J Biol Chem, 1998 Apr 3, 273(14), 8447 - 53 Replication origin of the broad host range plasmid RK2 . Positioning of various motifs is critical for initiation of replication; Doran KS et al.; The 393-base pair minimal origin, oriV, of plasmid RK2 contains three iterated motifs essential for initiation of replication: consensus sequences for binding the bacterial DnaA protein, DnaA boxes, which have recently been shown to bind the DnaA protein; 17-base pair direct repeats, iterons, which bind the plasmid encoded replication protein, TrfA; and A + T-rich repeated sequences, 13-mers, which serve as the initial site of helix destabilization . To investigate how the organization of the RK2 origin contributes to the mechanism of replication initiation, mutations were introduced into the minimal origin which altered the sequence and/or spacing of each particular region relative to the rest of the origin . These altered origins were analyzed for replication activity in vivo and in vitro, for localized strand opening and for DnaB helicase mediated unwinding . Mutations in the region between the iterons and the 13-mers which altered the helical phase or the intrinsic DNA curvature prevented strand opening of the origin and consequently abolished replication activity . Insertions of more or less than one helical turn between the DnaA boxes and the iterons also inactivated the replication origin . In these mutants, however, strand opening appeared normal but the levels of DnaB helicase activity were substantially reduced . These results demonstrate that correct helical phasing and intrinsic DNA curvature are critical for the formation of an open complex and that the DnaA boxes must be on the correct side of the helix to load DnaB helicase. J Biol Chem, 1998 Apr 3, 273(14), 8419 - 24 The protein translocation apparatus contributes to determining the topology of an integral membrane protein in Escherichia coli; Prinz WA et al.; The assembly of integral membrane proteins is determined by features of these proteins and the protein translocation apparatus . We used alkaline phosphatase fusions to the membrane protein MalF to investigate the role of the protein translocation machinery in the arrangement of proteins in the cytoplasmic membrane of Escherichia coli . In particular, we studied the effects of prlA mutations on membrane protein topology . These mutations lie in the secY gene, which encodes a core component of the protein translocation apparatus . We find that the topology of some of the fusion proteins is changed and, in one case, is completely inverted in prlA mutants . We discuss the mechanism of prlA-mediated export and the role of the protein translocation apparatus in contributing to membrane protein topology. J Biol Chem, 1998 Apr 3, 273(14), 8294 - 300 Cooperative binding properties of restriction endonuclease EcoRII with DNA recognition sites; Reuter M et al.; EcoRII is a member of the expanding group of type IIe restriction endonucleases that share the distinguishing feature of requiring cooperativity between two recognition sites in their substrate DNA . To determine the stoichiometry of the active DNA-enzyme complex and the mode of cooperative interaction, we have investigated the dependence of EcoRII cleavage on the concentration of EcoRII dimers . Maximal restriction was observed at dimer/site ratios of 0.25 and 0 . 5 . The molecular weight of the DNA-enzyme complex eluted from a gel filtration column also corresponds to a dimeric enzyme structure bound to two substrate sites . We conclude that one EcoRII dimer is sufficient to interact cooperatively with two DNA recognition sites . A Lac repressor "barrier" bound between two normally reactive EcoRII sites did not inhibit restriction endonuclease activity, indicating that cooperativity between EcoRII sites is achieved by bending or looping of the intervening DNA stretch . Comparative cleavage of linear substrates with differently spaced interacting sites revealed an inverse correlation between cleavage rate and site distance . At the optimal distance of one helical turn, EcoRII cleavage is independent of the orientation of the recognition sequence in the DNA double strand. J Biol Chem, 1998 Apr 3, 273(14), 8193 - 202 Molecular cloning of human GDP-mannose 4,6-dehydratase and reconstitution of GDP-fucose biosynthesis in vitro; Sullivan FX et al.; We have cloned the cDNA encoding human GDP-mannose 4,6-dehydratase, the first enzyme in the pathway converting GDP-mannose to GDP-fucose . The message is expressed in all tissues and cell lines examined, and the cDNA complements Lec13, a Chinese Hamster Ovary cell line deficient in GDP-mannose 4,6-dehydratase activity . The human GDP-mannose 4,6-dehydratase polypeptide shares 61% identity with the enzyme from Escherichia coli, suggesting broad evolutionary conservation . Purified recombinant enzyme utilizes NADP+ as a cofactor and, like its E . coli counterpart, is inhibited by GDP-fucose, suggesting that this aspect of regulation is also conserved . We have isolated the product of the dehydratase reaction, GDP-4-keto-6-deoxymannose, and confirmed its structure by electrospray ionization-mass spectrometry and high field NMR . Using purified recombinant human GDP-mannose 4,6-dehydratase and FX protein (GDP-keto-6-deoxymannose 3,5-epimerase, 4-reductase), we show that the two proteins alone are sufficient to convert GDP-mannose to GDP-fucose in vitro . This unequivocally demonstrates that the epimerase and reductase activities are on a single polypeptide . Finally, we show that the two homologous enzymes from E . coli are sufficient to carry out the same enzymatic pathway in bacteria. J Biol Chem, 1998 Apr 3, 273(14), 7949 - 56 Thermodynamic evidence for conformational coupling between the B and C domains of the mannitol transporter of escherichia coli, enzyme IImtl; Meijberg W et al.; The transport across the cytoplasmic membrane and concomitant phosphorylation of mannitol in Escherichia coli is catalyzed by the mannitol-specific transport protein from the phosphoenolpyruvate-dependent phosphotransferase system, enzyme IImtl . Interactions between the cytoplasmic B and the membrane embedded C domain play an important role in the catalytic cycle of this enzyme, but the nature of this interaction is largely unknown . We have studied the thermodynamics of binding of (i) mannitol to enzyme IImtl, (ii) the substrate analog perseitol to enzyme IImtl, (iii) perseitol to phosphorylated enzyme IImtl, and (iv) mannitol to enzyme IImtl treated with trypsin to eliminate the cytoplasmic domains . Analysis of the heat capacity increment of these reactions showed that approximately 50-60 residues are involved in the binding of mannitol and perseitol, but far less in the phosphorylated state or after removal of the B domain . A model is proposed in which binding of mannitol leads to the formation of a contact interface between the two domains, either by folding of unstructured parts or by docking of preexisting surfaces, thus positioning the incoming mannitol close to the phosphorylation site on the B domain to facilitate the transfer of the phosphoryl group. J Biol Chem, 1998 Apr 3, 273(14), 7865 - 72 Molecular cloning, sequencing, and heterologous expression of the vaoA gene from Penicillium simplicissimum CBS 170.90 encoding vanillyl-alcohol oxidase; Benen JA et al.; The cDNA encoding vanillyl-alcohol oxidase (EC 1.1.3.7) was selected from a cDNA library constructed from mRNA isolated from Penicillium simplicissimum CBS 170.90 grown on veratryl alcohol by immunochemical screening . The vaoA-cDNA nucleotide sequence revealed an open reading frame of 1680 base pairs encoding a 560-amino acid protein with a deduced mass of 62,915 Da excluding the covalently bound FAD . The deduced primary structure shares 31% sequence identity with the 8alpha-(O-tyrosyl)-FAD containing subunit of the bacterial flavocytochrome p-cresol methyl hydroxylase . The vaoA gene was isolated from a P . simplicissimum genomic library constructed in lambdaEMBL3 using the vaoA-cDNA as a probe . Comparison of the nucleotide sequence of the vaoA gene with the cDNA nucleotide sequence demonstrated that the gene is interrupted by five short introns . Aspergillus niger NW156 prtF pyrA leuA cspA transformed with the pyrA containing plasmid and a plasmid harboring the complete vaoA gene including the promoter and terminator was able to produce vaoA mRNA and active vanillyl-alcohol oxidase when grown on veratryl alcohol and anisyl alcohol . A similar induction of