov, 61(1-2), 109 - 22 The stress-related production of the active Photinus pyralis and Luciola mingrelica firefly luciferases in Escherichia coli; Leont'eva O et al.; The kinetics of Photinus pyralis and Luciola mingrelica luciferase gene expression was studied on plasmids with the thermoinducible lambda PR promoter in Escherichia coli by SDS-gel electrophoresis of cell lysates to follow luciferase protein-synthesized, enzyme immunoassay (EIA) to follow native enzyme conformer, and the luciferase activity assay . E . coli cells were cultivated at temperature schemes 28-42-21 degrees C or 28-21 degrees C, or at alkali pH shift . In the cases of thermoinduction and pH shift, the luciferase expressions have similar features . The 3-h thermoinduction (42 degrees C) followed by the incubation at 21 degrees C, for 10 h resulted in the maximal amount of the luciferase protein of 4-5% of the total cell proteins . The yield did not change further . The amount of native luciferase conformer and the luciferase activity started to grow after incubation for 10 h at 21 degrees C and reached the maximum after 50-60 h when the synthesized luciferase protein adopted the native-like conformation . At the same time, only 50% of the latter appeared to be catalytically active . An increase in the enzymatic activity correlates with an increase in the intracellular pH and ATP content . Intracellular metabolic reactions were shown to play a role in the conformational changes of the enzyme in a postthermoinduction period, and a possible mechanism of this effect is proposed. Appl Biochem Biotechnol, 1996 Oct-Nov, 61(1-2), 97 - 107 HIV-I protease . Cloning, expression, and purification; Dergousova NI et al.; A new method for obtaining HIV-I protease was suggested . Fusion proteins composed of the N-terminal fragment of human gamma-interferon and HIV-I protease connected with (Asp)4Lys (protein I) or Asp-Pro (protein II) linkers were expressed in Escherichia coli cells . The fusion proteins were produced as insoluble inclusion bodies in the 20% yield of total cell protein . Protein I was cleaved by enterokinase . The solubility of protein I was increased by treating with Na-sulfite/Na-tetrathionate under denaturing conditions . Optimal conditions for efficient acidic hydrolysis of protein II at Asp-Pro bond were found . The hydrolysis products were separated by reversed-phase FPLC . The amount of tryptophan and cysteine residues in the enzyme obtained was estimated . The activity of HIV-I protease was determined using the chromogenic peptide . AlaArgVal NleNphGluAlaNleNH2 and a high-mol-wt substrate consisting of beta-galactosidase and a fragment of gag proteins, including p17-p24 processing site. Appl Biochem Biotechnol, 1996 Oct-Nov, 61(1-2), 85 - 96 Protein engineering of de novo protein with predesigned structure and activity; Dolgikh DA et al.; The de novo protein albebetin has been engineered (J . Mol . Biol . 1992, 225, 927-931) to form a predesigned tertiary fold that has not yet been observed in natural proteins . Analysis of albebetin expressed in a cell-free system and in Escherichia coli revealed its compactness, relative stability, and the secondary structure close to the predesigned one . The blast-transforming biological activity of human interferon was grafted to albebetin by attachment of an eight amino acid interferon fragment to the N-terminus of albebetin next to its first methionine residue . The chimeric protein was expressed in a wheat germ cell-free translation system and tested for its structural properties, receptor binding, and biological activity . According to the tests, albebetin incorporating the active interferon fragment has a compact and relatively stable structure, and binds the murine thymocyte receptor effectively . It activates the blast transformation reaction of thymocyte cells even more efficiently than human interferon at low concentrations. Appl Biochem Biotechnol, 1996 Oct-Nov, 61(1-2), 47 - 56 D-glyceraldehyde-3-phosphate dehydrogenase . Properties of the enzyme modified at arginine residues; Nagradova NK et al.; Examination of the properties of Escherichia coli and rabbit muscle D-glyceraldehyde-3-phosphate dehydrogenase (GPDHs) modified by 2,3-butanedione has shown that both tetrameric enzymes are stabilized, on selective modification of arginine residues (probably Arg 231), in an asymmetric state with only two active centers capable of performing the