RNA, 2000 Mar, 6(3), 434 - 48 In vivo selection of lethal mutations reveals two functional domains in arginyl-tRNA synthetase; Geslain R et al.; Using random mutagenesis and a genetic screening in yeast, we isolated 26 mutations that inactivate Saccharomyces cerevisiae arginyl-tRNA synthetase (ArgRS) . The mutations were identified and the kinetic parameters of the corresponding proteins were tested after purification of the expression products in Escherichia coli . The effects were interpreted in the light of the crystal structure of ArgRS . Eighteen functional residues were found around the arginine-binding pocket and eight others in the carboxy-terminal domain of the enzyme . Mutations of these residues all act by strongly impairing the rates of tRNA charging and arginine activation . Thus, ArgRS and tRNA(Arg) can be considered as a kind of ribonucleoprotein, where the tRNA, before being charged, is acting as a cofactor that activates the enzyme . Furthermore, by using different tRNA(Arg) isoacceptors and heterologous tRNA(Asp), we highlighted the crucial role of several residues of the carboxy-terminal domain in tRNA recognition and discrimination. Bioorg Med Chem Lett, 2000 Mar 6, 10(5), 407 - 9 Irreversible inhibition of type I dehydroquinase by substrates for type II dehydroquinase; Bello CG et al.; Mechanistic differences between type I and type II dehydroquinases have been exploited in the design of type specific inhibitors . (2R)-2-Bromo-3-dehydroquinic acid (3), (2R)-2-fluoro-3-dehydroquinic acid (5) and 2-bromo-3-dehydroshikimic acid (4), all excellent substrates for type II dehydroquinase, are shown to be irreversible inhibitors of type I dehydroquinase. Plant J, 2000 Jan, 21(2), 189 - 98 Molecular cloning, functional expression and characterisation of RCC reductase involved in chlorophyll catabolism; Wuthrich KL et al.; Red chlorophyll catabolite (RCC) reductase (RCCR) and pheophorbide (Pheide) a oxygenase (PaO) catalyse the key reaction of chlorophyll catabolism, porphyrin macrocycle cleavage of Pheide a to a primary fluorescent catabolite (pFCC) . RCCR was purified from barley and a partial gene sequence was cloned (pHvRCCR) . The gene was expressed at all stages of leaf development and in roots . By comparison with different databases, genomic sequences and expressed sequence tags similar to RCCR were found in phylogenetically diverse species, and activity of RCCR was demonstrated in two of them, Arabidopsis thaliana and Marchantia polymorpha . The gene of A . thaliana (AtRCCR) was employed for molecular cloning, heterologous expression and the production of polyclonal antibodies . With recombinant RCCR, the major product of RCC reduction was pFCC-1, but small quantities of its C1 epimer, pFCC-2, also accumulated . The reaction required reduced ferredoxin and was sensitive to oxygen . AtRCCR encoded a 35 kDa protein which was used for chloroplast import experiments . Upon transport, it was processed to a mature form of 31 kDa . The significance of cloning of RCCR is discussed in respect to the evolution of chlorophyll catabolism and to the cloning of PaO. Mol Biochem Parasitol, 2000 Feb 25, 106(1), 121 - 9 Molecular cloning, expression analysis and iron metal cofactor characterisation of a superoxide dismutase from Toxoplasma gondii; Odberg-Ferragut C et al.; A genomic region of 12 kb encompassing the gene encoding the superoxide dismutase (SOD) of Toxoplasma gondii has been cloned . The gene contains four exons of 121, 42, 381 and 59 bp which are separated by three introns of 321, 202, and 577 bp, respectively . The open reading frame can be translated into a protein of 201 amino acids with a molecular mass of 22.6 kDa . Alignment indicated that it is a FeSOD, a type only found in bacteria, protozoa and chloroplast of higher plants . Recombinant SOD was expressed in a Escherichia coli double mutant lacking both MnFeSOD and FeSODs . The presence of iron as metal cofactor was confirmed by measurements of iron by absorption mass spectrometry and electron paramagnetic resonance studies . Semi-quantitative reverse transcribed polymerase chain reaction experiments showed a similar amount of SOD transcripts in two developmental stages of T . gondii . Antibodies raised against the purified recombinant protein detected SOD protein in both bradyzoite and tachyzoite forms suggesting this SOD might be essential for the intracellular growth of both developmental stages . Southern blot analysis indicated that SOD occured as a single copy gene in T . gondii genome. Mol Biochem Parasitol, 2000 Feb 25, 106(1), 83 - 91 Comparative study of Leishmania mexicana and Trypanosoma brucei NAD-dependent glycerol-3-phosphate dehydrogenase; Marche S et al.; The NAD-dependent glycerol-3-phosphate dehydrogenases (G3PDH, EC 1.1.1.8) of Trypanosoma brucei and Leishmania mexicana are thought to have different roles in carbohydrate metabolism . Here the physicochemical and kinetic properties of natural G3PDH from T . brucei with the recombinant homologue of L . mexicana which share 63% positional identity are compared . Despite their supposed different functions in energy metabolism of the parasites the two G3PDHs have remarkably similar properties, including pH optima and K(m) value for dihydroxyacetone phosphate (DHAP) and NADH in the formation of glycerol 3-phosphate (G3P) and for NAD+ and G3P in the reverse reaction . Both enzymes are subject inhibition by dihydroxyacetone phosphate at concentrations above 0.2 mM and are inhibited by the trypanocidal drugs suramin and melarsen oxide at sub-micromolar concentrations. Rev Inst Med Trop Sao Paulo, 2000 Jan-Feb, 42(1), 9 - 15 Asymptomatic infections by diarrheagenic Escherichia coli in children from Misiones, Argentina, during the first twenty months of their lives; Quiroga M et al.; Diarrheagenics Escherichia coli are the major agents involved in diarrheal disease in developing countries . The aim of this study was to evaluate the time of appearance of the first asymptomatic infection by the different categories of diarrheagenic E . coli in 44 children since their birth and during the first 20 months of their lives . In all of the children studied, we detected at least one category of diarrheagenic E . coli through the 20 months of the study . 510 diarrheagenic E . coli (33.5%) were obtained from the 1,524 samples collected from the 44 children during the time of the study (31.4% EAggEC, 28.8% EPEC, 27.1% DAEC, and 12.7% ETEC) . Neither EHEC nor EIEC were identified . The median age for diarrheagenic E . coli colonization was 7.5 months . The mean weaning period was 12.8 months and the mean age for introduction of mixed feeding (breast fed supplemented) was 3.8 months . A significantly lower incidence of diarrheal disease and asymptomatic infections was recorded among the exclusively breast-fed rather than in the supplemented and non breast-fed infants . For ETEC, EPEC and EAggEC the introduction of weaning foods and complete termination of breast-feeding were associated with an increase of asymptomatic infections. J Membr Biol, 2000 Mar 15, 174(2), 135 - 40 The melibiose carrier of Escherichia coli: cysteine substitutions for individual residues in helix XI; Ding PZ et al.; The melibiose carrier from Escherichia coli is a sugar-cation cotransport system . Previously evidence was obtained that this integral membrane protein consists of 12 transmembrane helices . Starting with the cysteine-less melibiose carrier, cysteine has been substituted individually for amino acids 374-396, which includes all of the residues in the proposed helix XI . The carriers with cysteine substitutions were studied for their transport activity and the effect of the water soluble sulfhydryl reagent p-chloromercuribenzenesulfonic acid (PCMBS) . Studies were carried out on both intact cells and inside out vesicles . Cysteine substitution caused loss of transport activity in seven of the mutants (K377C, G379C, A383C, F385C, L391C, G395C and Y396C) . PCMBS produced more than 50% inhibition in six of the mutants (S380C, A381C, A384C, F387C, A388C and L391C) . Preincubation of the cells with melibiose protected five of these residues from the inhibitory action of PCMBS . It was concluded that the residues whose cysteine derivatives were inhibited by PCMBS probably faced the aqueous channel. Br J Pharmacol, 2000 Apr, 129(7), 1309 - 14 The role of lipopolysaccharide injected systemically in the reactivation of collagen-induced arthritis in mice; Yoshino S et al.; 1 . We investigated the role of bacterial lipopolysaccharide (LPS) in the reactivation of autoimmune disease by using collagen-induced arthritis (CIA) in mice in which autoimmunity to the joint cartilage component type II collagen (CII) was involved . 2 . CIA was induced by immunization with CII emulsified with complete Freund's adjuvant at the base of the tail (day 0) followed by a booster injection on day 21 . Varying doses of LPS from E . coli were i.p . injected on day 50 . 3 . Arthritis began to develop on day 25 after immunization with CII and reached a peak on day 35 . Thereafter, arthritis subsided gradually but moderate joint inflammation was still observed on day 50 . An i.p . injection of LPS on day 50 markedly reactivated arthritis on a dose-related fashion . Histologically, on day 55, there were marked oedema of synovium which had proliferated by the day of LPS injection, new formation of fibrin, and intense infiltration of neutrophils accompanied with a large number of mononuclear cells . The reactivation of CIA by LPS was associated with increases in anti-CII IgG and IgG2a antibodies as well as various cytokines including IL-12, IFN-gamma, IL-1beta, and TNF-alpha . LPS from S . enteritidis, S . typhimurium, and K . neumoniae and its component, lipid A from E . coli also reactivated the disease . Polymyxin B sulphate suppressed LPS- or lipid A-induced reactivation of CIA . 4 . These results suggest that LPS may play an important role in the reactivation of autoimmune joint inflammatory diseases such as rheumatoid arthritis in humans. Appl Environ Microbiol, 2000 Apr, 66(4), 1734 - 6 Genetic and biochemical characterization of a highly thermostable alpha-L-arabinofuranosidase from Thermobacillus xylanilyticus; Debeche T et al.; The gene encoding an alpha-L-arabinofuranosidase from Thermobacillus xylanilyticus D3, AbfD3, was isolated . Characterization of the purified recombinant alpha-L-arabinofuranosidase produced in Escherichia coli revealed that it is highly stable with respect to both temperature (up to 90 degrees C) and pH (stable in the pH range 4 to 12) . On the basis of amino acid sequence similarities, this 56, 071-Da enzyme could be assigned to family 51 of the glycosyl hydrolase classification system . However, substrate specificity analysis revealed that AbfD3, unlike the majority of F51 members, displays high activity in the presence of polysaccharides. Appl Environ Microbiol, 2000 Apr, 66(4), 1680 - 4 Engineering desiccation tolerance in Escherichia coli; Billi D et al.; Recombinant sucrose-6-phosphate synthase (SpsA) was synthesized in Escherichia coli BL21DE3 by using the spsA gene of the cyanobacterium Synechocystis sp . strain PCC 6803 . Transformants exhibited a 10,000-fold increase in survival compared to wild-type cells following either freeze-drying, air drying, or desiccation over phosphorus pentoxide . The phase transition temperatures and vibration frequencies (P==O stretch) in phospholipids suggested that sucrose maintained membrane fluidity during cell dehydration. Appl Environ Microbiol, 2000 Apr, 66(4), 1393 - 9 Glutathione-dependent conversion of N-ethylmaleimide to the maleamic acid by Escherichia coli: an intracellular detoxification process; McLaggan D et al.; The electrophile N-ethylmaleimide (NEM) elicits rapid K(+) efflux from Escherichia coli cells consequent upon reaction with cytoplasmic glutathione to form an adduct, N-ethylsuccinimido-S-glutathione (ESG) that is a strong activator of the KefB and KefC glutathione-gated K(+) efflux systems . The fate of the ESG has not previously been investigated . In this report we demonstrate that NEM and N-phenylmaleimide (NPM) are rapidly detoxified by E . coli . The detoxification occurs through the formation of the glutathione adduct of NEM or NPM, followed by the hydrolysis of the imide bond after which N-substituted maleamic acids are released . N-ethylmaleamic acid is not toxic to E . coli cells even at high concentrations . The glutathione adducts are not released from cells, and this allows glutathione to be recycled in the cytoplasm . The detoxification is independent of new protein synthesis and NAD(+)-dependent dehydrogenase activity and entirely dependent upon glutathione . The time course of the detoxification of low concentrations of NEM parallels the transient activation of the KefB and KefC glutathione-gated K(+) efflux systems. Appl Environ Microbiol, 2000 Apr, 66(4), 1311 - 20 Properties of engineered poly-3-hydroxyalkanoates produced in recombinant Escherichia coli strains; Ren Q et al.; To prepare medium-chain-length poly-3-hydroxyalkanoates (PHAs) with altered physical properties, we generated recombinant Escherichia coli strains that synthesized PHAs with altered monomer compositions . Experiments with different substrates (fatty acids with different chain lengths) or different E . coli hosts failed to produce PHAs with altered physical properties . Therefore, we engineered a new potential PHA synthetic pathway, in which ketoacyl-coenzyme A (CoA) intermediates derived from the beta-oxidation cycle are accumulated and led to the PHA polymerase precursor R-3-hydroxyalkanoates in E . coli hosts . By introducing the poly-3-hydroxybutyrate acetoacetyl-CoA reductase (PhbB) from Ralstonia eutropha and blocking the ketoacyl-CoA degradation step of the beta-oxidation, the ketoacyl-CoA intermediate was accumulated and reduced to the PHA precursor . Introduction of the phbB gene not only caused significant changes in the monomer composition but also caused changes of the physical properties of the PHA, such as increase of polymer size and loss of the melting