the vaoA gene was found for P . simplicissimum, indicating that similar regulatory systems are involved in the induction of the vaoA gene in these fungi . Introduction of a consensus ribosome binding site, AGAAGGAG, in the vaoA-cDNA resulted in elevated expression levels of active vanillyl-alcohol oxidase from the lac promoter in Escherichia coli TG2 . The catalytic and spectral properties of the purified recombinant enzyme were indistinguishable from the native enzyme. J Biol Chem, 1998 Apr 3, 273(14), 7818 - 27 Introduction of a tryptophan reporter group into the ATP binding motif of the Escherichia coli UvrB protein for the study of nucleotide binding and conformational dynamics; Hildebrand EL et al.; The DNA-dependent ATPase activity of UvrB is required to support preincision steps in nucleotide excision repair in Escherichia coli . This activity is, however, cryptic . Elicited in nucleotide excision repair by association with the UvrA protein, it may also be unmasked by a specific proteolysis eliminating the C-terminal domain of UvrB (generating UvrB*) . We introduced fluorescent reporter groups (tryptophan replacing Phe47 or Asn51) into the ATP binding motif of UvrB, without significant alteration of behavior, to study both nucleotide binding and those conformational changes expected to be essential to function . The inserted tryptophans occupy moderately hydrophobic, although potentially heterogeneous, environments as evidenced by fluorescence emission and time-resolved decay characteristics, yet are accessible to the diffusible quencher acrylamide . Activation, via specific proteolysis, is accompanied by conformational change at the ATP binding site, with multiple changes in emission spectra and a greater shielding of the tryptophans from diffusible quencher . Titration of tryptophan fluorescence with ATP has revealed that, although catalytically incompetent, UvrB can bind ATP and bind with an affinity equal to that of the active UvrB* form (Kd of approximately 1 mM) . The ATP binding site of UvrB is therefore functional and accessible, suggesting that conformational change either brings amino acid residues into proper alignment for catalysis and/or enables response to effector DNA. J Biol Chem, 1998 Apr 3, 273(14), 7787 - 90 A single BIR domain of XIAP sufficient for inhibiting caspases; Takahashi R et al.; The inhibitor of apoptosis proteins (IAPs) constitute an evolutionarily conserved family of homologous proteins that suppress apoptosis induced by multiple stimuli . Some IAP family proteins, including XIAP, cIAP-1, and cIAP-2, can bind and directly inhibit selected caspases, a group of intracellular cell death proteases . These caspase-inhibiting IAP family proteins all contain three tandem BIR domains followed by a RING zinc finger domain . To determine the structural basis for caspase inhibition by XIAP, we analyzed the effects of various fragments of this IAP family protein on caspase activity in vitro and on apoptosis suppression in intact cells . The RING domain of XIAP failed to inhibit the activity of recombinant caspases-3 or -7, whereas a fragment of XIAP encompassing the three tandem BIR domains potently inhibited these caspases in vitro and blocked Fas (CD95)-induced apoptosis when expressed in cells . Further dissection of the XIAP protein demonstrated that only the second of the three BIR domains (BIR2) was capable of binding and inhibiting these caspases . The apparent inhibition constants (Ki) for BIR2-mediated inhibition of caspases-3 and -7 were 2-5 nM, indicating that this single BIR domain possesses potent anti-caspase activity . Expression of the BIR2 domain in cells also partially suppressed Fas-induced apoptosis and blocked cytochrome c-induced processing of caspase-9 in cytosolic extracts, whereas BIR1 and BIR3 did not . These findings identify BIR2 as the minimal caspase-inhibitory domain of XIAP and indicate that a single BIR domain can be sufficient for binding and inhibiting caspases. Arch Biochem Biophys, 1998 Apr 1, 352(1), 144 - 52 Overexpression, single-step purification, and site-directed mutagenetic analysis of casbene synthase; Huang K et al.; Casbene synthase is a diterpene cyclase isolated from castor bean (Ricinus communis L), which catalyzes the cyclization of geranylgeranyl diphosphate to form the phytoalexin casbene . We here report the overexpression of casbene synthase in Escherichia coli in soluble form using a thioredoxin fusion system . The amplified DNA by PCR carried on pCS7 was inserted into the expression vector pET32b(+) to form pCAS.2 . The resulting transformants of pCAS . 2/BL21(DE3) produced a thioredoxin casbene synthase fusion protein (20-30% of total soluble protein) when induced with isopropyl beta-d-thiogalactopyranoside at 20 degrees C . Recombinant casbene synthase was purified to homogeneity in a single step with a His-binding metal-affinity column . Casbene synthase has a conserved aspartate-rich region {amino acids 355-359 (DDTID)}, one cysteine, and three histidines with several prenyl transferases and terpene cyclases . Seven mutants were constructed by site-directed mutagenesis . The importance of Asp 355 and Asp 356 for catalysis was established by an increase in Km as well as a reduction in kcat in the corresponding glutamate mutants . These results indicate that the first and the second aspartate are involved in catalysis, while the third aspartate and the conserved cysteine and histidine residues selected for mutagenesis appear not to be involved in catalysis . Proc Natl Acad Sci U S A, 1998 Mar 31, 95(7), 3627 - 31 Regulation of the Escherichia coli water channel gene aqpZ; Calamita G et al.; Osmotic movement of water across bacterial cell membranes is postulated to be a homeostatic mechanism for maintaining cell turgor . The molecular water transporter remained elusive until discovery of the Escherichia coli water channel, AqpZ, however the regulation of the aqpZ gene expression and physiological function of the AqpZ protein are unknown . Northern analysis revealed a transcript of 0.7 kb, confirming the monocistronic nature of aqpZ . Regulatory studies performed with an aqpZ::lacZ low copy plasmid demonstrate enhanced expression during mid-logarithmic growth, and expression of the gene is dependent upon the extracellular osmolality, which increased in hypoosmotic environments but strongly reduced in hyperosmolar NaCl or KCl . While disruption of the chromosomal aqpZ is not lethal for E . coli, the colonies of the aqpZ knockout mutant are smaller than those of the parental wild-type strain . When cocultured with parental wild-type E . coli, the aqpZ knockout mutant exhibits markedly reduced colony formation when grown at 39 degrees C . Similarly, the aqpZ knockout mutant also exhibits greatly reduced colony formation when grown at low osmolality, but this phenotype is reversed by overexpression of AqpZ protein . These results implicate AqpZ as a participant in the adaptive response of E . coli to hypoosmotic environments and indicate a requirement for AqpZ by rapidly growing cells. Proc Natl Acad Sci U S A, 1998 Mar 31, 95(7), 3578 - 82 Oxidized, deaminated cytosines are a source of C --> T transitions in vivo; Kreutzer DA et al.; The most common base substitution arising from oxidative damage of DNA is a GC --> AT transition . In an effort to determine the oxidized lesion(s) that gives rise to this mutation, the mutagenicity of three oxidized cytosines, 5-hydroxycytosine, 5-hydroxyuracil, and uracil glycol, were investigated in Escherichia coli . An M13 viral genome was constructed to contain a single oxidized cytosine at a specific site . Replication in vivo of the single-stranded genomes yielded mutation frequencies of 0.05%, 83%, and 80% for 5-hydroxycytosine, 5-hydroxyuracil, and uracil glycol, respectively . The predominant mutation observed was C --> T . A model for C --> T oxidative mutagenesis is suggested in which initial cytosine oxidation is followed by deamination to a poorly repaired uracil derivative that is strongly miscoding during replication. Proc Natl Acad Sci U S A, 1998 Mar 31, 95(7), 3555 - 60 Epoxidation of olefins by cytochrome P450: evidence from site-specific mutagenesis for hydroperoxo-iron as an electrophilic oxidant; Vaz AD et al.; P450 cytochromes (P450) catalyze many types of oxidative reactions, including the conversion of olefinic substrates to epoxides by oxygen insertion . In some instances epoxidation leads to the formation of products of physiological importance from naturally occurring