dehydrogenase reaction . The functionally incompetent active centers can be alkylated by iodoacetate or iodoacetamide in the case of E . coli enzyme, but are inaccessible for these reagents in the case of rabbit muscle D-GPDH . These results are consistent with the idea that the two homologous enzymes share common principles of the protein design, but differ somewhat in their active centers geometries . Modification of the arginine procedures marked changes in the shape of the charge transfer complex spectrum in the region of 300-370 nm, suggestive of the alterations in the microenvironment of the nicotinamide ring of NAD(+), although the coenzyme binding characteristics remain largely unaltered . On arginine modification, the enzyme becomes insensitive to the effect of AMP on the kinetic parameters of p-nitrophenyl acetate hydrolysis reaction. Appl Biochem Biotechnol, 1996 Oct-Nov, 61(1-2), 13 - 23 Wild-type and mutant forms of recombinant horseradish peroxidase C expressed in Escherichia coli . Substrate specificity and stability under irradiation; Mareeva EA et al.; Two horseradish peroxidase C (HRPC) mutants with substitutions in the active center, i.e., Phe41-->His and Phe143-->Glu, were compared to the wild-type recombinant enzyme expressed in Escherichia coli in terms of the enzymatic activity and stability under irradiation . Both mutations caused a significant decrease in activity, but it was still possible to follow the effect of mutations on the key steps of the reaction mechanism . Phe41 can be considered a nonpolar barrier separating histidine residues in the active center and providing a firm noncovalent binding with the highly hydrophobic porphyrin ring . The replacement of Phe41 with the ionizable His residue destabilizes the enzyme . The Phe143-->Glu replacement creates a negative charge at the entrance of the heme-binding pocket, and protects the latter from both donor substrates and free radicals . The radiolytic inactivation of the wild-type and mutant forms of recombinant HRP suggested different binding sites for iodide, 2,2'-bis(3-ethylbenzothiasoline-6-sulfonate (ABTS), guaiacol, and O-phenylene diamine . The study of kinetics and inactivation is in agreement with the direct binding of iodide to the heme porphyrin ring . The results also suggest that the ABTS binding site is less accessible than that for O-phenylene diamine. Genetika, 1996 Oct, 32(10), 1326 - 32 {Identification and expression of plasmid genes participating in the synthesis of microcin C51}; Fomenko DE et al.; A structural gene of heptapeptide, which is a component of the microcin C51 molecule, was identified by hybridization of plasmid DNA fragments and a mixture of synthesized oligonucleotides with the sequence corresponding to that of amino acids in the peptide . Sequence analysis of the structural gene of microcin peptide and its promoter region was performed . The data obtained indicate that the peptide of microcin C51 is synthesized on ribosomes . Four polypeptides of 67, 39, 16, and 14 kDa were identified using the system of minicells . These polypeptides are specified by a DNA fragment responsible for microcin synthesis and immunity in a producer cell . Apparently, three of these polypeptides with molecular masses of 67, 39, and 16 kDa are responsible for microcin production and immunity . The 67 kDa polypeptide is involved in the expression of immunity to microcin and, probably, in microcin production. Mol Immunol, 1996 Oct, 33(15), 1171 - 6 Ezrin, radixin and moesin are possible auto-immune antigens in rheumatoid arthritis; Wagatsuma M et al.; In order to deduce which cellular molecules react with the sera from patients with rheumatoid arthritis (RA), human and mouse cellular extracts were fractionated stepwise, by ethanol precipitation and their reactivity analysed by Western blotting . It was found that three cytoplasmic molecules with molecular weights of 80,000, 81,000 and 77,000 were immunoreactive and they were identified as ezrin (E), radixin (R), and moesin (M), respectively, by partial amino acid sequencing . Using cDNA clones of these human molecules, recombinant proteins were produced in Escherichia coli and used to enable the antigens to detect the antibodies in the sera of patients with RA . Of 71 sera tested, 24 sera (33.8%) reacted with at least one of three recombinant antigens, although there was no significant correlation between the presence of the antibodies and