point . The present study demonstrates that pathway engineering can be a useful approach for producing PHAs with engineered physical properties. Nat Struct Biol, 2000 Apr, 7(4), 298 - 303 Structural insights into substrate binding by the molecular chaperone DnaK; Pellecchia M et al.; How substrate affinity is modulated by nucleotide binding remains a fundamental, unanswered question in the study of 70 kDa heat shock protein (Hsp70) molecular chaperones . We find here that the Escherichia coli Hsp70, DnaK, lacking the entire alpha-helical domain, DnaK(1-507), retains the ability to support lambda phage replication in vivo and to pass information from the nucleotide binding domain to the substrate binding domain, and vice versa, in vitro . We determined the NMR solution structure of the corresponding substrate binding domain, DnaK(393-507), without substrate, and assessed the impact of substrate binding . Without bound substrate, loop L3,4 and strand beta3 are in significantly different conformations than observed in previous structures of the bound DnaK substrate binding domain, leading to occlusion of the substrate binding site . Upon substrate binding, the beta-domain shifts towards the structure seen in earlier X-ray and NMR structures . Taken together, our results suggest that conformational changes in the beta-domain itself contribute to the mechanism by which nucleotide binding modulates substrate binding affinity. Extremophiles, 2000 Feb, 4(1), 43 - 51 MJ1647, an open reading frame in the genome of the hyperthermophile Methanococcus jannaschii, encodes a very thermostable archaeal histone with a C-terminal extension; Li WT et al.; All archaeal histones studied to date have similar lengths, 66 to 69 amino acid residues that form three alpha-helices separated by two beta-strand loop regions which together constitute a histone fold . In contrast, the eukaryal nucleosome core histones are larger, 102 to 135 residues in length, with N-terminal and C-terminal extensions flanking the histone fold that participate in gene regulation and higher-order chromatin assembly . In the Methanococcus jannaschii genome, MJ1647 was annotated as an open reading frame predicted to encode an archaeal histone with an approximately 27-amino-acid C-terminal extension, and we here document the DNA binding and assembly properties and thermodynamic stability parameters of the recombinant product of MJ1647 synthesized in Escherichia coli with (rMJ1647) and without (rMJ1647delta) the C-terminal extension . The presence of the C-terminal extension did not prevent homodimer formation or inhibit DNA binding, but the complexes formed by rMJ1647, presumably archaeal nucleosomes containing a (rMJ1647)4 tetramer, were apparently less stable than those formed by (rMJ1647delta)4 . The presence of the C-terminal extension increased the thermostability of rMJ1647 when compared with rMJ1647delta in 0.2 M KCl at pH 4 but not in the absence of KCl at pH 1 . Based on thermal unfolding transitions, rMJ1647 and rHAfB generated by expression of AF0337 cloned from the genome of the related hyperthermophile Archaeoglobus fulgidus in E . coli were found to have higher thermodynamic stabilities than all previously studied archaeal histones. J Membr Biol, 2000 Mar 1, 174(1), 31 - 40 Roles of charged residues in the conserved motif, G-X-X-X-D/E-R/K-X-G-{X}-R/K-R/K, of the lactose permease of Escherichia coli; Pazdernik NJ et al.; The lactose permease is a polytopic membrane protein that has a duplicated conserved motif, GXXX(D/E)(R/K)XG{X}(R/K)(R/K), located in cytoplasmic loops 2/3 and 8/9 . In the current study, the roles of the basic residues and the acidic residue were investigated in greater detail . Neutral substitutions of two positive charges in loop 2/3 were tolerated, while a triple mutant resulted in a complete loss of expression . Neutral substitutions of a basic residue in loop 8/9 (i.e., K289I) also diminished protein stability . By comparison, neutral substitutions affecting the negative charge in loop 2/3 had normal levels of expression, but were defective in transport . A double mutant (D68T/N284D), in which the aspartate of loop 2/3 was moved to loop 8/9, did not have appreciable activity, indicating that the negative charge in the conserved motif could not be placed in loop 8/9 to recover lactose transport activity . An analysis of site-directed mutants in loop 7/8 and loop 8/9 indicated that an alteration in the charge distribution across transmembrane segment 8 was not sufficient to alleviate a defect caused by the loss of a negative charge in loop 2/3 . To further explore this phenomenon, the double mutant, D68T/N284D, was used as a parental strain to isolate suppressor mutations which restored function . One mutant was obtained in which an acidic residue in loop 11/12 was changed to a basic residue (i.e., Glu374 --> Lys) . Overall, the results of this study suggest that the basic residues in the conserved motif play a role in protein insertion and/or stability, and that the negative charge plays a role in conformational changes. Kansenshogaku Zasshi, 2000 Feb, 74(2), 134 - 42 {The prevalence of virulence-related genes, eaeA, aggR and astA, of localized and aggregative-adherent Escherichia coli (EPEC and EAggEC) in healthy children and age-matched patients with diarrhea}; Moriya K et al.; The prevalence of virulence-related genes of localized- and aggregated-adherent Escherichia coli (EPEC and EAggEC), such as eaeA, aggR and astA was compared between E . coli isolated from 0 to 5 year old children with and without diarrhea in Saga Prefecture . In the case of eaeA, 233 cases in Aichi Prefecture were included . The subjects were 74 diarrheal patients from which no diarrheagenic bacteria were detected besides E . coli . The control subjects were 304 nursery school children without diarrhea, and E . coli was isolated from 278 children in which 105 strains were of 0-serotype . EaeA-positive E . coli was isolated from nine (12.2%) Saga cases, 19 (8.2%) Aichi cases and 6 (5.7%) control subjects; aggR-positive E . coli was isolated from 10 (13.5%) cases and 6 (5.7%) control subjects and astA-positive E . coli from 10 (13.5%) cases and 14 (13.3%) control subjects . No significant difference (p > 0.05) was observed in the prevalence of eaeA, aggR and astA between healthy and diarrheal children, even in age-matched and 0-serotypable E . coli limited comparisons . The pathogenicity of EPEC and EAggEC should be investigated, considering other known or unidentified factors. Anal Chem, 2000 Mar 15, 72(6), 1134 - 43 Site-specific mass tagging with stable isotopes in proteins for accurate and efficient protein identification; Chen X et al.; Proteolytic peptide mass mapping as measured by mass spectrometry provides a major approach for the identification of proteins . A protein is usually identified by the best match between the measured and calculated m/z values of the proteolytic peptides . A unique identification is, however, heavily dependent upon the mass accuracy and sequence coverage of the fragment ions generated by peptide ionization . Without ultrahigh instrumental accuracy, it is possible to increase the specificity of the assignments of particular proteolytic peptides by the incorporation of selected amino acid residue(s) enriched with stable isotope(s) into the protein sequence . Here we report this novel method of generating residue-specific mass-tagged proteolytic peptides for accurate and efficient protein identification . Selected amino acids are labeled with 13C/15N/2H and incorporated into proteins in a sequence-specific manner during cell culturing . Each of these labeled amino acids carries a defined mass change encoded in its monoisotopic distribution pattern . Through their characteristic patterns, the peptides with mass tags can then be readily distinguished from other peptides in mass spectra . This method of identifying unique proteins can also be extended to protein complexes and will significantly increase data search specificity, efficiency, and accuracy for protein identifications. J Biochem (Tokyo), 2000 Apr, 127(4), 665 - 71 Mitochondrial targeting signal-induced conformational change and repression of the peroxisomal targeting signal of the precursor for rat liver serine:pyruvate/alanine:glyoxylate aminotransferase; Oda T et al.; In the rat liver, two mRNAs for serine:pyruvate (or alanine:glyoxylate) aminotransferase are generated from a single gene by alternative transcription initiation . The longer mRNA encodes a precursor of a mitochondrial enzyme that has a mitochondrial targeting signal at the N-terminus and is translocated into mitochondria . The shorter mRNA encodes a peroxisomal enzyme of mature size that is imported into peroxisomes . We have been interested in the mechanism of selective targeting to mitochondria of the precursor protein that also contains a peroxisomal targeting signal in the molecule . In this study, we examined the effect of the mitochondrial targeting signal on the conformation of the protein and on the function of the peroxisomal targeting signal in the precursor molecule . The results suggest that the mitochondrial targeting signal causes the conformation of the protein to become unfolded and that this conformational change in turn causes repression of the putative peroxisomal targeting signal contained in the precursor protein. J Biochem (Tokyo), 2000 Apr, 127(4), 627 - 33 A new approach to gene mutation analysis using "GFP-Display"; Aoki T et al.; The unique behavior of green fluorescent protein (GFP) on SDS-PAGE was applied to the detection of a single amino acid substitution in GFP-tagged polypeptides . This simple detection method using SDS/urea gels was designated GFP-display . The N-terminal 18 or 37 amino acids of K-Ras was used as a model GFP-tagged polypeptide . K-ras exon 1 was fused to a gfp cDNA at each end and expressed in Escherichia coli . Amino acid number 12 of K-Ras (wild type; Gly) was changed to Ser, Arg, Cys, Asp, Ala, or Val, and the mobility shift of the greenish fluorescent bands in the SDS/urea gel was analyzed . These mutants were easily detected by GFP-display; however, detection depended strongly on the urea concentration and electrophoresis temperature . Subsequently, GFP-display was applied to the 36 amino acids encoding human p53 exon 7 . Amino acid number 248 (wild type; Arg) was changed to Gly, Trp, Gln, Pro, or Leu, and similar mobility shifts were observed . GFP-display could be coupled with an in vitro translation system . Fluorescent active GFP and GFP-Ras fusion proteins were synthesized within a few hours . GFP-display shows potential as a modern approach to gene mutation analysis at the protein level, and is a useful method for protein engineering studies. J Biochem (Tokyo), 2000 Apr, 127(4), 559 - 67 Kinetic and mutational studies of three NifS homologs from Escherichia coli: mechanistic difference between L-cysteine desulfurase and L-selenocysteine lyase reactions; Mihara H et al.; We have purified three NifS homologs from Escherichia coli, CSD, CsdB, and IscS, that appear to be involved in iron-sulfur cluster formation and/or the biosynthesis of selenophosphate . All three homologs catalyze the elimination of Se and S from L-selenocysteine and L-cysteine, respectively, to form L-alanine . These pyridoxal 5'-phosphate enzymes were inactivated by abortive transamination, yielding pyruvate and a pyridoxamine 5'-phosphate form of the enzyme . The enzymes showed non-Michaelis-Menten behavior for L-selenocysteine and L-cysteine . When pyruvate was added, they showed Michaelis-Menten behavior for L-selenocysteine but not for L-cysteine . Pyruvate significantly enhanced the activity of CSD toward L-selenocysteine . Surprisingly, the enzyme activity toward L-cysteine was not increased as much by pyruvate, suggesting the presence of different rate-limiting steps or reaction mechanisms for L-cysteine desulfurization and the degradation of L-selenocysteine . We substituted Ala for each of Cys358 in CSD, Cys364 in CsdB, and Cys328 in IscS, residues that correspond to the catalytically essential Cys325 of Azotobacter vinelandii NifS . The enzyme activity toward L-cysteine was almost completely abolished by the mutations, whereas the activity toward L-selenocysteine was much less affected . This indicates that the reaction mechanism of L-cysteine desulfurization is different from that of L-selenocysteine decomposition, and that the conserved cysteine residues play a critical role only in L-cysteine desulfurization. J Biochem (Tokyo), 2000 Apr, 127(4), 537 - 41 The iteron regions necessary for the RepE-iteron interaction in vivo in mini-F plasmids of Escherichia coli; Uga H et al.; We have determined the nucleotide positions of an incC iteron essential for RepE binding by analyzing mutated incC iterons defective in exerting incompatibility towards mini-F plasmids . The mutations affecting this incompatibility occurred mostly at two positions within the incC iteron, i.e . an iteron conserved position and a mini-F specific position . Most of the iterons with a base-change at either of these two positions had lost the binding affinity for RepE . This agrees with the crystallographic structure of the RepE-iteron complex which showed that the N and C terminal domains of RepE interact with the two major grooves on one face of the iteron DNA . These grooves contain the iteron conserved and mini-F specific positions necessary for RepE binding . Thus the binding mode may be common to in the case of mini-F like plasmids. Acta Crystallogr D Biol Crystallogr, 2000 Apr, 56 ( Pt 4), 512 - 5 Crystallization and preliminary X-ray analysis of shikimate dehydrogenase from Escherichia coli; Maclean J et al.; Shikimate dehydrogenase from Escherichia coli has been crystallized by the vapour-diffusion method using ammonium sulfate as a precipitant . Mass spectrometry confirmed the purity of the enzyme and dynamic light scattering was used to find the appropriate additives to yield a monodisperse enzyme solution . The crystals are monoclinic, space group C2, with unit-cell parameters a = 110.0, b = 