substrates, such as arachidonic acid, and to the toxicity, carcinogenicity, or teratogenicity of foreign compounds, including drugs . In the present mechanistic study, the rates of oxidation of model olefins were determined with N-terminal-truncated P450s 2B4 and 2E1 and their respective mutants in which the threonine believed to facilitate proton delivery to the active site was replaced by alanine . Styrene epoxidation, cyclohexene epoxidation and hydroxylation to give 1-cyclohexene-3-ol, and cis- or trans-butene epoxidation (without isomerization) and hydroxylation to give 2-butene-1-ol were all significantly decreased by the 2B4 T302A mutation . Reduced proton delivery in this mutant is believed to interfere with the activation of dioxygen to the oxenoid species, as shown earlier by decreased hydroxylation of several substrates and enhanced aldehyde deformylation via a presumed peroxo intermediate . Of particular interest, however, the T303A mutation of P450 2E1 resulted in enhanced epoxidation of all of the model olefins along with decreased allylic hydroxylation of cyclohexene and butene . These results and a comparison of the ratios of the rates of epoxidation and hydroxylation support the concept that two different species with electrophilic properties, hydroperoxo-iron (FeO2H)3+ and oxenoid-iron (FeO)3+, can effect olefin epoxidation . The ability of cytochrome P450 to use several different active oxidants generated from molecular oxygen may help account for the broad reaction specificity and variety of products formed by this versatile catalyst. Proc Natl Acad Sci U S A, 1998 Mar 31, 95(7), 3531 - 6 The domain organization of NaeI endonuclease: separation of binding and catalysis; Colandene JD et al.; NaeI is a remarkable type II restriction endonuclease . It must bind two recognition sequences to cleave DNA, forms a covalent protein-DNA intermediate, and is only 1 aa change away from topoisomerase and recombinase activity . The latter activities apparently derive from reactivation of a cryptic DNA ligase active site . Here, we demonstrate that NaeI has two protease-resistant domains, involving approximately the N-terminal and C-terminal halves of the protein, linked by a protease-accessible region of 30 aa . The domains were purified by cloning . The C-terminal domain was shown by gel mobility-shift assay to have approximately 8-fold lower DNA-binding ability than intact NaeI . Analytical ultracentrifugation showed this domain to be a monomer in solution . The N-terminal domain, which contains the catalytic region defined by random mutagenesis, did not bind DNA and was a mixture of different-sized complexes in solution implying that it mediates self-association . DNA greatly inhibited proteolysis of the linker region . The results identify the DNA-binding domain, imply that DNA cleavage and recognition are independent and separable, and lead us to speculate about a cleft-like structure for NaeI. Proc Natl Acad Sci U S A, 1998 Mar 31, 95(7), 3525 - 30 23S rRNA positions essential for tRNA binding in ribosomal functional sites; Bocchetta M et al.; rRNA plays an important role in function of peptidyl transferase, the catalytic center of the ribosome responsible for the peptide bond formation . Proper placement of the peptidyl transferase substrates, peptidyl-tRNA and aminoacyl-tRNA, is essential for catalysis of the transpeptidation reaction and protein synthesis . In this report, we define a small set of rRNA nucleotides that are most likely directly involved in binding of tRNA in the functional sites of the large ribosomal subunit . By binding biotinylated tRNA substrates to randomly modified large ribosomal subunits from Escherichia coli and capturing resulting complexes on the avidin resin, we identified four nucleotides in the large ribosomal subunit rRNA (positions G2252, A2451, U2506, and U2585) whose modifications prevent binding of a peptidyl-tRNA analog in the P site and one residue (U2555) whose modification interferes with transfer of peptidyl moiety to puromycin . These nucleotides represent a subset of positions protected by tRNA analogs from chemical modification and significantly narrow the number of 23S rRNA nucleotides that may be directly involved in tRNA binding in the ribosomal functional sites. Proc Natl Acad Sci U S A, 1998 Mar 31, 95(7), 3478 - 82 Thiolate ligands in metallothionein confer redox activity on zinc clusters; Maret W et al.; We postulate a novel and general mechanism in which the redox-active sulfur donor group of cyst(e)ine confers oxidoreductive characteristics on stable zinc sites in proteins . Thus, the present, an earlier, and accompanying manuscripts {Maret, W., Larsen, K . S . & Vallee, B . L . (1997) Proc . Natl . Acad . Sci . USA 94, 2233-2237; Jiang, L.-J., Maret, W . & Vallee, B . L . (1998) Proc . Natl . Acad . Sci . USA 95, 3483-3488; and Jacob, C., Maret, W . & Vallee, B . L . (1998) Proc . Natl . Acad . Sci . USA 95, 3489-3494} demonstrate that the interactive network featuring multiple zinc/sulfur bonds as found in the clusters of metallothionein (MT) constitutes a coordination unit critical for the concurrent oxidation of cysteine ligands and the ensuing release of zinc . The low position of MT (<-366 mV) on a scale of redox reagents allows its effective oxidation by relatively mild cellular oxidants, in particular disulfides . When MT is exposed to an excess of dithiodipyridine, all of its 20 cysteines are oxidized within 1 hr with the concomitant release of all 7 zinc atoms; similarly, the thiol/disulfide oxidoreductase DsbA reacts stoichiometrically with MT to release zinc . Zinc and sulfur ligands in the clusters are in a spatial arrangement that seemingly favors disulfide bond formation . Jointly, this and the above-mentioned manuscripts conclude that the control of cellular zinc distribution as a function of the energy state of the cell is the long sought role of MT . This specific MT function renders dubious the widely held belief that MT primarily scavenges radicals or detoxifies metals and is consistent with the frequent use of cysteine as a zinc ligand in proteins as a means of both tight and weak zinc binding of thiols and disulfides, respectively . Thus, we relate changes in the reducing power of the cell to the stability of the zinc/sulfur network in MT and the relative mobility of zinc and its control. Proc Natl Acad Sci U S A, 1998 Mar 31, 95(7), 3472 - 7 Structure and mechanism of a proline-specific aminopeptidase from Escherichia coli; Wilce MC et al.; The structure of the proline-specific aminopeptidase (EC 3.4.11.9) from Escherichia coli has been solved and refined for crystals of the native enzyme at a 2.0-A resolution, for a dipeptide-inhibited complex at 2.3-A resolution, and for a low-pH inactive form at 2.7-A resolution . The protein crystallizes as a tetramer, more correctly a dimer of dimers, at both high and low pH, consistent with observations from analytical ultracentrifuge studies that show that the protein is a tetramer under physiological conditions . The monomer folds into two domains . The active site, in the larger C-terminal domain, contains a dinuclear manganese center in which a bridging water molecule or hydroxide ion appears poised to act as the nucleophile in the attack on the scissile peptide bond of Xaa-Pro . The metal-binding residues are located in a single subunit, but the residues surrounding the active site are contributed by three subunits . The fold of the protein resembles that of creatine amidinohydrolase (creatinase, not a metalloenzyme) . The C-terminal catalytic domain is also similar to the single-domain enzyme methionine aminopeptidase that has a dinuclear cobalt center. Proc Natl Acad Sci U S A, 1998 Mar 31, 95(7), 3402 - 7 A single side chain prevents Escherichia coli DNA polymerase I (Klenow fragment) from incorporating ribonucleotides; Astatke M et al.; Although nucleic acid polymerases from different families show striking similarities in structure, they maintain stringent specificity for the sugar structure of the incoming nucleoside triphosphate . The Klenow fragment of E . coli DNA polymerase I selects its natural substrates, deoxynucleotides, over ribonucleotides by several thousand fold . Analysis of mutant Klenow fragment derivatives indicates that discrimination is provided by the Glu-710 side chain which sterically blocks the 2'-OH of an incoming rNTP . A nearby aromatic side chain, at position 762, plays an important role in constraining the nucleotide so that the Glu-710 "steric