clinical manifestations, such as disease duration or stage . There was also no discernible relationship to other auto-antibodies such as antinuclear antibodies (ANA) and rheumatoid factor . The results suggest that ERM proteins are possible novel auto-immune target antigens for RA. Proc Natl Sci Counc Repub China B, 1996 Oct, 20(4), 110 - 6 Expression of polyphosphate kinase inhibits the glucose uptake in Escherichia coli; Liu JK et al.; This paper examines the effects of phosphate pool and expression of polyphosphate kinase on glucose uptake by expressing the polyphosphate kinase under the control of lac promoter . The E . coli transformant of pL1, containing an IPTG controllable element for polyphosphate kinase expression, showed that the total intracellular phosphate significantly increased . However, the rate of glucose uptake by the resting plasmid-bearing cells with IPTG induction significantly decreased . These findings suggest that the polyphosphate can not directly function as an energy source in E . coli or at least not as a good energy supplier. Mol Immunol, 1996 Oct, 33(14), 1119 - 25 The conserved lymphokine element-0 in the IL5 promoter binds to a high mobility group-1 protein; Marrugo J et al.; The conserved lymphokine elements-0 (CLE0) in the IL5 promoter is essential for the expression of IL-5 . Here, we report the cloning and expression of a cDNA encoding a novel CLE0-binding protein, CLEBP-1 from a mouse Th2 clone, D10.G4.1 . Interestingly, it was found that the CLEBP1 cDNA sequence was almost identical to the sequences of known high mobility group-1 (HMG1) cDNAs . When expressed as a recombinant fusion protein in Escherichia coli, CLEBP-1 was shown to bind to the IL5-CLE0 element in electrophoretic mobility-shift assays (EMSA) and southwestern blot analysis . The CLEBP-1 fusion protein cross-reacts with and-HMG-1/2 in Western blot analysis . It also binds to the CLE0 elements of IL4, GMCSF and GCSF genes . CLEBP-1 and closely related HMG-1 and HMG-2 proteins may play key roles in facilitating the expression of the lymphokine genes that contain CLE0 elements. Mol Immunol, 1996 Oct, 33(14), 1113 - 8 Expression, purification and immunochemical characterization of recombinant bovine beta-lactoglobulin, a major cow milk allergen; Chatel JM et al.; The immunological characteristics of a recombinant beta-lactoglobulin were studied using monoclonal antibodies, polyclonal antiserum and sera from allergic patients . Recombinant beta-lactoglobulin (rBLG) was expressed in Escherichia coli strain DH5alpha and purified as described previously {Cho et al . (1994) J . Biol . Chem . 269, 11 102-11 107} . The method has been modified by adding an immunoaffinity purification step . A quantity of 5-10mg of purified rBLG per liter of medium culture can be produced . rBLG shared the same molecular weight as the natural BLG (nBLG) and also possessed at least one intrachain disulfide bridge . In HPLC, rBLG appeared as a single peak, and the purity was estimated to be greater than 95% . All the monoclonal antibodies (mAbs) used in this study recognized different epitopes of the BLG and presented compatible binding . No differences could be detected between rBLG and nBLG when tested in a Western blot with rabbit polyclonal antiserum or with three mAbs that bound preferentially the reduced and S-carboxymethylated form of BLG . In a competitive enzyme immunoassay (EIA) using either a rabbit polyclonal antiserum or four mAbs that recognized conformational epitopes, we could not distinguish between rBLG or nBLG . In direct ELISA using nBLG or rBLG as the immobilized allergen, we measured a similar concentration of specific anti-BLG IgE in five sera from allergic patients . The results of this study indicate that we have obtained a rBLG with biochemical and immunological properties very similar to nBLG. Can J Physiol Pharmacol, 1996 Oct, 74(10), 1141 - 8 Muscle aspartyl protease (cathepsin D) activity: detection using a chromophoric substrate and relation to wasting in DBA/2 mice implanted with leukemic L1210 tumor cells; Bolger GT et al.; The relationship between skeletal muscle aspartyl protease activity (APA) and wasting was investigated in male DBA/2 mice inoculated with L1210 tumor cells . Using the peptidic substrate H-Pro-Thr-Glu-Phe-Phe(NO2)-Arg-Leu-OH, which is specific for aspartyl proteases, proteases, proteolytic activity was detected in a number of tissues including muscle by