139.8, c = 102.6 A, beta = 122.2 degrees (at 100 K) . Native crystals diffract to 2.3 A in-house on a rotating-anode X-ray source . The asymmetric unit is likely to contain four molecules, related by 222 symmetry, corresponding to a packing density of 2.86 A(3) Da(-1). Acta Crystallogr D Biol Crystallogr, 2000 Apr, 56 ( Pt 4), 509 - 11 Crystallization and preliminary crystallographic studies of a new crystal form of Escherichia coli L--asparaginase II (Ser58Ala mutant); Kozak M et al.; Periplasmic Escherichia coli L-asparaginase II with an Ser58Ala mutation in the active-site cavity has been crystallized in a new orthorhombic form (space group P2(1)2(1)2) . Crystals of this polymorph suitable for X-ray diffraction have been obtained by vapour diffusion using two sets of conditions: (i) 1% agarose gel using MPD as precipitant (pH 4.8) and (ii) liquid droplets using PEG-MME 550 (pH 9.0) . The crystals grown in agarose gel are characterized by unit-cell parameters a = 226.9, b = 128.4, c = 61.9 A and diffract to 2.3 A resolution . The asymmetric unit contains six protein molecules arranged into one pseudo-222-symmetric homotetramer and an active-site competent dimer from which another homotetramer is generated by crystallographic symmetry. Acta Crystallogr D Biol Crystallogr, 2000 Apr, 56 ( Pt 4), 501 - 3 Crystallization and preliminary X-ray analysis of squid neuronal Sec1; Bracher A et al.; Sec1 protein family members are involved in the regulation of all intracellular SNARE-mediated (SNARE = soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor) vesicle-fusion processes in a step preceding membrane fusion and have been shown to interact with t-SNAREs . To better understand the structural basis and the role of Sec1 in the regulation of the SNARE-complex formation, neuronal Sec1 from the squid Loligo pealei has been expressed and crystallized; this invertebrate protein shows a high sequence homology to the human neuronal Sec1, Munc18a . Here, the production of diffraction-quality native crystals, which belong to space group P3(1)21 and diffract to 3.3 A resolution, is described . In addition, selenomethionyl n-Sec1 crystals in space groups P3(1)21 and P2(1) have been generated . Preliminary analysis of the monoclinic space group indicates that these crystals diffract to a resolution higher than 2.5 A. Acta Crystallogr D Biol Crystallogr, 2000 Apr, 56 ( Pt 4), 489 - 91 The NADP(H)-binding component (dIII) of human heart transhydrogenase: crystallization and preliminary crystallographic analysis; Peake SJ et al.; Transhydrogenase is a membrane protein which uses the energy of the proton motive force to drive the reduction of NADP(+) by NADH . The enzyme has three domains: dII spans the membrane, while dI and dIII protrude from the membrane and contain the binding sites for NAD(H) and NADP(H), respectively . DIII from human heart transhydrogenase has been expressed in Escherichia coli . The purified protein has been crystallized with bound NADP(+) using the hanging-drop vapour-diffusion method with ammonium sulfate as a precipitant . The crystals belong to the tetragonal space group P4(1)22 or P4(3)22, with unit-cell parameters a = b = 58.1, c = 251.0 A . A 2.1 A resolution native data set has been collected with an R(merge) of 6 . 8%. Acta Crystallogr D Biol Crystallogr, 2000 Apr, 56 ( Pt 4), 472 - 4 Crystallization of native and selenomethionyl yeast orotidine 5'-phosphate decarboxylase; Miller BG et al.; Crystals of the Saccharomyces cerevisiae pyrimidine biosynthetic enzyme orotidine 5'-phosphate decarboxylase (ODCase) were grown by the hanging-drop vapor-diffusion technique at 277 K using polyethylene glycol 4000 as the precipitant . Crystals of native and selenomethionyl ODCase diffract to less than 2.2 A and belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 90.1, b = 116.2, c = 117.0 A . Crystals of ODCase grown in the presence of the postulated transition-state analog inhibitor 6-hydroxyuridine 5'--phosphate (BMP) diffract to less than 2.5 A and belong to space group P2(1), with unit-cell parameters a = 79.9, b = 80.0, c = 98.2 A, beta = 108.6 degrees. Acta Crystallogr D Biol Crystallogr, 2000 Apr, 56 ( Pt 4), 469 - 71 Crystallization and X-ray diffraction studies of the fatty-acid responsive transcription factor FadR from Escherichia coli; van Aalten DM et al.; FadR, an acylCoA-dependent Escherichia coli transcription factor controlling the expression of genes involved in fatty-acid degradation and synthesis, has been crystallized . Crystals of two binary complexes were obtained . The FadR-CoA complex crystallized in space group C222(1), with unit-cell parameters a = 61.3, b = 102.0, c = 91.3 A . The FadR-octanoyl-CoA complex crystallized in space group P6(5)22, with unit-cell parameters a = b = 59.7, c = 296.2 A . Both crystal forms diffracted to 3.5 A on a rotating-anode generator . In both crystal forms, the asymmetric unit contains one subunit . The protein is known to be a homodimer; each subunit consists of two domains of unknown fold . For the acyl-CoA-binding domain, a previously undetected sequence homology to PAS domains, in particular the photoactive yellow protein, is reported. Exp Cell Res, 2000 Apr 10, 256(1), 282 - 90 Heat-inducible expression of a reporter gene detected by transient assay in zebrafish; Adam A et al.; Heat-inducibility of two reporter constructs expressing lacZ gene under the control of mouse and Xenopus hsp70 promoters was tested in zebrafish (Danio rerio) embryos using a transient expression system . Cells expressing beta-galactosidase were stained blue by histochemical staining and their average number per embryo was used as an indicator of the expression level of the reporter gene . Both constructs were heat-inducible in the embryonic tissues and showed similar heat dependence (increasing expression levels from 35-36 degrees C up to 39 degrees C with an apparent decrease at 40 degrees C), resembling that of the zebrafish hsp70 genes . However, their induction kinetics were different, which might be due to differences in their 5' UTRs . Spatial expression patterns of the two hsp/lacZ constructs and an endogenous hsp70 gene were mostly similar on the RNA level . These results indicate that our approach is applicable for in vivo analysis of the heat-shock response and that exogenous heat-shock promoters may be useful for inducible expression of transgenes in fish . Biochemistry (Mosc), 2000 Mar, 65(3), 388 - 92 Engineering a new magnesium binding site in the subunit contact region of Escherichia coli inorganic pyrophosphatase; Parfenyev AN et al.; Three Gln-80 residues belonging to different subunits of homohexameric Escherichia coli pyrophosphatase are separated by only one water molecule to which they are hydrogen bonded . Substitution of Glu for Gln-80 stabilizes quaternary structure of the enzyme but has only a small effect on enzyme activity . The substitution stimulates Mg2+ binding and changes the appearance of the Mg2+ concentration dependence of the rate constant for the trimer --> hexamer transition . These data suggest that a new Mg2+ binding site is formed in the intersubunit contact region as a result of the substitution . Three-dimensional modeling of the mutated protein showed that a chelate complex might form involving two of the three Glu-80 residues. Biochemistry (Mosc), 2000 Mar, 65(3), 373 - 87 Mechanism of Ca2+-induced inhibition of Escherichia coli inorganic pyrophosphatase; Avaeva SM et al.; The causes of inhibition of Escherichia coli inorganic pyrophosphatase (PPase) by Ca2+ were investigated . The interactions of several mutant pyrophosphatases with Ca2+ in the absence of substrate were analyzed by equilibrium dialysis . The kinetics of Ca2+ inhibition of hydrolysis of the substrates MgPPi and LaPPi by the native PPase and three mutant enzymes (Asp-42-Asn, Ala, and Glu) were studied . X-Ray data on E . coli PPase complexed with Ca2+ or CaPPi solved at atomic resolution were analyzed . It was shown that, in the course of the catalytic reaction, Ca2+ replaces Mg2+ at the M2 site, which shows higher affinity for Ca2+ than for Mg2+ . Different properties of these cations account for active site deformation . Our findings indicate that the filling of the M2 site with Ca2+ is sufficient for PPase inhibition . This fact proves that Ca2+ is incapable of properly activating the H2O molecule for nucleophilic attack on PPi . It was also demonstrated that Ca2+, as a constituent of the non-hydrolyzable substrate analog CaPPi, competes with MgPPi at the M3 binding site . As a result, Ca2+ is a powerful inhibitor of all known PPases . Other possible reasons for the inhibitory effect of Ca2+ on the enzyme activity are also considered. Biochemistry (Mosc), 2000 Mar, 65(3), 361 - 72 Active site interactions in oligomeric structures of inorganic pyrophosphatases; Avaeva SM; Recent progress in studies of the mode of action of cytoplasmic inorganic pyrophosphatases is mainly due to the analysis of a dozen and a half structures of the apoenzyme, its complexes, and mutants . However, despite considerable research on the mechanism of action of these enzymes, many important problems remain unclear . Among them is the problem of active site interactions in oligomeric structures and their role in catalysis; this review focuses on this problem . The abundant experimental data requires generalization and comprehensive analysis . A characteristic feature of the spatial structure of inorganic pyrophosphatases is a flexible system of noncovalent interactions between protein groups penetrating the whole molecule of the oligomeric enzyme . Binding of metal ions, sulfate (an analog of the product of the enzymatic reaction), and affinity phosphorus-containing inhibitors at the active site or site-directed mutagenesis induce rearrangements in the set of hydrogen and ionic interactions, which change active site properties and in some instances, cause molecule asymmetry . In the trimeric form of Escherichia coli pyrophosphatase obtained by dissociation of a hexamer, active sites also interact with each other, which is manifested by negative cooperativity upon substrate binding . The association of trimers into the hexamer leads to perfect organization of active sites and to their coordinated functioning, probably due to the restoration of communication channels between the trimers. Biochemistry (Mosc), 2000 Mar, 65(3), 315 - 23 Inorganic polyphosphate and polyphosphate kinase: their novel biological functions and applications; Shiba T et al.; In this review, we discuss the following two subjects: 1) the physiological function of polyphosphate (poly(P)) as a regulatory factor for gene expression in Escherichia coli, and 2) novel functions of E . coli polyphosphate kinase (PPK) and their applications . With regard to the first subject, it has been shown that E . coli cells in which yeast exopolyphosphatase (poly(P)ase), PPX1, was overproduced reduced resistance to H2O2 and heat shock as did a mutant whose polyphosphate kinase gene is disrupted . Sensitivity to H2O2 and heat shock evinced by cells that overproduce PPX1 is attributed to depressed levels of rpoS expression . Since rpoS is a central element in a regulatory network that governs the expression of stationary-phase-induced genes, poly(P) affects the expression of many genes through controlling rpoS expression . Furthermore, poly(P) is also involved in expression of other stress-inducible genes that are not directly regulated by rpoS . The second subject includes the application of novel functions of PPK for nucleoside triphosphate (NTP) regeneration . Recently E . coli PPK has been found to catalyze the kination of not only ADP but also other nucleoside diphosphates using poly(P) as a phospho-donor, yielding NTPs . This nucleoside diphosphate kinase-like activity of PPK was confirmed to be available for NTP regeneration essential for enzymatic oligosaccharide synthesis using the sugar nucleotide cycling method . PPK has also been found to express a poly(P):AMP phosphotransferase activity by coupling with adenylate kinase (ADK) in E . coli . The ATP-regeneration system consisting of ADK, PPK, and poly(P) was shown to be promising for practical utilization of poly(P) as ATP substitute. Biochemistry (Mosc), 2000 Mar, 65(3), 309 - 14 Polyphosphates and enzymes of polyphosphate metabolism in Escherichia coli; Nesmeyanova MA; This review summarizes the results of our study of polyphosphate and enzymes of polyphosphate metabolism in E . coli and their regulation by exogenous orthophosphate and other physiological and genetic factors. Protein Sci, 2000 Jan, 9(1), 64 - 72 Conformational substates in different crystal forms of the photoactive yellow protein--correlation with theoretical and experimental flexibility; van Aalten DM et al.; The conformational changes during the photocycle of the photoactive yellow protein have been the subject of many recent studies . Spectroscopic measurements have shown that the photocycle also occurs in a crystalline environment, and this has been the basis for subsequent Laue diffraction and cryocrystallographic studies . These studies have shown that conformational changes during the photocycle are limited to the chromophore and its immediate environment . However, spectroscopic studies suggest the presence of large conformational changes in the protein . Here, we address this apparent discrepancy in two ways . First, we obtain a description of large concerted motions in the ground state of the yellow protein from NMR data and theoretical calculations . Second, we describe the high-resolution structure of the yellow protein crystallized in a different space group . The structure of the yellow protein differs significantly between the two crystal forms . We show that these differences can be used to obtain a description of the flexibility of the protein that is consistent with the motions observed in solution. Protein Sci, 2000 Jan, 9(1), 53 - 63 The use of nucleotide analogs to evaluate the mechanism of the heterotropic response of Escherichia coli aspartate transcarbamoylase; Sakash JB et al.; As an alternative method to study the heterotropic mechanism of Escherichia coli aspartate transcarbamoylase, a series of nucleotide analogs were used . These nucleotide analogs have the advantage over site-specific mutagenesis experiments in that interactions between the backbone of the protein and the nucleotide could be evaluated in terms of their importance for function . The ATP analogs purine 5'-triphosphate (PTP), 6-chloropurine 5'-triphosphate (Cl-PTP), 6-mercaptopurine 5'-triphosphate (SH-PTP), 6-methylpurine 5'-triphosphate (Me-PTP), and 1-methyladenosine 5'-triphosphate (Me-ATP) were partially synthesized from their corresponding nucleosides . Kinetic analysis was performed on the wild-type enzyme in the presence of these ATP analogs along with GTP, ITP, and XTP . PTP, Cl-PTP, and SH-PTP each activate the enzyme at subsaturating concentrations of L-aspartate and saturating concentrations of carbamoyl phosphate, but not to the same extent as does ATP . These experiments suggest that the interaction between N6-amino group of ATP and the backbone of the regulatory chain is important for orienting the nucleotide and inducing the displacements of the regulatory chain backbone necessary for initiation of the regulatory response . Me-PTP and Me-ATP also activate the enzyme, but in a more complex fashion, which suggests differential binding at the two sites within each regulatory dimer . The purine nucleotides GTP, ITP, and XTP each inhibit the enzyme but to a lesser extent than CTP . The influence of deoxy and dideoxynucleotides on the activity of the enzyme was also investigated . These experiments suggest that the 2' and 3' ribose hydroxyl groups are not of significant importance for binding and orientation of the nucleotide in the regulatory binding site . 