gate" can be fully effective . Even with the E710A mutation, which is extremely permissive for addition of a single ribonucleotide to a DNA primer, Klenow fragment does not efficiently synthesize pure RNA, indicating that additional barriers prevent the incorporation of successive ribonucleotides. J Mol Biol, 1998 Mar 6, 276(4), 819 - 27 A single mutation disrupts the pH-dependent dimerization of glycinamide ribonucleotide transformylase; Mullen CA et al.; Monomeric GART reversibly associates into a dimeric form as a function of decreasing solution pH . The transition is consistent with a three-proton transfer reaction with an apparent pKa near 7 . We now report that a single mutation, which replaces a glutamic acid at position 70 in the dimer interface with alanine (E70A), disrupts the pH-dependent dimerization of GART based on dynamic light scattering and gel filtration studies . A comparison of data obtained from UV-absorbance difference spectroscopy for both the wild-type and mutant forms of GART indicates that a tyrosine residue(s) undergoes a change in solvent exposure over the pH range 6.55 to 8.19 . A conformational change in tertiary structure that accompanies dimerization accounts for 60% of the observed optical difference, while the remaining 40% can be attributed to a pH-dependent process unrelated to dimerization . In addition, fluorescence studies of the mutant protein indicate that a pH-dependent change in tryptophan fluorescence exhibited by the wild-type protein is unrelated to quaternary structural changes and is likely a result of simple fluorescence quenching by nearby protonated histidine side-chains . Taken together, our results indicate that a single amino acid change at the dimer interface is sufficient to interrupt the highly specific, pH-dependent assembly reaction of GART, although pH-dependent conformational changes present in the wild-type protein also occur in E70A GART . This work is a first application of structure-based site-directed mutagenesis to the analysis of this pH-dependent assembly reaction . J Mol Biol, 1998 Mar 6, 276(4), 689 - 703 Structure of the Escherichia coli primase/single-strand DNA-binding protein/phage G4oric complex required for primer RNA synthesis; Sun W et al.; Escherichia coli primase/SSB/single-stranded phage G4oric is a simple system to study how primase interacts with DNA template to synthesize primer RNA for initiation of DNA replication . By a strategy of deletion analysis and antisense oligonucleotide protection on small single-stranded G4oric fragments, we have identified the DNA sequences required for binding primase and the critical location of single-strand DNA-binding (SSB) protein . Together with the previous data, we have defined the structure of the primase/SSB/G4oric priming complex . Two SSB tetramers bind to the G4oric secondary structure, which dictates the spacing of 3' and 5' bound adjacent SSB tetramers and leaves SSB-free regions on both sides of the stem-loop structure . Two primase molecules then bind separately to specific DNA sequences in the 3' and 5' SSB-free G4oric regions . Binding of the 3' SSB tetramer, upstream of the primer RNA initiation site, is also necessary for priming . The generation of a primase-recognition target by SSB phasing at DNA hairpin structures may be applicable to the binding of initiator proteins in other single-stranded DNA priming systems . Novel techniques used in this study include antisense oligonucleotide protection and RNA synthesis on an SSB-melted, double-stranded DNA template . Development, 1998 Mar, 125(5), 949 - 58 Functional analysis of eve stripe 2 enhancer evolution in Drosophila: rules governing conservation and change; Ludwig MZ et al.; Experimental investigations of eukaryotic enhancers suggest that multiple binding sites and trans-acting regulatory factors are often required for wild-type enhancer function . Genetic analysis of the stripe 2 enhancer of even-skipped (eve), an important developmental gene in Drosophila, provides support for this view . Given the importance of even-skipped expression in early Drosophila development, it might be predicted that many structural features of the stripe 2 enhancer will be evolutionarily conserved, including the DNA sequences of protein binding sites and the spacing between them . To test this hypothesis, we compared sequences of the stripe 2 enhancer between four species of Drosophila: D . melanogaster, D . yakuba, D . erecta and D . pseudoobscura . Our analysis revealed a large number of nucleotide substitutions in regulatory protein binding sites for bicoid, hunchback, Kruppel and giant, as well as a systematic change in the size of the enhancer . Some of the binding sites in D . melanogaster are either absent or modified in other species . One functionally important bicoid-binding site in D . melanogaster appears to be recently evolved . We, therefore, investigated possible functional consequences of sequence differences among these stripe 2 enhancers by P-element-mediated transformation . This analysis revealed that the eve stripe 2 enhancer from each of the four species drove reporter gene expression at the identical time and location in D . melanogaster embryos . Double staining of native eve protein and transgene mRNA in early embryos showed that the reporter gene mimicked native eve expression and, in every case, produced sharply defined stripes at the blastoderm stage that were coincident with eve stripe 2 protein . We argue that stripe 2 eve expression in Drosophila evolution can be viewed as being under constant stabilizing selection with respect to the location of the anterior and posterior borders of the stripe . We further hypothesize that the stripe 2 enhancer is functionally robust, so that its evolution may be governed by the fixation of both slightly deleterious and adaptive mutations in regulatory protein binding sites as well as in the spacing between binding sites . This view allows for a slow but continual turnover of functionally important changes in the stripe 2 enhancer. Neurochem Res, 1998 Jun, 23(6), 907 - 11 Mycobacterium leprae binds to a 25-kDa phosphorylated glycoprotein of human peripheral nerve; Suneetha LM et al.; Mycobacterium leprae, the causative agent of leprosy, specifically invades and destroys the peripheral nerve, which results in the main clinical manifestation of the disease . Little is known about the bacteria-nerve protein interaction . We show in the present work that M leprae binds to a 25 kDa glycoprotein from human peripheral nerve . This protein is phosphorylatable and it binds to lectins which have alpha-mannose specificity . This M leprae-protein interaction could be of importance in the pathogenesis of leprosy. Neurosci Lett, 1998 Mar 13, 244(2), 112 - 4 Are the capsaicin-sensitive structures of ventral medulla involved in the temperature response to endotoxin in rats? Koulchitsky SV. In chronic experiments on rats pretreated with bilateral microinjection of 25 nl 1% capsaicin to the caudal ventrolateral medulla under ketamine-xylazine-acepromazine anesthesia, an enhancement of the temperature response to intraperitoneal application of 3 microg/kg E . coli lipopolysaccharide as compared to animals who received vehicle to the caudal ventrolateral medulla was found . This is indicative of the involvement of the capsaicin-sensitive bulbar structures in thermoregulatory processes during endotoxemia. Neurosci Lett, 1998 Mar 13, 244(2), 81 - 4 Efficient mutagenesis of zebrafish by a DNA cross-linking agent; Ando H et al.; We developed a novel procedure for efficient mutagenesis of zebrafish using a DNA cross-linking agent 4,5',8-trimethylpsoralen (TMP), which is known to frequently induce small deletions in Escherichia coli and Caenorhabditis elegans . A specific-locus test and pilot screenings indicated that the TMP mutagenesis procedure was efficient . To confirm the successful mutagenesis by TMP, we characterized mutants with selective impairments in the nervous system . The no tectal neuron mutation hindered the development of the tectal neurons, while the edawakare mutation resulted in the enhancement of the extension and branching of the peripheral axons of trigeminal ganglion and Rohon-Beard sensory neurons . These results suggest that the TMP mutagenesis will provide an efficient method to isolate and characterize zebrafish mutants at molecular level. Arch Virol, 1998, 143(3), 467 - 74 Transient marker stabilisation: a general procedure to construct marker-free recombinant