using a crude extraction procedure for isolation of lysosomal enzymes . Biochemical characterization and increased muscle levels following either fasting or injection of endotoxin (ETX) suggest that the enzyme is likely cathepsin D . The wasting syndrome accompanying the tumor was measured by comparing the weight of the skinned hind limb in treated and control animals . DBA/2 mice inoculated intraperitoneally with L1210 cells developed multiple solid tumors in the peritoneum and ascites; maximal tumor burden was reached by 16 days . There was a significant reduction in hind limb weight (16 +/- 2%; mean +/- SE) and significant increase (31 +/- 8%) in muscle APA associated with the development of ascites and solid tumors . Plasma APA activity was substantially increased (240 +/- 33%), while liver and spleen APA were increased (10-20%) but not significantly . Chronic pepstatin administration, 30 mg.kg-1.day-1, for 7 days concurrent with the initiation of observable ascites and solid tumor formation (7 days post-inoculation), completely inhibited hind limb weight loss and alleviated the tumor-dependent increase of APA in both plasma and muscle without altering tumor development . Delaying the administration of pepstatin by 3 days resulted in less of an inhibition (33 +/- 13%) of hind limb weight loss . Thus, cathepsin D or a similar aspartyl protease appears to be of key importance in the wasting syndrome associated with cachexia. Food Chem Toxicol, 1996 Oct, 34(10), 959 - 62 Lethality induced by stannous chloride on Escherichia coli AB1157: participation of reactive oxygen species; Dantas FJ et al.; Stannous chloride (SnCl2) has been widely used in nuclear medicine as a reducing agent of pharmaceutical products radiolabelled with technetium-99m . To verify whether the lethality induced by this salt could be mediated by reactive oxygen species (ROS), Escherichia coli cultures were treated with SnCl2 in the presence of catalase, ROS scavengers or metal-ion chelators . The inactivation effect, as measured by survival determination, was abolished by thiourea, sodium benzoate, dipyridyl or catalase . The results suggest the participation of ROS, generated by a Fenton-like reaction, in the lethal effect induced by SnCl2. Biokhimiia, 1996 Oct, 61(10), 1758 - 71 {Expression of functionally active hyman cytochrome P-450c21 (CYPXXIA2) in Escherichia coli and single-stage purification of it using metal-affinity chromatography}; Guzov VM et al.; Active human cytochrome P-450c21 was expressed in Escherichia coli and purified to homogeneity . To increase expression, cDNA encoding for the N-terminal fragment of cytochrome P-450c21 was modified . Four histidine codons were added to cDNA encoding for the C-terminus of the protein; thus, recombinant protein could have been rapidly and effectively purified by metal-affinity chromatography . Modified human cytochrome P-450c21 was expressed (40-50 nmoles/l of culture according to spectrophotometry) which was able to bind to bacterial membrane . Modifications of N- and C-terminal regions of cytochrome P-450c21 did not change Km and Vmax for hydroxylation of progesterone and 17 alpha-hydroxyprogesterone in reconstituted system . Recombinant cytochrome P-450c21 was purified to apparent homogeneity from Escherichia coli membrane extract by metal-affinity chromatography . Purified cytochrome P-450c21 migrates as a single 54 kD band on polyacrylamide gel and exhibits type I spectral changes during interaction with progesterone and 17 alpha-hydroxyprogesterone . Activity of purified cytochrome P-450c21 was reconstituted with mouse liver microsomal NADPH-cytochrome P-450-reductase and NADPH-regenerating system . Purified enzyme had Km 12.2 and 3.21 microM and Vmax 192.9 and 198 nmoles/min/nmole of P-450c21 for 17 alpha-hydroxyprogesterone and progesterone, respectively . According to titration spectra, dissociation constants for progesterone and 17 alpha-hydroxy-progesterone were 14.7 and 31.1 microM, respectively. An Esp Pediatr, 1996 Oct, 45(4), 398 - 402 {Risk and outcome factors in necrotizing enterocolitis}; Carbonell Estrany X et al.; OBJECTIVE: The objective of this study was to identify risk and outcome factors in necrotizing enterocolitis (NEC) . PATIENTS AND METHODS: We have studied 72 cases of NEC collected from 1987 until 1994 in the three hospitals of the integrated Unit . A case-control study matched for gestational age and center was performed