2'-dCTP inhibits the enzyme to the same extent as CTP, indicating that the interactions of the enzyme to the O2-carbonyl of CTP are critical for CTP binding, inhibition, and the ability of the enzyme to discriminate between ATP and CTP . Examination of the electrostatic surface potential of the nucleotides and the regulatory chain suggest that the complimentary electrostatic interactions between the nucleotides and the regulatory chain are important for binding and orientation of the nucleotide necessary to induce the local conformational changes that propagate the heterotropic effect. Protein Sci, 2000 Jan, 9(1), 37 - 48 Expression, purification, and structural analysis of the trimeric form of the catalytic domain of the Escherichia coli dihydrolipoamide succinyltransferase; Knapp JE et al.; The dihydrolipoamide succinyltransferase (E2o) component of the alpha-ketoglutarate dehydrogenase complex catalyzes the transfer of a succinyl group from the S-succinyldihydrolipoyl moiety to coenzyme A . E2o is normally a 24-mer, but is found as a trimer when E2o is expressed with a C-terminal {His}6 tag . The crystal structure of the trimeric form of the catalytic domain (CD) of the Escherichia coli E2o has been solved to 3.0 A resolution using the Molecular Replacement method . The refined model contains an intact trimer in the asymmetric unit and has an R-factor of 0.257 (Rfree = 0.286) for 18,699 reflections between 10.0 and 3.0 A resolution . The core of tE2oCD (residues 187-396) superimposes onto that of the cubic E2oCD with an RMS difference of 0.4 A for all main-chain atoms . The C-terminal end of tE2oCD (residues 397-404) rotates by an average of 37 degrees compared to cubic E2oCD, disrupting the normal twofold interface . Despite the alteration of quaternary structure, the active site of tE2oCD shows no significant differences from that of the cubic E2oCD, although several side chains in the active site are more ordered in the trimeric form of E2oCD . Analysis of the available sequence data suggests that the majority of E2 components have active sites that resemble that of E . coli E2oCD . The remaining E2 components can be divided into three groups based on active-site sequence similarity . Analysis of the surface properties of both crystal forms of E . coli E2oCD suggests key residues that may be involved in the protein-protein contacts that occur between the catalytic and lipoyl domains of E2o. Protein Sci, 2000 Jan, 9(1), 20 - 8 NMR analysis of cleaved Escherichia coli thioredoxin (1-73/74-108) and its P76A variant: cis/trans peptide isomerization; Yu WF et al.; Inspection of high resolution three-dimensional (3D) structures from the protein database reveals an increasing number of cis-Xaa-Pro and cis-Xaa-Yaa peptide bonds . However, we are still far from being able to predict whether these bonds will remain cis upon single-site substitution of Pro or Yaa and/or cleavage of a peptide bond close to it in the sequence . We have chosen oxidized Escherichia coli thioredoxin (Trx), a member of the Trx superfamily with a single alpha/beta domain and cis P76 to determine the effect of single-site substitution and/or cleavage on this isomer . Standard two-dimensional (2D) NMR analysis were performed on cleaved Trx (1-73/74-108) and its P76A variant . Analysis of the NOE connectivities indicates remarkable similarity between the secondary and supersecondary structure of the noncovalent complexes and Trx . Analysis of the 2D version of the HCCH-TOCSY and HMQC-NOESY-HMQC and 13C-filtered HMQC-NOESY spectra of cleaved Trx with uniformly 13C-labeled 175 and P76 shows surprising conservation of both cis P76 and packing of 175 against W31 . A similar NMR analysis of its P76A variant provides no evidence for cis A76 and shows only subtle local changes in both the packing of 175 and the interstrand connectivities between its most protected hydrophobic strands (beta2 and beta4) . Indeed, a molecular simulation model for the trans P76A variant of Trx shows only subtle local changes around the substitution site . In conclusion, cleavage of R73 is insufficient to provoke cis/trans isomerization of P76, but cleavage and single-site substitution (P76A) favors the trans isomer. Anal Chem, 2000 Mar 1, 72(5), 1006 - 14 A method for the chemical generation of N-terminal peptide sequence tags for rapid protein identification; Hoving S et al.; We describe a method for generating multiple small sequences from the N terminal of peptides in unseparated protein digests by stepwise thioacetylation and acid cleavage . The mass differences between a series of N-terminally degraded peptides give short sequences of defined length . Such short "sequence tags" together with the mass of the parent peptide can be used to identify the protein in a database . The sequence ladders are generated without the use of chain terminators or sample aliquoting and the degradation reagents are water soluble so that the chemistry can be carried out on peptides immobilized on C-18 reversed-phase supports without any peptide loss due to washing with organic solvents as occurs in Edman type sequencing . The entire procedure can be automated, and we describe a prototype device for the parallel analysis of multiple samples . We demonstrate the effectiveness of this chemical tagging method in a comparison with Edman sequencing, peptide mass fingerprinting, and MS/MS analysis of crude protein fractions obtained from an HPLC separation of the Escherichia coli ribosome complex which consists of 57 proteins . We show that chemical tagging is a viable first-pass high-throughput identification method to be used prior to an in depth MS/MS analysis. Pharmacogenetics, 2000 Feb, 10(1), 59 - 66 Characterization of human polymorphic DNA repair methyltransferase; Inoue R et al.; The O6-methylguanine-DNA methyltransferase (MGMT) is a critical defence against alkylation-induced mutagenesis and carcinogenesis . More than a 20-fold interindividual difference in the MGMT activity is known to exist among human cultured fibroblasts . We previously reported three allelic variants of the human MGMT gene, namely V1, V2, and V3 . Both V1 and V2 carry amino acid substitutions, Leu84Phe and Trp65Cys, respectively, while V3 has a silent mutation . In order to reveal the pharmacogenetic and ecogenetic significance of polymorphism in the human MGMT gene, we investigated the in-vivo characteristics of V1 and V2 methyltransferase enzyme . Escherichia coli strain KT233 (ogt-, ada-) and mer- HeLa MR cells carrying a V1 sequence exhibited almost the same level of sensitivity against N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), as did those with a wild-type sequence . The level of methyltransferase protein in those cells was essentially the same as for the wild-type and V1 samples . On the other hand, E . coli and human cells expressing V2 cDNA showed a significantly reduced level of survival . In these cells, V2 protein was hardly detected, even though mRNA was produced normally . An in-vitro translation experiment revealed that the V2 sequence had the potential to produce methyltransferase protein, as did the wild-type and V1 sequences . There was also evidence for a small amount of V2 protein being produced but rapidly degraded, thus implying that the V2 molecule is unstable in vivo . Using purified recombinant proteins, we estimated the kinetic values of wild-type and variant form of enzymes, which would support these views . From these results, we concluded that the wild-type and V1 protein have similar enzymatic and physicochemical properties, while V2 protein is considered to be unstable and rare. Pharmacogenetics, 2000 Feb, 10(1), 49 - 57 Discovery of a functional polymorphism in human glutathione transferase zeta by expressed sequence tag database analysis; Blackburn AC et al.; Analysis of the expressed sequence tag (EST) database by sequence alignment allows a rapid screen for polymorphisms in proteins of physiological interest . The human zeta class glutathione transferase GSTZ1 has recently been characterized and analysis of expressed sequence tag clones suggested that this gene may be polymorphic . This report identifies three GSTZ1 alleles resulting from A to G transitions at nucleotides 94 and 124 of the coding region, GSTZ1*A-A94A124; GSTZ1*B-A94G124; GSTZ1*C-G94G124 . Polymerase chain reaction/restriction fragment length polymorphism analysis of a control Caucasian population (n = 141) showed that all three alleles were present, with frequencies of 0.09, 0.28 and 0.63 for Z1*A, Z1*B and Z1*C, respectively . These nucleotide substitutions are non-synonymous, with A to G at positions 94 and 124 encoding Lys32 to Glu and Arg42 to Gly substitutions, respectively . The variant proteins were expressed in Escherichia coli as 6X His-tagged proteins and purified by Ni-agarose column chromatography . Examination of the activities of recombinant proteins revealed that GSTZ1a-1a displayed differences in activity towards several substrates compared with GSTZ1b-1b and GSTZ1c-1c, including 3.6-fold higher activity towards dichloroacetate . This report demonstrates the discovery of a functional polymorphism by analysis of the EST database. J Photochem Photobiol B, 2000 Jan, 54(1), 67 - 71 H2O2-induced cross-protection against UV-C killing in Escherichia coli is blocked in a lexA (Def) background; Asad LM et al.; Pretreatment with 2.5 mM H2O2 protects E . coli cells against UV-C killing, a phenomenon independent of LexA cleavage . In this paper, we observe that this cross-protection response is neither dependent on the dinY gene product nor on the system that controls dinY, since H2O2 is able to induce cross-protection but not to induce the dinY gene in a lexA-noninducible strain {lexA (Ind-)} . Moreover, this response is not induced in a lexA (Def) background, suggesting that the expression of the SOS regulon may inhibit this cross-protection response. J Hered, 2000 Jan-Feb, 91(1), 24 - 30 Detection of RFLP markers associated with antibody response in meat-type chickens: haplotype/genotype, single-band, and multiband analyses of RFLP in the major histocompatibility complex; Yonash N et al.; Improving disease resistance in poultry by direct selection or by selecting for immune response is hardly feasible due to the quantitative nature of these traits, their low heritability, and the difficulties associated with reliable measurements . In this situation, marker-assisted selection (MAS) is expected to be a more effective breeding approach . The major histocompatibility complex (MHC), known to affect immune response and disease resistance, was examined as a set of candidate genes for association between DNA markers and antibody response . Backcross (BC1) and F2 families were generated from a cross between lines divergently selected for high or low antibody response to Escherichia coli vaccination . Restriction fragment length polymorphism (RFLP) analysis of the highly polymorphic MHC class IV (B-G) region suggested an association with antibody response to several antigens (E . coli, SRBC, NDV) . The multiband data generated with the class IV probe were used to compare the efficacies of three alternative analyses: "single-band" (carriers versus noncarriers of each RFLP band separately), "multiband" (multiple regression on all RFLP bands), and "genotype" (determined from family analysis of RFLP patterns/haplotypes) . Groups of birds identified by the "multiband" analysis were identical to the haplotype-based genotypes, suggesting that the laborious step of haplotype determination can be omitted without unduly sacrificing power of analysis. Microbios, 2000, 101(399), 89 - 103 Regulation of the thdF gene, which is involved in thiophene oxidation by Escherichia coli K-12; Zabel MD et al.; The thdF gene of Escherichia coli encodes a 48 kD protein which is involved in the oxidation of derivatives of the sulphur-containing heterocycle thiophene and which appears to be induced during stationary phase . In this work the upstream regulatory region of the thdF gene was isolated by polymerase chain reaction and inserted in front of the lacZ structural gene . Examination of the resulting thdF-lacZ operon fusions showed that expression of the thdF gene increased as E . coli entered the stationary phase . However, the expression of thdF was not dependent on RpoS (KatF), the