vaccinia virus; Scheiflinger F et al.; Recombinant vaccinia viruses based on the highly attenuated Modified Vaccinia Ankara (MVA) strain expressing HIV-1 antigen genes were constructed by a novel procedure involving the transient use of two marker genes . The selectable markers used, the Escherichia coli guanine phosphoribosyltransferase (gpt) and the beta-galactosidase (lacZ) genes, are not retained within the final recombinant virus . The transient marker stabilisation (TMS) procedure allows the generation of marker-free recombinant viruses in a series of simple plaque purification steps . HIV-1 gag pol genes were inserted into two loci of vaccinia MVA demonstrating the efficiency of the procedure. J Neurochem, 1998 May, 70(5), 1793 - 8 Molecular cloning and expression of a mouse brain cDNA encoding a novel protein target of calcyclin; Filipek A et al.; A protein target of mouse calcyclin, p30, which we call calcyclin-binding protein (CacyBP), was identified in mouse brain and Ehrlich ascites tumor (EAT) cells . The amino acid sequence of the CacyBP chymotryptic peptide was used to prepare synthetic oligonucleotides that served as a probe to screen the mouse brain cDNA library . A 1.4-kb positive clone was detected, isolated, and sequenced . The analyzed clone contains an open reading frame encoding a protein of a molecular mass of approximately 26 kDa . The nucleotide and predicted amino acid sequences indicate that CacyBP is a novel protein . The results obtained from northern blots show that the CacyBP gene is expressed predominantly in mouse brain and EAT cells . Using a pGEX vector the recombinant CacyBP was expressed in Escherichia coli, and its properties were analyzed . The recombinant protein interacts with calcyclin at a physiologically relevant range of Ca2+ in solution during affinity chromatography and on blots . Because CacyBP, like calcyclin, is present in the brain, the interaction of these two proteins might be involved in calcium signaling pathways in neuronal tissue. J Biotechnol, 1998 Feb 5, 60(1-2), 55 - 66 Estimation of biomass and specific growth rate in a recombinant Escherichia coli batch cultivation process using a chemical multisensor array; Bachinger T et al.; A chemical multisensor array is used in combination with an artificial neural network to estimate the biomass concentration and specific growth rate in a recombination Escherichia coli batch cultivation . It is shown that by providing sufficient information to the artificial neural network, an accuracy comparable to that of an established dry weight method can be achieved . The obtained prediction error (1 sigma) of 0.043 g l-1 for biomass compares well with the error of the dry weight method in this low biomass concentration range (0.1-3 g l-1) . The prediction for the specific growth rate is accurate during important parts of the cell growth (1 sigma = 0.025 h-1) . The results show that this non-invasive method is potentially useful for estimating biomass and specific growth rate on-line in bioprocesses. J Biotechnol, 1998 Feb 5, 60(1-2), 23 - 35 Enzymatic specificity and hydrolysis pattern of the catalytic domain of the xylanase Xynl from Rhodothermus marinus; Karlsson EN et al.; The catalytic domain of a xylanase from Rhodothermus marinus was produced in Escherichia coli . The catalytic domain belongs to glycosyl hydrolase family 10 . The produced protein has a 22-amino acid leader peptide followed by a 411-amino acid truncated xylanase . The molecular mass was 48 kDa and the recombinant xylanase had a pI of 4.9 . The pH and temperature optima for activity were determined to be 7.5 and 80 degrees C, respectively . At that temperature the enzyme had a half-life of 1 h 40 min . An addition of 1 mM calcium stabilized the activity of the enzyme at 80 degrees C . The xylanase had its highest specific activity on oat spelt xylan but was active also on other xylans and to a limited extent on some other polysaccharides (soluble glucans) . No exo- or endo-cellulase activity was observed . Hydrolysis of xylo-oligomers and oat spelt xylan was studied and the predominant products of hydrolysis were xylobiose and xylotriose . The enzyme was inactive on xylobiose, xylotriose and on the soluble fraction from oat spelt xylan . The R . marinus xylanase is shown to have a strong preference for internal linkages and is therefore classified as an endo-xylanase. Biosci Biotechnol Biochem, 1998 Mar, 62(3), 564 - 6 cDNA cloning, expression, and mutagenesis of scytalone dehydratase needed for pathogenicity of the rice blast fungus, Pyricularia oryzae; Motoyama T et al.; We purified scytalone dehydratase from the rice blast fungus, Pyricularia oryzae, and cloned its cDNA on the basis of its amino acid sequence . The deduced amino acid sequence was 62% identical to the scytalone dehydratase from Colletotrichum lagenarium . The expression of this gene was induced transcriptionally in the stationary phase when melanin synthesis occurs . We constructed a heterologous expression system for the enzyme in Escherichia coli, did deletion analysis with this system, and found that a C-terminal region is essential for the enzyme function. Biosci Biotechnol Biochem, 1998 Mar, 62(3), 448 - 52 Catalase catalyzes of peroxynitrite-mediated phenolic nitration; Kono Y et al.; Catalase catalyzed the peroxynitrite-mediated nitration of 4-hydroxyphenylacetic acid . The curve for the pH dependence of nitration was similar to that for the reaction between peroxynitrite and phenol . Cyanide, azide, and 3-amino-1,2,4-triazole inhibited the nitration in a dose-dependent way . When catalase was mixed with peroxynitrite, Compound I was detected as an intermediate . Because azide was an electron donor for the peroxidatic action of catalase, and because 3-amino-1,2,4-triazole inhibited catalase activity by binding with Compound I, peroxynitrite-mediated phenolic nitration was probably accompanied by Compound I formation . Both catalase and superoxide dismutase protected Escherichia coli from peroxynitrite toxicity. Mol Cells, 1998 Feb 28, 8(1), 96 - 100 Expression of C5 protein, the protein component of Escherichia coli RNase P, from the tac promoter; Park BH et al.; The accurate function of C5 protein, the protein component of Escherichia coli RNase P, is uncertain in vivo . A controllable expression system for C5 protein was constructed which can be used to investigate effects of C5 protein on various cellular functions including biosynthesis of RNase P in vivo . The semisynthetic rnpA gene encoding C5 protein was fused to the tac promoter of the pKK223-3 expression vector . This tac promoter expression system produced a high level of C5 protein upon induction with isopropyl-beta-D-thiogalacto-pyranoside . When the overexpressed C5 protein was purified and used for reconstitution of RNase P, the reconstituted enzyme was active . The N-terminal amino acid of the overexpressed C5 protein was leucine specified by the second codon of the rnpA gene . The more controllable expression system was constructed by introducing the lacIq gene into the vector sequence itself. Mol Cells, 1998 Feb 28, 8(1), 43 - 8 Characterization of protein interaction among subunits of protein kinase CKII in vivo and in vitro; Kim MS et al.; Protein kinase CKII (CKII) is a ubiquitous protein serine/threonine kinase . CKII usually exists in tetrameric complexes composed of two catalytic (CKII alpha and/or CKII alpha') and two regulatory (CKII beta) subunits . In the present study, using a combined in vivo and in vitro approach, we have investigated the role of CKII subunits in the formation of the tetrameric structure of CKII and the formation of the polymeric structure of CKII holoenzyme . Our in vivo experiments show that CKII beta interacts with either another CKII beta or CKII alpha and that CKII alpha does not interact with another CKII alpha (or CKII alpha') . Our in vitro experiments also show that CKII beta is able to associate with both CKII alpha and another CKII beta and that CKII alpha exists as a monomeric form in solution . These data indicate that CKII beta mediates the formation of a tetramer by both the dimerization of CKII beta and the interaction of CKII beta with CKII alpha . The results of this study also suggest that CKII beta may be involved in the formation of the polymeric structure of the CKII holoenzyme. Biochem Biophys Res Commun, 1998 Apr 17, 245(2), 478 - 82 Expression of a hepatitis C virus NS3 protease-NS4A fusion protein in Escherichia coli; Inoue H et al.; Both the NS3 