for 26 risk factors . Conditional logistic regression was used in significant bivariate variables . The 18 outcome factors had the same statical treatment, but without the paired design . RESULTS: Serous infections previous to NEC, apnea and feeding increments greater than 20 cc/kg/day have been identified as risk factors for preterm babies (p < 0.05) . Severe acidosis and pneumoperitoneum have been found significant outcome variables, but with very low discriminatory capacity . CONCLUSIONS: It has been found difficult to identify risk factors for NEC besides the gestational age . Outcome factors have very low sensitivity . Preventive treatment should be directed to decrease the effect of the inflammatory mediators in the gastrointestinal tract. Berl Munch Tierarztl Wochenschr, 1996 Oct, 109(10), 385 - 7 {Practical experiences in the therapy of postweaning edema disease in piglets}; Papp I et al.; In a field trial 23 weeks old weaned piglets were orally inoculated with E . coli O139 K12 H1 . The piglets received the same food and were kept under the same management regime . They were selected randomly into four groups and treated after the first clinical signs of CNS involvement of edema disease as follows during three days: Group 1: received a single daily i.m . application of 4 mg/kg body weight Melperone . Group 2: received a single daily i.m . application of 2 mg/kg body weight Amperozide . Group 3: was treated orally (intranasally) with 2 mg/kg body weight Amphetamin in a single daily application . Group 4: untreated control . The following parameters were evaluated before the begin of the therapy (day 0-4) . A: Occurrence of diarrhea in a group B: Food consumption per piglet per day C: Death of piglets per group After the begin of the therapy (day 5-32) D: Death of piglets per group E: Average daily feed intake per piglet The results showed that the Melperone and Amphetamin treatment was superior regarding all examined parameters when compared to Amperozide treatment and to the control group. Comput Appl Biosci, 1996 Oct, 12(5), 415 - 22 Syntactic recognition of regulatory regions in Escherichia coli; Rosenblueth DA et al.; MOTIVATION: One of the most common methodologies to identify cis-regulatory sites in regulatory regions in the DNA is that of weight matrices, as testified by several articles in this issue . An alternative to strengthen the computational predictions in regulatory regions is to develop methods that incorporate more biological properties present in such DNA regions . The grammatical implementation presented in this paper provides a concrete example in this direction . RESULTS: On the basis of the analysis of an exhaustive collection of regulatory regions in Escherichia coli, a grammatical model for the regulatory regions of sigma 70 promoters has been developed . The terminal symbols of the grammar represent individual sites for the binding of activator and repressor proteins, and include the precise position of sites in relation to transcription initiation . Combining these symbols, the grammar generates a large number of different sentences, each of which can be searched for matching against a collection of regulatory regions by means of weight matrices specific for each set of sites for individual proteins . On the basis of this grammatical model, a Prolog syntactic recognizer is presented here . Specific subgrammars for ArgR, LexA and TyrR were implemented . When parsing a collection of 128 sigma 70 promoter regions, the syntactic recognizer produces a much lower number of false-positive sites than the standard search using weight matrices. J Photochem Photobiol B, 1996 Oct, 36(1), 47 - 53 A mutant of Escherichia coli hyper-resistant to a number of DNA damaging agents: location of the mutational site; Ahmad SI; A mutant of Escherichia coli, isolated as hyper-resistant to UVC, is found to be hyper-resistant to UVA, H2O2, low concentrations of nalidixic acid, novobiocin, UVA plus H2O2 and UVA plus 8-methoxypsoralen . A mutational site (uvh) conferring the hyper-resistance phenotype to UVC, and presumably to other DNA damaging agents, has been mapped at the 89.9 min region on the chromosome . Complementation analysis with an F-prime uvh+/uvh- diploid strain showed that the uvh+ allele is dominant over uvh- in trans . Studies with a variety of plasmids, carrying various LexA regions, introduced into the UV hyper-resistant strain show that mutation at