stationary phase sigma factor . The thdF gene was subject to substantial catabolite repression by glucose and its expression was also greatly decreased in the absence of oxygen . The thdF-lacZ fusions were not significantly affected by elevated temperature or medium of high osmolarity, nor by mutations in thdA, fadR, arcA, arcB, or fnr . Both multicopy, plasmid-borne fusions and single-copy fusions gave similar results in all of the above cases except that the plasmid-borne fusions still showed substantial expression in the absence of oxygen . The heterocyclic compounds thiophene carboxylic acid, furan carboxylic acid and proline increased expression of the thdF gene by 2- to 3-fold, but only during the stationary phase . Tryptophan, indole, and several indole derivatives had no effect. J Gene Med, 1999 Jan-Feb, 1(1), 31 - 42 Selective uptake and sustained expression of AAV vectors following subcutaneous delivery; Donahue BA et al.; BACKGROUND: Recombinant adeno-associated viral (rAAV) vectors are capable of long-term expression of secreted and intracellular proteins following delivery to muscle, liver, and the central nervous system . In this study, we have evaluated subcutaneous injection of rAAV encoding a variety of transgenes as an alternative route of administration for the systemic delivery of therapeutic proteins . METHODS: rAAV vectors encoding the human factor IX, human interferon-alpha 2a, murine erythropoietin (epo), and Escherichia coli lacZ genes were used for subcutaneous delivery into mature immunocompetent mice . Expression of factor IX and interferon in mouse serum was measured by ELISA . Expression of Epo was monitored by an increase in hemotocrit and by RIA . The tissue tropism of AAV transduction was determined by histochemistry following administration of the lacZ vector . RESULTS: Long-term protein expression (at least one year) is demonstrated in the serum of immunocompetent mice following subcutaneous delivery of AAV vectors encoding the human factor IX and interferon genes . The murine epo gene delivered via this route resulted in levels of Epo that correlate with increased hematocrits of up to 90% for a duration of nine months . rAAV encoding the lacZ gene revealed that the panniculus carnosus, a skeletal muscle layer of the skin, was transduced upon subcutaneous administration . CONCLUSIONS: This study shows that long-term expression of secreted proteins can be achieved using rAAV vectors injected subcutaneously as a single administration . The observation that the panniculus carnosus is the primary tissue transduced by rAAV illustrates the high tropism of rAAV for skeletal muscle. J Gene Med, 1999 Mar-Apr, 1(2), 84 - 92 Expression of mucin (MUC-1) from a mini-Epstein-Barr virus in immortalized B-cells to generate tumor antigen specific cytotoxic T cells; Kilger E et al.; BACKGROUND: EBV immortalized B-cells can be used as antigen presenting cells (APC) to stimulate specific T-cell responses . Mini-Epstein-Barr virus (mini-EBV) plasmids contain all functional elements of Epstein-Barr virus (EBV) necessary to immortalize B-cells in vitro . These immortalized B-cells are incapable of releasing infectious virus in contrast to cells immortalized by wildtype EBV . In addition, mini-EBVs can be modified in E . coli to alter their genetic composition or adopt new genes . METHODS: We constructed a mini-EBV plasmid carrying an expression cassette for the human tumor antigen mucin encoded by the gene MUC-1 . Primary human B-cells were infected with the MUC-1 carrying mini-EBV plasmid packaged into an EBV coat and immortalized B-cell clones were expanded in vitro . These B-cells were analyzed by FACS analyses for the expression of mucin and co-stimulatory molecules and were subsequently used as antigen presenting cells (APC) to stimulate peripheral blood mononuclear cells from healthy donors . RESULTS: Several B-cell lines were established that were shown to be free of helper virus or wildtype EBV . These B-cells expressed the relevant tumor-specific epitopes of mucin and the co-stimulatory ligands B7.1 and B7.2 necessary for efficient T-cell activation . Using the mucin expressing B-cells as antigen presenting cells (APC) mucin-epitope specific cytotoxic T-cells were established . CONCLUSIONS: Virus-free B-cell lines expressing tumor-associated epitopes such as mucin or other antigens of interest provide an unlimited and safe source of APC to generate antigen specific T-cells which could be used for clinical trials in adoptive immune therapy or cancer vaccines. Mol Reprod Dev, 2000 May, 56(1), 34 - 44 New approach to cell lineage analysis in mammals using the Cre-loxP system; Sato M et al.; The Cre-loxP site-specific recombination system was used for cell lineage analysis in mammals . We constructed an expression plasmid, pCETZ-17, which consists of cytomegalovirus enhancer/chicken beta-actin promoter (CAG), a portion of the rabbit beta-globin gene, loxP-flanked DNA sequence (containing enhanced green fluorescent protein (EGFP) cDNA), and lacZ gene encoding E . coli beta-galactosidase (beta-gal) . When circular pCETZ-17 plasmid DNA was microinjected into the pronuclei of fertilized eggs and these eggs were allowed to develop to two-cell stage, 62.8% (59/94) of the two-cell embryos exhibited distinct fluorescence in one or both blastomeres, but never expressed lacZ protein, as evaluated by histochemical staining with X-Gal, a substrate for beta-gal . When both circular plasmids, pCETZ-17 and pCAG/NCre (containing CAG and DNA sequences encoding nuclear location signal and Cre), were co-injected into fertilized eggs, almost all (87.0%, 47/54) embryos exhibited low or no fluorescence, but 51.9% (27/52) exhibited positive staining for beta-gal activity . This indicates that transient expression of the Cre recombinase gene removed the loxP-flanked DNA sequence in pCETZ-17 and then caused expression of the downstream lacZ sequence . We next microinjected pCETZ-17 into the pronuclei of fertilized eggs, cultured these injected eggs for 1 day, and collected only two-cell embryos expressing EGFP in both blastomeres . One blastomere of the EGFP-expressing two-cell embryos was microinjected with pCAG/NCre, and these treated embryos were cultured for 1 day up to four-cell stage . When the developing four-cell embryos were subjected to staining with X-Gal, cell lineage-related staining pattern for beta-gal activity was observed in most (77.8%, 7/9) embryos . These findings were further confirmed using two-cell embryos derived from a transgenic mouse line carrying CETZ-17 transgene . Thus, our system, which is based on transient expression of the Cre recombinase gene directly introduced into nuclei of embryonic cells by microinjection, is a powerful means for cell lineage analysis in mammals . Arch Insect Biochem Physiol, 2000 Apr, 43(4), 165 - 72 Molecular cloning of a cDNA for a small GTP binding protein, BRho, from the embryo of Bombyx mori and its characterization after expression and purification; Uno T et al.; A cDNA clone encoding a small GTP binding protein (Brho) was isolated from an embryonic cDNA library of Bombyx mori that encoded a polypeptide with 202 amino acids sharing 60-80% similarity with the Rho1 family of GTP binding proteins . The effector site and one of the guanine nucleotide binding sites differed from other members of the Rho family . To characterize the biochemical properties of Brho, the clone was expressed in Escherichia coli as a glutathione S-transferase (GST) fusion protein . The recombinant protein was purified to homogeneity with glutathione S-Sepharose . The fusion protein bound {(35)S} GTPgammaS and {(3)H} GDP with association constants of 11x10(6) M(-1) and 6.2x10(6) M(-1), respectively . The binding of {(35)S} GTPgammaS was inhibited by GTP and GDP, but by no other nucleotides . The calculated GTP-hydrolysis activity was 89.6 m mol/min/mol of Brho . Bound {(35)S} GTPgammaS and {(3)H} GDP were exchanged with GTPgammaS most efficiently in the presence of 6 mM MgCl(2) . These results suggest that Brho has a higher affinity for GTP than GDP, converts from the GTP-bound state into the GDP-bound state by intrinsic GTP hydrolytic activity, and returns to the GTP-bound state with the exchange of GDP with GTP . Arch . Arch Insect Biochem Physiol, 2000 Apr, 43(4), 147 - 64 Hematopoiesis in larval Pseudoplusia includens and Spodoptera frugiperda; Gardiner EM et al.; Maintenance of circulating hemocytes in larval Lepidoptera has been attributed to both mitosis of hemocytes already in circulation and the release of hemocytes from hematopoietic organs . In this study, we compared hematopoiesis in the noctuids Pseudoplusia includens and Spodoptera frugiperda . For both species, hemocyte densities per microl of blood increased with instar . Differential hemocyte counts indicated that plasmatocytes were the most abundant hemocyte type during early instars but granular cells were the most abundant hemocyte type in the last instar . Hematopoietic organs were located in the meso- and metathorax of S . Frugiperda and P . Includens . These organs contained large numbers of hemocytes in S . Frugiperda, but contained few hemocytes in P . Includens . The majority of the hemocytes recovered from hematopoietic organs were identified as plasmatocytes . Using hemocyte type-specific markers and bromodeoxyuridine (BrdU) incorporation experiments, we determined that all hemocyte types with the exception of oenocytoids synthesize DNA . BrdU labeling indices for both species also fluctuated with the molting cycle . Ligation experiments suggested that hematopoietic organs are an important source of circulating plasmatocytes in S . Frugiperda but not in P . Includens . Injection of heat killed bacteria into larvae induced higher levels of BrdU labeling than injection of sterile saline, suggesting that infection and wounding induce different levels of hemocyte proliferation . Arch . J Cell Physiol, 2000 May, 183(2), 182 - 95 Interaction between CD44 and the repeat domain of ankyrin promotes hyaluronic acid-mediated ovarian tumor cell migration; Zhu D et al.; The adhesion molecule, CD44, interacts with ankyrin within its cytoplasmic domain and binds to hyaluronic acid (HA) at its extracellular domain . In this study, we focused on the functional domain in ankyrin (in particular, the ankyrin repeat domain {ARD}) responsible for CD44 binding and its role in regulating HA-mediated ovarian tumor cell function . Using recombinant fragments of ankyrin (e.g., ARD and subdomain 1 {S1, aa1-aa217}, subdomain 2 {S2, aa218-aa381}, subdomain 3 {S3, aa382-aa612}, and subdomain 4 {S4, aa613-aa834}) and in vitro binding assays, we determined that the S2 but not S1, S3, or S4 of ARD is the primary ankyrin binding region for CD44 . Microinjection of antiglutathione S-transferase (GST)-tagged S2 or GST-tagged ARD fusion protein into CD44-positive ovarian tumor cells (e.g., SKOV3 cell line) promotes ankyrin association with CD44 in plaque-like structures and membrane projections . Additionally, we demonstrated that transfection of SKOV3 cells with S2cDNA or ARD cDNA results in an upregulation of HA-mediated tumor cell migration . Taken together, we believe that the S2 of the ARD plays a pivotal role in the direct binding to CD44 and promotes the cytoskeleton activation required for HA-mediated function such as ovarian tumor cell migration . Avian Dis, 2000 Jan-Mar, 44(1), 185 - 91 Iss from a virulent avian Escherichia coli; Foley SL et al.; No single characteristic of virulent avian Escherichia coli has been identified that can be exploited in colibacillosis detection protocols . Research in our lab suggests a strong association between the presence of an iss DNA sequence with an isolate's disease-causing ability . The study presented here focuses on the techniques used in the expression, purification, and characterization of avian E . coli Iss protein . In brief, iss was cloned into an expression vector, the construct was transformed into a protease-deficient E . coli, and expression was induced . The protein was expressed as a glutathione-S-transferase (GST) fusion and purified by affinity chromatography . The GST portion was cleaved from Iss, Iss was harvested by affinity chromatography, and the identity of Iss was confirmed by N-terminal sequencing . Currently, purified Iss is being used to prepare hybridomas for production of monoclonal antibodies with the goal of evaluating anti-Iss as a reagent for the detection of virulent avian E . coli. Avian Dis, 2000 Jan-Mar, 44(1), 179 - 84 Cloning and sequencing of the iss gene from a virulent avian Escherichia coli; Horne SM et al.; Control of colibacillosis is important to the poultry industry . We have found that the presence of a gene for increased serum survival, iss, is strongly correlated with Escherichia coli isolated from birds with colibacillosis . Therefore, the iss gene and its protein product, Iss, are potential targets for detection and control of avian colibacillosis . The iss gene was amplified from a virulent avian E . coli isolate and sequenced . The sequences of the gene and the predicted protein product were compared with those of iss from a human E . coli isolate and lambda bor . The iss gene from the avian E . coli isolate has 96.8% identity with the iss gene from the human E . coli isolate and 89.4% identity with lambda bor . The Iss protein from the avian isolate has 87% identity with Iss from the human isolate and 90% identity with Bor . The low identity between the two Iss proteins is because of a frame-shift in their respective coding sequences . In sum, iss from this avian E . coli isolate is very similar to iss from a human E . coli isolate, but because of a frameshift mutation in the coding sequence of iss from the human E . coli isolate, Iss proteins from avian and human E . coli isolates have only 87% identity . The strong association of iss with E . coli isolated from birds with colibacillosis, suggests that this sequence be studied for its value as a marker or target to be used in colibacillosis control. Avian Dis, 2000 Jan-Mar, 44(1), 170 - 8 Cloning and expression