protease and the NS4A protein are required for efficient cleavage of the nonstructural protein region of the hepatitis C virus polyprotein . The NS3 protease domain was fused at its C-terminal end with full length NS4A and expressed in Escherichia coli . This protein (NS3 delta-NS4A) was purified to apparent homogeneity after refolding from extracts recovered from inclusion bodies . During the expression and purification process, NS3 delta-NS4A was not auto-processed in either a cis or trans manner at NS3/NS4A junction site . When the kcat/K(m) values and thermostability of NS3 delta-NS4A were compared with those for maltose binding protein-NS3 fusion protein (MBP-NS3), which contains only NS3 region, the single-chain NS3 delta-NS4A showed enhanced proteolytic activities and thermostability. Biochem Biophys Res Commun, 1998 Apr 17, 245(2), 319 - 24 In vitro and in vivo potentiation of radiosensitivity of malignant gliomas by antisense inhibition of the RAD51 gene; Ohnishi T et al.; The mammalian RAD51 gene is a homologue of the yeast RAD51 and E . coli RecA genes, which are related to the repair of DNA double-strand breaks and are also involved in recombination repair and various SOS responses to DNA damage by gamma-irradiation and alkylating reagents . In this study, we investigated both in vitro and in vivo whether inhibition of the RAD51 gene by antisense oligonucleotides (ODNs) enhances the radiosensitivity of mouse malignant gliomas . A volume of 100 nM of RAD51 antisense ODNs inhibited the level of mRNA by more than 95% and reduced the protein expression by about 70% . Treatment of mouse 203G glioma cells with 100 nM of RAD51 antisense ODNs significantly enhanced the radiation-induced cell kill compared to control cells, and cells treated with sense or scrambled ODNs . When the glioma cells were implanted in the cisterna magna of mice followed by treatment with RAD51 antisense ODNs, the survival time of the mice was markedly prolonged compared to that of the untreated group (p < 0.001, logrank test) . In addition, the combination of antisense ODNs and irradiation extended the survival time of the glioma-bearing mice much longer than could be achieved with radiation alone (p < 0.0001, logrank test) . These results suggest that inhibition of RAD51 can be expected to serve as a novel potentiator for radiation therapy in malignant gliomas by inhibiting DNA double-strand break repair. Gen Comp Endocrinol, 1998 May, 110(2), 201 - 11 Rainbow trout glucocorticoid receptor overexpression in Escherichia coli: production of antibodies for western blotting and immunohistochemistry; Tujague M et al.; Fragments of cDNA that encode the N-terminal and DNA-binding domains (DBD) of the rainbow trout glucocorticoid receptor (rtGR) were expressed in Escherichia coli as fusion proteins with glutathione-S-transferase (GST) . The fusion proteins induced by IPTG could readily be detected as 45- and 40-kDa bands, respectively, in crude extracts, as well as in proteins purified on glutathione-agarose . These purified hybrid proteins were used to immunize rabbits . The antisera produced were tested for specificity by Western blot analysis using extracts from COS-1 cells transfected with an rtGR expression vector and from trout liver cells . The antisera raised against the DBD domain did not detect any bands on Western blots, even at low antiserum dilution . However, the purified DBD fusion protein specifically bound GRE-containing DNA fragments in gel-shift assays, and the retarded complexes were supershifted by these antibodies . The antisera raised against the N-terminal domain consistently detected two protein bands at 104 and 100 kDa in the two cell extracts and allowed specific immunohistochemical staining in fish brain and pituitary . For the first time in fish, these antibodies will allow analysis of GR expression in different cortisol target tissues. Anal Biochem, 1998 May 1, 258(2), 305 - 10 Detection of lectins using ligand blotting and polyacrylamide-type glycoconjugate probes; Kamemura K et al.; A sensitive and convenient method for detection of the carbohydrate-binding activity of lectins was established using the combination of blotting of lectins on polyvinylidene difluoride membranes, carbohydrate-conjugated biotinylated polyacrylamide-type probes (carbohydrate-bp probes), horseradish peroxidase-streptavidin, and detection by enhanced chemiluminescence of the enzyme reaction . This method was tested for detection of four plant lectins blotted on the membrane: concanavalin A was detectable down to 100 ng by mannose-bp probe, Ricinus communis agglutinin 120 to as low as 5 ng by N-acetyllactosamine-bp probe, soybean agglutinin to 1 microgram by beta-N-acetyl-D-galactosamine-bp probe, and wheat germ agglutinin to 5 ng by beta-N-acetyl-D-glucosamine-bp probe . All four lectins were detectable on an electroblotted membrane after SDS-polyacrylamide gel electrophoresis . This method was used to detect recombinant human galectin-3 in Escherichia coli cell lysates and mannan-binding protein in human serum . These results indicate that this method is widely applicable to convenient detection and characterization of lectins in crude samples. Mol Microbiol, 1998 Mar, 27(6), 1247 - 60 EspB and EspD require a specific chaperone for proper secretion from enteropathogenic Escherichia coli; Wainwright LA et al.; Enteropathogenic Escherichia coli uses a type III secretion apparatus to deliver proteins essential for pathogenesis to the host epithelium . Several proteins have been detected in culture supernatants of the prototype EPEC strain E2348/69 and three of these, EspA, EspB, and EspD, use type III machinery for export . Here, we report the identification and characterization of CesD, a protein required for proper EspB and EspD secretion . CesD shows sequence homology to chaperone proteins from other type III secretion pathways . Based on this, we hypothesize that CesD may function as a secretion chaperone in EPEC . A mutation in cesD abolished EspD secretion into culture supernatants and reduced the amount of secreted EspB, but had little effect on the amount of secreted EspA . The mutant strain was negative for both FAS and Tir phosphorylation, consistent with the previously described roles for EspB and EspD in EPEC pathogenesis . CesD was shown to interact with EspD but not EspB or EspA . CesD was detected in the bacterial cytosol, and, surprisingly, a substantial amount of the protein was also found to be associated with the inner membrane . Thus, although CesD has some attributes that are similar to other type III secretion chaperones, its membrane localization separates it from previously described members of this family. Mol Microbiol, 1998 Mar, 27(6), 1213 - 21 Synthesis of Escherichia coli O9a polysaccharide requires the participation of two domains of WbdA, a mannosyltransferase encoded within the wb* gene cluster; Kido N et al.; WbdA (previously MtfA) is one of the mannosyltransferases encoded within the Escherichia coli O9a wb* gene cluster . It is composed of two domains of similar size, connected by an alpha-helix chain . Elimination of the C-terminal half by transposon insertion or gene deletion caused synthesis of an altered structural O-polysaccharide consisting only of alpha-1,2-linked mannose . O9a polysaccharide synthesis was restored by the C-terminal half of WbdA in trans . No membrane incorporation of mannose from GDP mannose was observed in a strain carrying only the gene for truncated WbdA . For mannose incorporation, it was necessary to introduce both wbdB and wbdC genes into the strain . Therefore, it is likely that the N-terminal half of truncated WbdA synthesizes the altered O-polysaccharide together with other mannosyltransferases which participate in the initial reactions of the O9a polysaccharide synthesis . Both N- and C-terminal domains of WbdA are required for the synthesis of the complete E . coli O9a polysaccharide . The chi sequence location between the two domains and homology plot analyses of the wbdA and the WbdA protein suggested that the wbdA gene might have arisen by fusion of two independent genes. Mol Microbiol, 1998 Mar, 27(6), 1141 - 56 Influence of FIS on the transcription from closely spaced and non-overlapping divergent promoters for an aminoacyl-tRNA synthetase gene (gltX) and a tRNA operon (valU) in Escherichia coli; Champagne N et al.; The gltX gene, encoding the glutamyl-tRNA synthetase (GluRS), and the valU operon, whose transcripts contain three tRNAVal/UAC and one