the uvh locus may be responsible for derepression of the SOS inducible repair system . Based on the results, it is suggested that uvh is a part of the SOS inducible system . A plausible explanation for the hyper-resistance phenotypes for various DNA damaging agents and a model for the genetic control of a second set of putative SOS regulons are presented. Biosci Biotechnol Biochem, 1996 Oct, 60(10), 1681 - 5 A novel gene that interferes with the phosphotransfer signal transduction mediated by the EnvZ osmosensor in Escherichia coli; Hirokawa K et al.; In Escherichia coli, expression of the major outer membrane proteins, OmpC and OmpF, is regulated in response to the medium osmolarity and other environmental stimuli . A two-component signal transduction system, mediated by EnvZ and OmpR, is crucially responsible for this osmotic regulation of the ompC and ompF genes . In this study, an E . coli gene was cloned, which interferes with expression of both the ompC and ompF genes at the level of transcription, provided that the cloned gene was introduced in E . coli cells by a multicopy plasmid . The gene product was identified as F107, which was previously characterized as a hypothetical protein in E . coli genome databases . F107 containing 107 amino acids appears to be highly hydrophobic, and has a sequence similarity to the eukaryotic type of cytochrome-c oxidase subunit III . The mechanism by which F107 inhibits transcription of ompC and ompF was examined extensively, mainly by using a set of envZ and ompR mutants . These results suggested that F107 interferes specifically with a function of the EnvZ osmosensory kinase . Possible mechanisms by which F107 affects the EnvZ function are discussed. Int J Androl, 1996 Oct, 19(5), 271 - 7 Influence of Escherichia coli on motility parameters of human spermatozoa in vitro; Diemer T et al.; The influence of E . coli on human sperm motility was studied in vitro . Semen samples were prepared by a swim-up technique and adjusted to 22 x 10(6) spermatozoa/ml . Samples were then inoculated with different concentrations of a uropathogenic strain of E . coli, serotype 06, with initial sperm/bacteria ratios varying between 10:1 and 10000:1 . Motion parameters were analysed by computer-aided motility analysis directly, and 2, 4 and 6 h after inoculation . In a second series of experiments, bacterial replication was inhibited by addition of chloramphenicol . In a third series, the effect of E . coli culture filtrates on sperm motility was investigated . The direct inhibitory effect of E . coli on progressive motility of spermatozoa was found to depend upon the bacterial concentration . A distinct inhibitory effect was observed only at a sperm/bacteria ratio of approximately 1, achieved by growth of E . coli during the experiments . For modality of motion, no distinct changes were observed . When growth of bacteria was prevented by chloramphenicol, no inhibitory effect on sperm motility was detected . Sperm motility was not inhibited by E . coli culture filtrates . Analysis by electron microscopy revealed multiple adhesions of E . coli to spermatozoa, causing variable ultrastructural damage as probable morphological correlates of immobilization. Eur J Haematol, 1996 Oct, 57(4), 278 - 85 Gene transduction into murine primitive hematopoietic cells with 2-gene retroviral vectors using a Transwell coculture system; Asami N et al.; The present study aims at expressing a reporter gene in hematopoietic cells in vivo by introducing it into primitive hematopoietic cells with a 2-gene retroviral vector . Various constructs of retroviral vectors containing the human IL-2 receptor alpha chain gene (TAC) as the reporter and the neomycin phosphotransferase gene (neo) as a selectable marker were engineered, and the effectiveness of these vectors for expression of the reporter gene was evaluated after transfection into the packaging cell line GP + E86 . It was found that the highest levels of reporter gene expression were attained with constructs ordered 5' long terminal repeat (LTR)-TAC-internal promoter-neo-3' LTR . In experiments investigating the expression of a reporter gene in hematopoietic cells, we used the Escherichia coli beta-galactosidase gene (lacZ) instead of TAC, because a very sensitive detection method was available for lacZ . For transduction of hematopoietic progenitors, packaging cell lines producing recombinant viruses were cultured in a Transw