of the VP2 gene of an infectious bursal disease virus; Yu L et al.; A serotype I infectious bursal disease virus (IBDV) strain HZ96 was isolated in Hangzhou, China, in 1996 and attenuated by adaptation to chicken embryo fibroblast cells . The VP2 gene of strain HZ96 was amplified by reverse transcription-polymerase chain reaction, cloned, and sequenced . Compared with the VP2 sequences of other IBDV strains, HZ96 is most related to two attenuated strains, CU-1 and PBG98, and two attenuated Chinese strains, Harbin and CJ801bkf . HZ96 shares nucleotide sequence homology 98.9% with CU-1 and PBG98, 98.5% with Harbin, and 98.6% with CJ801bkf . Most of the sequence variations observed between HZ96 and other strains are located in the middle variable region from nucleotides 637 to 996 . Similar to other attenuated IBDVs, HZ96 has unique substitutions at residues 279 (Asp to Asn) and 284 (Ala to Thr), suggesting that these two substitutions may be directly related to adaptation of the virus to cell culture and attenuation of its virulence . As part of our effort to develop a submit vaccine for IBDV, the VP2 gene of HZ96 was cloned into a heat-inducible expression vector and expressed in Escherichia coli system . A protein band with expected molecular weight of 52 kD was detected by direct protein staining and western blotting. Avian Dis, 2000 Jan-Mar, 44(1), 59 - 65 Alterations in macrophage-produced cytokines and nitrite associated with poult enteritis and mortality syndrome; Heggen CL et al.; Poult enteritis and mortality syndrome (PEMS) is an acute, transmissible, infectious intestinal disease associated with high mortality and morbidity in turkey poults . Earlier studies demonstrated immune dysfunction, involving both humoral and cell-mediated immunity, associated with PEMS . The current study examined cytokines and metabolites produced by macrophages from poults exposed to PEMS agent(s) . Six trials were conducted with six separate hatches of poults . Poults in the PEMS group were exposed to PEMS agent(s) via contact exposure at 7 days of age whereas uninfected poults served as control poults . Abdominal macrophages were harvested from control (uninfected) and PEMS poults at various times postexposure and cultured for 18-24 hr in the presence of Escherichia coli lipopolysaccharide . Interleukin-1 (IL-1), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-alpha) bioactivities and nitrite levels in macrophage culture supernatants were quantified . Macrophage supernatants from PEMS poults had greater IL-1-mediated stimulation index compared with the macrophage supernatants from uninfected control poults in both trials . However, this increase was significant only in trial 1 . IL-6 activity tested in three separate trials was significantly higher in PEMS macrophage supernatants over the controls . On the contrary, TNF-alpha production by macrophages was decreased in PEMS macrophage culture supernatants . Nitrite levels in PEMS macrophage culture supernatants were significantly higher in two out of three trials . These findings suggest that the enhanced production of proinflammatory cytokine/metabolites by activated macrophages in PEMS poults may be responsible, at least in part, for the physiological intestinal inflammation, gut motility, and anorexia that characterize this disease. Proc R Soc Lond B Biol Sci, 2000 Mar 7, 267(1442), 515 - 22 Pervasive compensatory adaptation in Escherichia coli; Moore FB et al.; To investigate compensatory adaptation (CA), we used genotypes of Escherichia coli which were identical except for one or two deleterious mutations . We compared CA for (i) deleterious mutations with large versus small effects, (ii) genotypes carrying one versus two mutations, and (iii) pairs of deleterious mutations which interact in a multiplicative versus synergistic fashion . In all, we studied 14 different genotypes, plus a control strain which was not mutated . Most genotypes showed CA during 200 generations of experimental evolution, where we define CA as a fitness increase which is disproportionately large relative to that in evolving control lines, coupled with retention of the original deleterious mutation(s) . We observed greater CA for mutations of large effect than for those of small effect, which can be explained by the greater benefit to recovery in severely handicapped genotypes given the dynamics of selection . The rates of CA were similar for double and single mutants whose initial fitnesses were approximately equal . CA was faster for synergistic than for multiplicative pairs, presumably because the marginal gain which results from CA for one of the component mutations is greater in that case . The most surprising result in our view, is that compensation should be so readily achieved in an organism which is haploid and has little genetic redundancy This finding suggests a degree of versatility in the E . coil genome which demands further study from both genetic and physiological perspectives. Br J Cancer, 2000 Mar, 82(5), 1035 - 40 Decreased GTPase activity of K-ras mutants deriving from human functional adrenocortical tumours; Lin SR et al.; Our previous studies have shown that seven out of 15 patients with adrenocortical tumours contained K-ras gene mutation . In addition, the mutation type was a multiple-site mutation, and the hot spots were located at codons 15, 16, 18 and 31, which were different from those reported before (codons 12, 13 and 61) . To understand whether the mutation hot spots in human adrenocortical tumours were associated with activation of K-Ras oncogene and the alterations of its biocharacteristics, mutant K-Ras genes were cloned from tumour tissues and then constructed with expression vector pBKCMV . Mutant K-Ras genes were expressed at high levels in Escherichia coli and the resultant K-Ras proteins were shown to be functional with respect to their well-known specific, high-affinity, GDP/GTP binding . The purified K-Ras protein from E . coli were then measured for their intrinsic GTPase activity and the GTPase activity in the presence of GTPase-activating protein for Ras . The results showed that the wild-type cellular K-Ras protein (p21BN) exhibits about ten times higher intrinsic GTPase activity than the activated protein (p21BM3) encoded by mutant K-Ras gene, which mutated at codon 60 . With regards to the codon 15, 16, 18 and 31 mutant K-Ras proteins (p21BM2), the GTPase activity in the presence of GAP is much lower than that of the normal K-Ras protein, whereas the intrinsic GTPase activity is nearly the same as that of the normal K-Ras protein . These results indicated that mutations at these hot spots of K-Ras gene were indeed activated K-Ras oncogene in adrenocortical tumours; however, their association with tumors needs further experiments to prove. Glycoconj J, 1999 Aug, 16(8), 457 - 63 Sulfated sialic acid-polymers inhibit the cytotoxic action of bee and snake venom; Oda Y et al.; Colominic acid is an alpha2,8-linked sialic acid polymer produced by Escherichia coli . We found that synthetic sulfated-colominic acids (SC) remarkably inhibited the cytotoxicity of bee and snake venom toward mouse fibroblast cells, but colominic acids showed no inhibition themselves, indicating the important role of sulfate groups in the inhibitory activity of SC . Other sulfated carbohydrates such as chondroitin sulfates, heparin and heparan sulfate showed no inhibition . SC also exhibited potent inhibition of melittin, a highly basic peptide, which is a major cytotoxic component of bee venom . SC did not inhibit phospholipase A2 activity in bee venom . This suggests that the inhibition of bee and snake venom by SC is due to inhibition of melittin and cardiotoxin, which is a cytolytic peptide in snake venom, respectively . SC with a higher sulfur content and a larger molecular mass showed more potent activity . The interaction between SC and melittin basically seems an ionic one, however, the conformation of SC is also likely important . For the binding of SC to melittin leading loss of its cytotoxic activity, the sulfate groups of SC must be properly arranged to interact with lysine and arginine residues of melittin molecules, which play an important role in the cytolytic activity . A higher molecular mass of SC substituted with more sulfate groups is required for more obvious inhibition of the cytotoxic activity. Biosci Biotechnol Biochem, 2000 Feb, 64(2), 348 - 54 Inhibitory effects of bovine lactoferrin on the adherence of enterotoxigenic Escherichia coli to host cells; Kawasaki Y et al.; Adherence is an essential and prerequisite step for the colonization of mucosal surfaces by enterotoxigenic Escherichia coli (ETEC) . We studied the effect of bovine lactoferrin (BLF) on the adherence of ETEC to human epithelial cells in vitro, and to intestinal mucosa of ICR germfree mice in vivo . In the in vitro study, BLF was found to inhibit the adherence of ETEC . This adhesion-inhibiting activity of BLF was found to lessen with decreasing BLF concentration, but the data obtained suggest a positive inhibitory effect of BLF against the adhesion of ETEC cells . In the in vivo study, the counts of adherent bacteria in various sections of the intestinal tract (duodenum, jejunoileum, and large intestine) were lower in the BLF group than in the control group, suggesting the possible action of BLF as an intestinal tract adherence-blocking agent with regards to ETEC. Int J Biochem Cell Biol, 2000 May, 32(5), 499 - 508 Interaction of mitochondrial phosphate carrier with fatty acids and hydrophobic phosphate analogs; Zackova M et al.; Mitochondrial transporters, in particular uncoupling proteins and the ADP/ATP carrier, are known to mediate uniport of anionic fatty acids (FAs), allowing FA cycling which is completed by the passive movement of FAs across the membrane in their protonated form . This study investigated the ability of the mitochondrial phosphate carrier to catalyze such a mechanism and, furthermore, how this putative activity is related to the previously observed HgCl(2)-induced uniport mode . The yeast mitochondrial phosphate carrier was expressed in Escherichia coli and then reconstituted into lipid vesicles . The FA-induced H(+) uniport or Cl(-) uniport were monitored fluorometrically after HgCl(2) addition . These transport activities were further characterized by testing various inhibitors of the two different transport modes . The phosphate carrier was found to mediate FA cycling, which led to H(+) efflux in proteoliposomes . This activity was insensitive to ATP, mersalyl or N-ethylmaleimide and was inhibited by methylenediphosphonate and iminodi(methylenephosphonate), which are new inhibitors of mitochondrial phosphate transport . Also, the HgCl(2) induced Cl(-) uniport mediated by the reconstituted yeast PIC, was found to be inhibited by these reagents . Both methylenediphosphonate and iminodi(methylenephosphonate) blocked unidirectional Cl(-) uptake, whereas Cl(-) efflux was inhibited by iminodi(methylenephosphonate) and phosphonoformic acid only . These results suggest that a hydrophobic domain, interacting with FAs, exists in the mitochondrial phosphate carrier, which is distinct from the phosphate transport pathway . This domain allows for FA anion uniport via the phosphate carrier and consequently, FA cycling that should lead to uncoupling in mitochondria . This might be considered as a side function of this carrier. Int J Biochem Cell Biol, 2000 May, 32(5), 481 - 8 Determination of interactions between human thrombopoietin and its receptor MPL by yeast two-hybrid system and affinity biosensor; Hsieh DP et al.; The binding of human thrombopoietin to the extracellular domain of its receptor MPL prompts a cascade transduction of intracellular signals, leading to the development of megakaryocyte precursors and the production of circulating platelets . We have used a yeast two-hybrid system to reveal, via in vivo interactions between different deletion constructs of MPL and thrombopoietin, that the extracellular subunit 1 of MPL is the ligand binding site and the N-terminal domain of thrombopoietin alone is sufficient for the binding . The extracellular portion of MPL was heterologously expressed in E . coli and its specific affinity with thrombopoietin was visualized in vitro by resonance mirror biosensor technique. J Mol Biol, 2000 Apr 7, 297(4), 947 - 59 Crystallization of the yeast MATalpha2/MCM1/DNA ternary complex: general methods and principles for protein/DNA cocrystallization; Tan S et al.; We describe our efforts to crystallize binary MCM1/DNA and ternary MATalpha2/MCM1/DNA complexes, including the unsuccessful attempts to crystallize MCM1/DNA complexes and the successful design of DNA crystal packing that resulted in high-resolution crystals of the MATalpha2/MCM1/DNA complex . We detail general procedures useful for preparing protein/DNA cocrystals, including improved methods for producing and purifying DNA-binding proteins and DNA fragments, for purifying protein/DNA complexes, and for controlling pH conditions during crystallization . We also describe the rational design of DNA for protein/DNA cocrystallization attempts, based on our analysis of how straight and bent DNA with single base-pair overhangs can pack end-to-end in a crystal . J Mol Biol, 2000 Apr 7, 297(4), 933 - 45 Molecular basis for the polyamine-ompF porin interactions: inhibitor and mutant studies; Iyer R et al.; By testing the sensitivity of Escherichia coli OmpF porin to various natural and synthetic polyamines of different lengths, charge and other molecular characteristics, we were able to identify the molecular properties required for compounds to act as inhibitors of OmpF in the nanomolar range . Inhibitors require at least two amine groups to be effective . For diamines, the optimum length of the hydrocarbon spacer was found to be of eight to ten methylene groups . Triamine molecules based on a 12-carbon motif were found to be more effective that spermidine, an eight-carbon trivalent derivative . But differences in inhibition efficiencies were also found for trivalent compounds depending on the relative position of the internal secondary amine group with respect to the terminal groups . Finally, quaternary ammonium derivatives had no effect, suggesting that the nature of the terminal amine is important for the interaction . From these observations, we deduce that inhibition efficiency in the nanomolar range requires a 12-carbon chain triamine with terminal primary amine groups and replacement of the eighth methylene by a secondary amine . The need for this type of molecular architecture suggests that inhibition is governed by interactions between specific amine groups and protein residues, and that this is not simply due to the accumulation of charges into the pore . Together with previous observations from site-directed mutagenesis studies and inspection of the crystal structure of OmpF, these results allowed us to propose three residues (D113, D121 and Y294) as putative sites of interaction between the channel and spermine . Alanine substitution at each of these three residues resulted in a loss of inhibition by spermine, while mutations of only D113 and D121 affected inhibition by spermidine . Based on these observations, we suggest a model for the molecular determinants involved in the porin-polyamine interaction . Biochemistry, 2000 Apr 4, 39(13), 3856 - 60 Site-directed spin-labeling of the catalytic sites yields insight into structural changes within the F0F1-ATP synthase of Escherichia coli; Kersten MV et al.; Electron spin resonance (ESR) spectroscopy using site-specific cysteine spin-labeling of the catalytic nucleotide binding sites of F(1)-ATPase was employed to investigate conformational changes within the nucleotide binding sites of the enzyme . Mutant Escherichia coli F(1) that had been modified at position beta-Y331C with a spin label showed almost normal catalytic activity and enabled us to study the effects of binding of different nucleotides and of the F(o) subunit b on the conformation of the catalytic binding sites . The ESR spectra of the spin-labeled, nucleotide-depleted F(1) indicate asymmetry within the sites as is expected from the structural models of the enzyme . Nucleotide binding to the enzyme clearly affects the conformation of the sites; the most pronounced feature upon nucleotide binding is the formation of catalytic site(s) in a very open conformation . Using the same beta-331 spin-labeled F(1) and a truncated form of F(o) subunit b, b(24)(-)(156), we found that binding of b(24)(-)(156) to spin-labeled F(1) significantly changes the conformation of the catalytic sites . In this paper we present data that for the first time directly show that a conformational binding change takes place upon binding of nucleotides to the nucleotide binding sites and that also show that binding of b(24)(-)(156) strongly affects the conformation of the catalytic sites, most likely by increasing the population of binding sites that are in the open conformation. Biochemistry, 2000 Apr 4, 39(13), 3827 - 34 Calcium binding properties of recombinant calcium binding protein 40, a major calcium binding protein of lower eukaryote Physarum polycephalum; Nakamura A et al.; Calcium binding protein 40 (CBP40) is a Ca(2+)-binding protein abundant in the plasmodia of Physarum polycephalum . CBP40 consists four EF-hand domains in the COOH-terminal half and a putative alpha-helix domain in the NH(2)-terminal half . We expressed recombinant proteins of CBP40 in Escherichia coli to investigate its Ca(2+)-binding properties . Recombinant proteins of CBP40 bound 4 mol of Ca(2+) with much higher affinity (pCa(1/2) = 6.5) than that of calmodulin . When residues 1-196 of the alpha-helix domain were deleted, the affinity for Ca(2+) decreased to pCa(1/2) = 4.6 . A chimeric calmodulin was generated by conjugating the alpha-helix domain of CBP40 with calmodulin . The affinity of Ca(2+) for the chimeric calmodulin was higher than that for calmodulin, suggesting that the alpha-helix domain is responsible for the high affinity of CBP40 for Ca(2+) . CBP40 forms large aggregates reversibly in a Ca(2+)-dependent manner . A mutant protein with a deletion of NH(2)-terminal 32 residues, however, could not aggregate, indicating the importance of these residues for the aggregation . The aggregation occurs above micromolar levels of Ca(2+) concentration, so it may only occur when CBP40 is secreted out of the plasmodial cells. Biochemistry, 2000 Apr 4, 39(13), 3817 - 26 Divalent metal binding properties of the methionyl aminopeptidase from Escherichia coli; D'souza VM et al.; The metal-binding properties of the methionyl aminopeptidase from Escherichia coli (MetAP) were investigated . Measurements of catalytic activity as a function of added Co(II) and Fe(II) revealed that maximal enzymatic activity is observed after the addition of only 1 equiv of divalent metal ion . Based on these studies, metal binding constants for the first metal binding event were found to be 0.3 +/- 0.2 microM and 0.2 +/- 0.2 microM for Co(II)- and Fe(II)-substituted MetAP, respectively . Binding of excess metal ions (>50 equiv) resulted in the loss of approximately 50% of the catalytic activity . Electronic absorption spectral titration of a 1 mM sample of MetAP with Co(II) provided a binding constant of 2.5 +/- 0.5 mM for the second metal binding site . Furthermore, the electronic absorption spectra of Co(II)-loaded MetAP indicated that both metal ions reside in a pentacoordinate geometry . Consistent with the absorption data, electron paramagnetic resonance (EPR) spectra of {CoCo(MetAP)} also indicated that the Co(II) geometries are not highly constrained, suggesting that each Co(II) ion in MetAP resides in a pentacoordinate geometry . EPR studies on {CoCo(MetAP)} also revealed that at pH 7.5 there is no significant spin-coupling between the two Co(II) ions, though a small proportion ( approximately 5%) of the sample exhibited detectable spin-spin interactions at pH values > 9.6 . EPR studies on {Fe(III)_(MetAP)} and {Fe(III)Fe(III)(MetAP)} also suggested no spin-coupling between the two metal ions . (1)H nuclear magnetic resonance (NMR) spectra of {Co(II)_(MetAP)} in both H(2)O and D(2)O buffer indicated that the first metal binding site contains the only active-site histidine residue, His171 . Mechanistic implications of the observed binding properties of divalent metal ions to the MetAP from E . coli are discussed. Biochemistry, 2000 Apr 4, 39(13), 3751 - 62 A three-step kinetic mechanism for peptide binding to MHC class II proteins; Joshi RV et al.; Peptide binding reactions of class II MHC proteins exhibit unusual kinetics, with extremely slow apparent rate constants for the overall association (<100 M(-)(1) s(-)(1)) and dissociation (<10(-)(5) s(-)(1)) processes . Various linear and branched pathways have been proposed to account for these data . Using fluorescence resonance energy transfer between tryptophan residues in the MHC peptide binding site and aminocoumarin-labeled peptides, we measured real-time kinetics of peptide binding to empty class II MHC proteins . Our experiments identified an obligate intermediate in the binding reaction . The observed kinetics were consistent with a binding mechanism that involves an initial bimolecular binding step followed by a slow unimolecular conformational change . The same mechanism is observed for different peptide antigens . In addition, we noted a reversible inactivation of the empty MHC protein that competes with productive binding . The implications of this kinetic mechanism for intracellular antigen presentation pathways are discussed. Biochemistry, 2000 Apr 4, 39(13), 3745 - 50 The glucose transporter of the Escherichia coli phosphotransferase system: linker insertion mutants and split variants; Beutler R et al.; The IICB(Glc) subunit of the glucose transporter acts by a mechanism which couples vectorial translocation with phosphorylation of the substrate . It contains 8 transmembrane segments connected by 4 periplasmic, 2 short, 1 long (80 residues), cytoplasmic loops and an independently folding cytoplasmic domain at the C-terminus . Random DNase I cleavage, EcoRI linker insertion, and screening for transport-active mutants afforded 12 variants with between 46% and 116% of wild-type sugar phosphorylation activity . They carried inserts of up to 29 residues and short deletions in periplasmic loops 1, 2, and 3, in the long cytoplasmic loop 3, and in the linker region between the membrane spanning IIC(Glc) and the cytoplasmic IIB(Glc) domains . Disruption of the gene at the sites of linker insertion decreased the expression level and diminished phosphotransferase activity to between 7% and 32% . IICB(Glc) with a discontinuity in the cytoplasmic loop was purified to homogeneity as a stable complex . It was active only if encoded by a dicistronic operon but not if encoded by two genes on two different replicons, suggesting that spatial proximity of the nascent polypeptide chains is important for folding and membrane assembly. Biochemistry, 2000 Apr 4, 39(13), 3690 - 8 Investigation of spectroscopic intermediates during copper-binding and TPQ formation in wild-type and active-site mutants of a copper-containing amine oxidase from yeast; Dove JE et al.; Copper amine oxidases possess the unusual ability to generate autocatalytically their organic cofactor, which is subsequently utilized in turnover . This cofactor, 2,4,5-trihydroxyphenylalanine quinone (TPQ), is formed within the active site of these enzymes by the oxidation of a single tyrosine residue . In vitro, copper(II) and oxygen are both necessary and sufficient for the conversion of tyrosine to TPQ . In this study, the biogenesis of TPQ has been characterized in an amine oxidase from Hansenula polymorpha expressed as the apo-enzyme in Escherichia coli . With the WT enzyme, optical absorbances which are copper or oxygen dependent are observed and characterized . Active-site mutants are used to investigate further the nature of these spectral species . Evidence is presented which suggests that tyrosine is activated for reaction with oxygen by liganding to Cu(II) . In the following paper in this issue {Schwartz, B., Dove, J . E., and Klinman, J . P . (2000) Biochemistry 39, 3699-3707}, the initial reaction of precursor protein with oxygen is characterized kinetically . Taken together, the available data suggest a mechanism for the oxidation of tyrosine to TPQ where the role of the copper is to activate substrate. Biochemistry, 2000 Apr 4, 39(13), 3656 - 65 Improving low-temperature catalysis in the hyperthermostable Pyrococcus furiosus beta-glucosidase CelB by directed evolution; Lebbink JH et al.; The beta-glucosidase from the hyperthermophilic archaeon Pyrococcus furiosus (CelB) is the most thermostable and thermoactive family 1 glycosylhydrolase described to date . To obtain more insight in the molecular determinants of adaptations to high temperatures and study the possibility of optimizing low-temperature activity of a hyperthermostable enzyme, we generated a library of random CelB mutants in Escherichia coli . This library was screened for increased activity on p-nitrophenyl-beta-D-glucopyranoside at room temperature . Multiple CelB variants were identified with up to 3-fold increased rates of hydrolysis of this aryl glucoside, and 10 of them were characterized in detail . Amino acid substitutions were identified in the active-site region, at subunit interfaces, at the enzyme surface, and buried in the interior of the monomers . Characterization of the mutants revealed that the increase in low-temperature activity was achieved in different ways, including altered substrate specificity and increased flexibility by an apparent overall destabilization of the enzyme . Kinetic characterization of the active-site mutants showed that in all cases the catalytic efficiency at 20 degrees C on p-nitrophenyl-beta-D-glucose, as well as on the disaccharide cellobiose, was increased up to 2-fold . In most cases, this was achieved at the expense of beta-galactosidase activity at 20 degrees C and total catalytic efficiency at 90 degrees C . Substrate specificity was found to be affected by many of the observed amino acid substitutions, of which only some are located in the vicinity of the active site . The largest effect on substrate specificity was observed with the CelB variant N415S that showed a 7.5-fold increase in the ratio of p-nitrophenyl-beta-D-glucopyranoside/p-nitrophenyl-beta-D-galactopyra noside hydrolysis . This asparagine at position 415 is predicted to interact with active-site residues that stabilize the hydroxyl group at the C4 position of the substrate, the conformation of which is equatorial in glucose-containing substrates and axial in galactose-containing substrates. Biochemistry, 2000 Apr 4, 39(13), 3647 - 55 Structural characterization of adenine nucleotides bound to Escherichia coli adenylate kinase . 2 . 31P and 13C relaxation measurements in the presence of cobalt(II) and manganese(II); Lin Y et al.; 13C spin-lattice relaxation rates have been measured for two complexes of Escherichia coli adenylate kinase (AKe), viz., AKe . {U-(13)C}ATP and AKe.{U-(13)C}AMP.GDP in the presence of the substituent activating paramagnetic cation Mn(II) for the purpose of determination of the enzyme-bound conformations of ATP and AMP . (GDP has been added to the AMP complex with the enzyme in order to hold the cation in the bound complex.) Measurements of relaxation times at three different (13)C frequencies, 181.0, 125.7, and 75.4 MHz, indicate that the relaxation times in the enzyme-nucleotide complexes with the paramagnetic cation are not exchange-limited; i.e . , they are larger than the effective lifetimes of cation binding to these complexes and are, therefore, dependent on the cation-(13)C distances . An analysis of the frequency-dependent relaxation data allowed all of the ten Mn(II)-(13)C distances to be determined in each of the complexes . Similar measurements of the (31)P relaxation rate made on AKe.ATP and AKe.AMP.GDP complexes in the presence of Co(II) as the activating cation yielded Co(II)-(31)P distances for each adenine nucleotide . These distances, together with the interproton distances determined previously from TRNOESY experiments {Lin, Y., and Nageswara Rao, B . D . (2000) Biochemistry 39, 3636-3646}, led to a complete characterization of both ATP and AMP conformations in AKe-bound complexes . These conformations differ significantly from the nucleotide conformations in crystals of AKe . AP(5)A and AKe.AMP.AMPPNP as determined by X-ray crystallography. Biochemistry, 2000 Apr 4, 39(13), 3636 - 46 Structural characterization of adenine nucleotides bound to Escherichia coli adenylate kinase . 1 . Adenosine conformations by proton two-dimensional transferred nuclear Overhauser effect spectroscopy; Lin Y et al.; Adenosine conformations of adenosine 5'-triphosphate (ATP) and adenosine 5'-monophosphate (AMP), and of an ATP analogue, adenylyl imidodiphosphate (AMPPNP), bound to Escherichia coliadenylate kinase (AKe) in the complexes of AKe.Mg(II)ATP, AKe.AMP.Mg(II)GDP, AKe . AMPPNP, and AKe.Mg(II)AMPPNP were determined by transferred two-dimensional nuclear Overhauser effect spectroscopy (TRNOESY) measurements and molecular dynamics simulations . The glycosidic torsion angles, chi, deduced for the adenine nucleotides in these complexes are 51 degrees, 37 degrees, 49 degrees, and 47 degrees, respectively, with an experimental error of about +/-5 degrees . These values are in general agreement with those previously measured for other ATP-utilizing enzymes, suggesting a possible common motif for adenosine recognition and binding . The pseudorotational phase angle, P, of the sugar puckers for the bound nucleotides varied between 50 degrees and 103 degrees . These solution-state conformations are significantly different from those in published data from X-ray crystallography . A computation of the ligand NOEs, made by using the program CORCEMA {Moseley, H . N . B., Curto, E . V., and Krishna, N . R . (1995) J . Magn . Reson . B108, 243-261} with the protein protons in the vicinity of nucleotide included, on the basis of the X-ray structure of the AKe.AMP.AMPPNP complex {Berry, M . B., Meador, B., Bilderback, T., Liang, P., Glaser, M., and Philips, G . N . , Jr . (1994) Proteins: Struct., Funct., Genet . 19, 183-198}, showed that polarization transfer to the protein protons does not produce significant errors in the structures determined by considering the ligand NOEs alone. Biochemistry, 2000 Apr 4, 39(13), 3525 - 32 Structural basis of cleavage by RNase H of hybrids of arabinonucleic acids and RNA; Minasov G et al.; The origins of the substrate specificity of Escherichia coli RNase H1 (termed RNase H here), an enzyme that hydrolyzes the RNA strand of DNA-RNA hybrids, are not understood at present . Although the enzyme binds double-stranded RNA, no cleavage occurs with such duplexes {Lima, W . F., and Crooke, S . T . (1997) Biochemistry 36, 390} . Therefore, the hybrid substrates may not adopt a canonical A-form geometry . Furthermore, RNase H is exquisitely sensitive to chemical modification of the DNA strands in hybrid duplexes . This is particularly relevant to the RNase H-dependent pathway of antisense action . Thus, only very few of the modifications currently being evaluated as antisense therapeutics are tolerated by the enzyme, among them phosphorothioate DNA (PS-DNA) . Recently, hybrids of RNA and arabinonucleic acid (ANA) as well as the 2'F-ANA analogue were shown to be substrates of RNase H {Damha, M . J., et al . (1998) J . Am . Chem . Soc . 120, 12976} . Using X-ray crystallography, we demonstrate here that ANA analogues, such as 2'F-ANA {Berger, I., et al . (1998) Nucleic Acids Res . 26, 2473} and {3.3.0}bicyclo-ANA (bc-ANA), may not be able to adopt sugar puckers that are compatible with pure A- or a B-form duplex geometries, but rather prefer the intermediate O4'-endo conformation . On the basis of the observed conformations of these ANA analogues in a DNA dodecamer duplex, we have modeled a duplex of an all-C3'-endo RNA strand and an all-O4'-endo 2'F-ANA strand . This duplex exhibits a minor groove width that is intermediate between that of A-form RNA and B-form DNA, a feature that may be exploited by the enzyme in differentiating between RNA duplexes and DNA-RNA hybrids . Therefore, the combination of the established structural and functional properties of ANA analogues helps settle existing controversies concerning the discrimination of substrates by RNase H . Knowlegde of the structure of an analogue that exhibits enhanced RNA affinity while not interfering with RNase H activity may prove helpful in the design of future antisense modifications. Scand J Immunol, 2000 Apr, 51(4), 392 - 9 CD14-dependent and independent pathways in lipopolysaccharide-induced activation of a murine B-cell line, CH12 . LX; Kimura S et al.; Using a lipopolysaccharide (LPS)-responsive murine B-cell line, CH12 . LX, we assessed the possible role of CD14 in LPS-induced activation of B cells . Flow cytometric analysis indicated that CH12.LX cells expressed the CD14 molecule with a lower intensity than did the macrophage cell line J774.1 . A reverse transcription-polymerase chain reaction and Northern blot analysis revealed low, but significant, levels of CD14 mRNA in CH12.LX cells, whose cDNA was identical to that of the mouse macrophage CD14 gene . After stimulation with LPS, CH12.LX cells proliferated, accompanied by up-regulations of CD14, transforming growth factor (TGF)-beta and interleukin (IL)-6 mRNA, and increased IgM and IgA secretion . In the absence of serum or with the addition of anti-CD14 monoclonal antibodies, however, LPS-stimulation induced neither the up-regulation of CD14 and TGF-beta mRNA nor an increase in IgA secretion . These findings indicate that CD14 expression is not restricted to myeloid cells, but is involved in some cellular activation events of murine B cells elicited by LPS . Furthermore, a CD14-independent pathway may also exist in the LPS-induced activation of B cells that leads to proliferation, IL-6 production and the enhancement of IgM (but not IgA) secretion. J Bacteriol, 2000 Apr, 182(8), 2285 - 91 Intrinsic polymerase activities of UmuD'(2)C and MucA'(2)B are responsible for their different mutagenic properties during bypass of a T-T cis-syn cyclobutane dimer; O'Grady PI et al.; In wild-type Escherichia coli, translesion replication is largely dependent upon the UmuD'(2)C complex (DNA polymerase V {polV}) or its plasmid-encoded homologs, such as MucA'(2)B . Interestingly, both the efficiency of translesion replication of a T-T cis-syn dimer and the spectra of mutations observed are different in Umu- and Muc-expressing strains . We have investigated whether the polIII core is responsible for these differences by measuring the frequency of dimer bypass, the error rate of bypass, and the resulting mutation spectrum in mutants carrying a deletion of dnaQ (epsilon subunit) or holE (theta subunit) or carrying the dnaQ allele mutD5, which is deficient in proofreading but is competent in the structural function of epsilon, or the dnaE antimutator allele spq-2 . The chromosomal copy of the umuDC operon was deleted in each strain, and the UmuDC, UmuD'C, MucAB, or MucA'B proteins were expressed from a low-copy-number plasmid . With only few exceptions, we found that the characteristically different mutation spectra resulting from Umu- and Muc-mediated bypass are maintained in all of the strains investigated, indicating that differences in the activity or structure of the polIII core are not responsible for the observed phenotype . We also demonstrate that the MucA'(2)B complex is more efficient in promoting translesion replication than the UmuD'(2)C proteins and show that, contrary to expectation, the T-T dimer is bypassed more accurately by MucA'(2)B than by UmuD'(2)C . These results are consistent with the view that in a wild-type cell, the polV-like enzymes are responsible for the spectra of mutations generated during translesion replication and that polIII may simply be required to fix the misincorporations as mutations by completing chromosomal replication . Our observations also show that the mutagenic properties of a lesion can depend strongly on the particular enzyme employed in bypass. J Bacteriol, 2000 Apr, 182(8), 2277 - 84 Characterization of a 12-kilodalton rhodanese encoded by glpE of Escherichia coli and its interaction with thioredoxin; Ray WK et al.; Rhodaneses catalyze the transfer of the sulfane sulfur from thiosulfate or thiosulfonates to thiophilic acceptors such as cyanide and dithiols . In this work, we define for the first time the gene, and hence the amino acid sequence, of a 12-kDa rhodanese from Escherichia coli . Well-characterized rhodaneses are comprised of two structurally similar ca . 15-kDa domains . Hence, it is thought that duplication of an ancestral rhodanese gene gave rise to the genes that encode the two-domain rhodaneses . The glpE gene, a member of the sn-glycerol 3-phosphate (glp) regulon of E . coli, encodes the 12-kDa rhodanese . As for other characterized rhodaneses, kinetic analysis revealed that catalysis by purified GlpE occurs by way of an enzyme-sulfur intermediate utilizing a double-displacement mechanism requiring an active-site cysteine . The K(m)s for SSO(3)(2-) and CN(-) were 78 and 17 mM, respectively . The apparent molecular mass of GlpE under nondenaturing conditions was 22.5 kDa, indicating that GlpE functions as a dimer . GlpE exhibited a k(cat) of 230 s(-1) . Thioredoxin 1 from E . coli, a small multifunctional dithiol protein, served as a sulfur acceptor substrate for GlpE with an apparent K(m) of 34 microM when thiosulfate was near its K(m), suggesting that thioredoxin 1 or related dithiol proteins could be physiological substrates for sulfurtransferases . The overall degree of amino acid sequence identity between GlpE and the active-site domain of mammalian rhodaneses is limited ( approximately 17%) . This work is significant because it begins to reveal the variation in amino acid sequences present in the sulfurtransferases . GlpE is the first among the 41 proteins in COG0607 (rhodanese-related sulfurtransferases) of the database Clusters of Orthologous Groups of proteins for which sulfurtransferase activity has been confirmed. J Bacteriol, 2000 Apr, 182(8), 2269 - 76 Construction and characterization of a recA mutant of Thiobacillus ferrooxidans by marker exchange mutagenesis; Liu Z et al.; To construct Thiobacillus ferrooxidans mutants by marker exchange mutagenesis, a genetic transfer system is required . The transfer of broad-host-range plasmids belonging to the incompatibility groups IncQ (pKT240 and pJRD215), IncP (pJB3Km1), and IncW (pUFR034) from Escherichia coli to two private T . ferrooxidans strains (BRGM1 and Tf-49) and to two collection strains (ATCC 33020 and ATCC 19859) by conjugation was analyzed . To knock out the T . ferrooxidans recA gene, a mobilizable suicide plasmid carrying the ATCC 33020 recA gene disrupted by a kanamycin resistance gene was transferred from E . coli to T . ferrooxidans ATCC 33020 by conjugation under the best conditions determined . The two kanamycin-resistant clones, which have retained the kanamycin-resistant phenotype after growth for several generations in nonselective medium, were shown to have the kanamycin resistance gene inserted within the recA gene, indicating that the recA::Omega-Km mutated allele was transferred from the suicide plasmid to the chromosome by homologous recombination . These mutants exhibited a slightly reduced growth rate and an increased sensitivity to UV and gamma irradiation compared to the wild-type strain . However, the T . ferrooxidans recA mutants are less sensitive to these physical DNA-damaging agents than the recA mutants described in other bacterial species, suggesting that RecA plays a minor role in DNA repair in T . ferrooxidans. J Bacteriol, 2000 Apr, 182(8), 2238 - 44 A second {2Fe-2S} ferredoxin from Sphingomonas sp . Strain RW1 can function as an electron donor for the dioxin dioxygenase; Armengaud J et al.; The first step in the degradation of dibenzofuran and dibenzo-p-dioxin by Sphingomonas sp . strain RW1 is carried out by dioxin dioxygenase (DxnA1A2), a ring-dihydroxylating enzyme . An open reading frame (fdx3) that could potentially specify a new ferredoxin has been identified downstream of dxnA1A2, a two-cistron gene (J . Armengaud, B . Happe, and K . N . Timmis, J . Bacteriol . 180:3954-3966, 1998) . In the present study, we report a biochemical analysis of Fdx3 produced in Escherichia coli . This third ferredoxin thus far identified in Sphingomonas sp . strain RW1 contained a putidaredoxin-type {2Fe-2S} cluster which was characterized by UV-visible absorption spectrophotometry and electron paramagnetic resonance spectroscopy . The midpoint redox potential of this ferredoxin (E'(0) = -247 +/- 10 mV versus normal hydrogen electrode at pH 8.0) is similar to that exhibited by Fdx1 (-245 mV), a homologous ferredoxin previously characterized in Sphingomonas sp . strain RW1 . In in vitro assays, Fdx3 can be reduced by RedA2 (a reductase similar to class I cytochrome P-450 reductases), previously isolated from Sphingomonas sp . strain RW1 . RedA2 exhibits a K(m) value of 3.2 +/- 0.3 microM for Fdx3 . In vivo coexpression of fdx3 and redA2 with dxnA1A2 confirmed that Fdx3 can serve as an electron donor for the dioxin dioxygenase. J Bacteriol, 2000 Apr, 182(8), 2218 - 29 Cellular responses to postsegregational killing by restriction-modification genes; Handa N et al.; Plasmids that carry one of several type II restriction modification gene complexes are known to show increased stability . The underlying mechanism was proposed to be the lethal attack by restriction enzyme at chromosomal recognition sites in cells that had lost the restriction modification gene complex . In order to examine bacterial responses to this postsegregational cell killing, we analyzed the cellular processes following loss of the EcoRI restriction modification gene complex carried by a temperature-sensitive plasmid in an Escherichia coli strain that is wild type with respect to DNA repair . A shift to the nonpermissive temperature