tRNALys/UUU, are adjacent and divergently transcribed . It is the only known case of adjacent genes encoding an aminoacyl-tRNA synthetase and a tRNA precursor in Escherichia coli . The gltX promoters (P1, P2 and P3) direct the synthesis of transcripts non-overlapping with and divergent from the one initiated at the valU promoter . We report that their promoter region (250 bp) contains three binding sites for the factor for inversion stimulation (FIS), centred at positions -71, -91 and -112 from the valU transcription initiation site, and that the destruction of any of these sites does not prevent the binding of FIS to the others . As FIS is one of the major positive regulators of stable RNA operons, we have studied its role on gltX and valU transcription . FIS stimulates valU transcription in vitro and about twofold in vivo during steady-state exponential growth . In contrast, gltX transcription is repressed by the presence of FIS in vitro and about twofold in vivo during growth acceleration when a decrease in GluRS concentration was observed . Under all conditions tested, most of the gltX transcripts start at the P3 promoter . Nested deletions of this regulatory region reveal that the FIS-dependent repression of the gltX-P3 promoter is abolished after the removal of the valU promoter, and is not altered by the additional removal of the FIS binding sites; moreover, in vivo transcription from gltX-P1 and/or gltX-P2 present on some of these regulatory region variants is modulated by the nature of the upstream region by FIS and is sometimes stronger than that from gltX-P3 . These results show that the strength and the site of gltX transcription initiation are influenced by the upstream region up to and including the valU promoter; furthermore, they indicate that although these adjacent genes are involved in the first step of protein biosynthesis and share cis and trans regulatory elements, their transcription is non-co-ordinate. Mol Microbiol, 1998 Mar, 27(6), 1119 - 27 Mutational analysis of the NH2-terminal arms of the trp repressor indicates a multifunctional domain; Mackintosh SG et al.; The NH2-terminal arms of the Escherichia coli trp repressor have been implicated in three functions: formation of repressor-operator complexes via association with non-operator DNA; stabilization of repressor oligomers bound to DNA; and oligomerization of the aporepressor in the absence of DNA . To begin to examine the structural aspects of the arms that are responsible for these varied activities, we generated an extensive set of deletion and substitution mutants and measured the activities of these mutants in vivo using reporter gene fusions . Deletion of any part of the arms resulted in a significant decrease in repressor activity at both the trp and the trpR operons . Positions 4, 5 and 6 were the most sensitive to missense changes . Most substitutions at these positions resulted in repressors with less than 5% of the activity of the wild-type trp repressor . A large percentage of the missense mutants were more active than the wild-type repressor in medium containing tryptophan and less active in medium without tryptophan . This phenotype can be explained in terms of altered oligomerization of both the repressor and the aporepressor . Also, nine super-repressor mutants, resulting from substitutions clustered at both ends of the arms, were found . Our results support the hypothesis that the NH2-terminal arm of the trp repressor is a multifunctional domain and reveal structural components likely to be involved in the various functions. RNA, 1998 Feb, 4(2), 231 - 8 Construction of an in vivo-regulated U6 snRNA transcription unit as a tool to study U6 function; Luukkonen BG et al.; U6 snRNA is the only spliceosomal snRNA transcribed by RNA polymerase III in yeast . We have constructed a regulated U6 snRNA transcription unit by introducing the binding site for the Escherichia coli lacI repressor protein in the U6 snRNA promoter . GAL-induced expression of lacI protein led to a decrease in U6 snRNA levels and blocked cell growth . lacI dissociation from the promoter, and consequent U6 snRNA transcription, could be induced by addition of IPTG and repression of lacI transcription . To test the usefulness of this system in studying spliceosomal U6 snRNA function, we conditionally expressed U6 snRNAs with a single base substitution in position A51 . We demonstrate that expression of the U6-A51 mutations confers a strong dominant negative phenotype as shown by severe reductions in growth rate . In these strains, splicing of endogenous pre-mRNAs was blocked before the second step. RNA, 1998 Feb, 4(2), 189 - 94 Mutational analysis of the donor substrate binding site of the ribosomal peptidyltransferase center; Saarma U et al.; Previous experiments have shown that the top of helix 90 of 23S rRNA is highly important for the ribosomal peptidyltransferase activity and might be part of the donor (P) site . Developing on these studies, mutations in the 23S rRNA at the highly conserved positions G2505, G2582, and G2583 were investigated . None of the mutations affected assembly, subunit association, or the capacity of tRNA binding to A and P sites . A "selective transpeptidation assay" revealed that the mutations specifically impaired peptide bond formation . Results with a modified "fragment" assay using the minimal donor substrate pA-fMet are consistent with a model where the nucleotides psiGG2582 form a binding pocket for C75 of the tRNA. Cancer Gene Ther, 1998 Mar-Apr, 5(2), 119 - 26 Transfection of primary tumor cells and tumor cell lines with plasmid DNA/lipid complexes; Stopeck AT et al.; Cancer vaccines that utilize genetically modified tumor cells require gene transfer methods capable of producing immunostimulatory doses of transgenes from fresh or short-term cultures of human tumor cells . Our studies optimize in vitro transfection of primary tumor cells using cationic lipids and a plasmid encoding the gene for human interleukin-2 (IL-2) . Established tumor cell lines produced 10- to 100-fold more IL-2 than did fresh or short-term tumor cultures as measured by enzyme-linked immunoabsorbent analysis . Importantly, transfection of primary tumor cells produced immunostimulatory levels of IL-2 as determined by increased thymidine incorporation by autologous peripheral blood mononuclear cells and lymphokine-activated killer cell activity . IL-2 secretion by tumor cells persisted for at least 30 days post-transfection and was unaffected by freeze thawing or irradiation to 8000 rads . Multiple solid tumor types were successfully transfected, but normal blood mononuclear cells and leukemic blasts were resistant to transfection . Enzyme-linked immunoabsorbent analysis of the amount of IL-2 secreted into the medium by transfected tumor cells correlated with the percentage of tumor cells expressing intracellular IL-2 as measured by flow cytometry . Plasmids utilizing a cytomegalovirus promoter yielded superior transfection efficiencies compared with plasmids containing a Rous sarcoma virus promoter . These results suggest that a clinical vaccine trial using autologous tumor cells genetically modified to secrete IL-2 is feasible in patients with solid tumors. Cancer Gene Ther, 1998 Mar-Apr, 5(2), 83 - 91 Phosphorylation and cytotoxicity of therapeutic nucleoside analogues: a comparison of alpha and gamma herpesvirus thymidine kinase suicide genes; Cazaux C et al.; Thymidine kinase (TK) genes from three alpha-herpesviruses (i.e., human herpes simplex type 1, varicella-zoster virus, equid herpesvirus 4) and two y-herpesviruses (i.e., Epstein-Barr virus and Saimiri herpesvirus 2) were cloned in expression vectors based on zeocin resistance by complementation of a TK-defective Escherichia coli strain . In vivo complementation of an appropriate yeast strain and in vitro enzymatic measurements demonstrated that all viral TKs possess a second phosphorylating activity corresponding to the thymidylate kinase function in contrast to the E coli TK, which is deprived of this activity . When expressed in an engineered E coli strain rendered resistant to purine and pyrimidine nucleoside analogs, the viral TKs sensitize host bacteria to 3'-azido-3'-deoxythymidine (AZT), 3'-deoxy-2',3'-didehydrothymidine (D4T), dideoxyinosine, or fluorodeoxyuridine (5-FUdR) . The extent of activation of all these analogs, in this bacterial assay, was found to be greatly superior for the two gamma-virus TKs, compared to the alpha-virus TKs, including the reference suicide gene, HSV1-TK . TK from the two gamma-Epstein-Barr and Saimiri 2 viruses were also found to be more efficient in sensitizing murine melanoma B16 tumor cells to pyrimide nucleoside analogs. Microbiol Immunol, 1998, 42(3), 195 - 202 Construction of the chimeric reverse transcriptase of simian immunodeficiency virus sensitive to nonnucleoside reverse transcriptase inhibitor; Isaka Y et al.; A number of structurally diverse compounds have been shown to be potent inhibitors of the DNA polymerase activity of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) . The compounds can be grouped into two broad classes; nucleoside analogs and nonnucleoside RT inhibitors . The nonnucleoside RT inhibitors are quite specific for HIV-1 RT but not human immunodeficiency virus type 2 (HIV-2) and simian immunodeficiency virus (SIV) RT . We have investigated the property of SIV/HIV-1 chimeric viruses in which portions of SIV(MAC) RT were exchanged with the corresponding domain of HIV-1 RT; amino acids 176-190, 176-383 and 176-495 of HIV-1 RT . The chimeric virus, which was substituted amino acids 176-190 of RT, had detectable RT activity, and this chimeric RT was sensitive to three nonnucleoside RT inhibitors {nevirapine, HEPT derivative (E-EBU-dM) and TIBO derivative (R82913)} . To further study this chimeric virus, we purified the chimeric RT enzyme expressed in Escherichia coli and determined its kinetic properties; the Km, and Vmax values, and the Ki value of HEPT derivative calculated for the DNA polymerase activity . This study reveals that amino acids 176-190 of SIV(MAC) RT were important for the enzymatic activity and the SIV/HIV-1 chimeric RT, which had amino acids 176-190 of HIV-1, was sensitive to the nonnucleoside RT inhibitor. Mol Hum Reprod, 1998 Mar, 4(3), 235 - 42 Expression of extracellular superoxide dismutase in the human male reproductive tract, detected using antisera raised against a recombinant protein; Williams K et al.; Mammalian spermatozoa are particularly susceptible to the deleterious effects of reactive oxygen species and lipid peroxidation, which ultimately lead to impaired fertility . A number of enzymes are present in the male reproductive tract which may play a role in preventing oxidative damage; in particular, the epididymis is the site of synthesis and secretion of large amounts of extracellular superoxide dismutase (eSOD) . In order to study the distribution of eSOD in the male reproductive tract, and distinguish it from other related superoxide dismutase isoenzymes (e.g . cytosolic SOD), polyclonal antisera have been raised against a recombinant human eSOD fusion protein, expressed in bacterial cells . This protein was expressed from a synthetic gene fragment, using preferred Escherichia coli codons, designed to overcome the problems associated with the high guanine+cytosine content of the natural human eSOD transcript . Using this antiserum, eSOD can be readily detected in a range of human reproductive tissues as well as in human seminal plasma . However, the presence of similar levels of eSOD in the seminal plasma of vasectomized men (probably of prostatic origin) precludes its use as a simple diagnostic indicator of eSOD activity levels in the epididymis. Int J Biochem Cell Biol, 1997 Dec, 29(12), 1475 - 83 The effect of a nested set of C-terminal substituted deletions on the function of the alpha subunit of Escherichia coli RNA polymerase; Thomas MS et al.; A genetic screen was devised to obtain plasmid-borne rpoA alleles exhibiting partial or no complementation of the chromosomal Escherichia coli rpoA341 allele responsible for a pleiotropic phenotype . Nine of the ten mutants obtained carried single base pair deletions within the 3' end of rpoA resulting in frameshifting into a 72 codon +1 orf extending from within codon L262 and terminating 16 bp downstream of the rpoA reading frame . These frameshifts give rise to a set of substituted alpha deletions that are all of the same size (334 aa) and carry segments of the Orf sequence replacing the alpha region from the C-terminus (residue 329) to various points between 272 > 319 . The in vivo properties of this nested set of nine C-terminal-substituted derivatives of the alpha subunit of RNA polymerase have been assessed in terms of their assembly and transcriptional proficiency . The results indicate: (i) replacement of as much as 42 C-terminal residues of the alpha subunit does not prevent formation of a transcriptionally proficient holoenzyme form of RNA polymerase capable of complementing rpoA112(Ts); (ii) the extreme C-terminal Orf region, like that of alpha itself, is exposed in holoenzyme; (iii) these substituted deletions are not commonly functional at class I activated promoters. FEMS Microbiol Lett, 1998 Apr 15, 161(2), 337 - 43 Isolation and heterologous expression of a gene encoding 4-hydroxyphenylpyruvate dioxygenase from the wheat leaf-spot pathogen, Mycosphaerella graminicola; Keon J et al.; We describe the isolation and sequence of a gene encoding 4-hydroxyphenylpyruvate dioxygenase (HPPD) (EC 1.13.11.27)) from the wheat leaf-spot fungal pathogen Mycosphaerella graminicola (Septoria tritici), that directs the synthesis of 2,5-dihydroxyphenylacetate (homogentisic acid, HGA) . The sequence of the deduced peptide showed homology to HPPDs from other organisms; the greatest identity was to a T-cell reactive protein, also identified as HPPD, from the human fungal pathogen Coccidioides immitis . As observed for HPPD from other sources, expression of the M . graminicola HPPD gene in Escherichia coli cells could be detected by the gradual development of a brown pigment in cultures as a result of the spontaneous oxidation and polymerisation of HGA . Pigment development in these cultures was prevented by the HPPD inhibitor sulcotrione. FEMS Microbiol Lett, 1998 Apr 15, 161(2), 325 - 30 Expression of membrane-associated proteins by strains of enteroaggregative Escherichia coli; Spencer J et al.; Certain strains of enteroaggregative Escherichia coli express an outer membrane-associated protein, involved with the adhesion of these bacteria to HEp-2 cells . Strains of enteroaggregative E . coli hybridising with DNA probes for aggregative adhesion, diffuse adhesion and aggregative adhesion fimbriac II expressed an outer membrane-associated protein of 18 kDa regulated by magnesium ions . Strains hybridising with the aggregative adhesion probe only expressed a 20-kDa outer membrane-associated protein regulated by calcium and magnesium . The present study describes two populations of enteroaggregative E . coli which appear to adhere to HEp-2 cells by expressing antigenically distinct, negatively charged membrane-associated proteins. Growth Factors, 1998, 15(3), 215 - 29 Purification and characterization of a recombinant human cripto-1 protein; Seno M et al.; Cripto-1 (CR-1) is a novel protein that contains a modified EGF-like motif and that does not directly bind to any of the known erb B type-1 receptor tyrosine kinase receptors . To more clearly define the biological effects of CR-1 and to more adequately compare the structure-function relationships of CR-1 with other members of the EGF family of growth factors, we have expressed a modified, full-length recombinant human CR-1 protein (rhCR-1) in E . coli and have devised a procedure for the solubilization, refolding and purification of a biologically active form of this protein . We have generated the mature form of hCR-1 from computer assisted predictions of potential signal peptide cleavage sites . Expression of the modified rhCR-1 protein in E . coli was limited to the inclusion bodies . The rhCR-1 protein was found to be expressed at high levels in bacterial cells when fused to a histidine-tag sequence . Refolding of rhCR-1 was found to be difficult because of the large number of cysteine residues in the protein which results in protein aggregation . By chemically modifying the cysteine residues in the rhCR-1 protein with 3-trimethylammoniopropyl methanethiosulfonate, additional positive charges have been introduced into the protein by this disulfiding reagent . This modification facilitates solubilization of the protein when rhCR-1 is denatured . The solubilized, denatured protein was then purified by CM cation exchange and C4 reverse phase HPLC chromatography and refolded in a redox buffer . The refolded, modified rhCR-1 protein was found to be biologically active by its ability to inhibit beta-casein expression, to stimulate the tyrosine phosphorylation of Shc and the activation of MAPK and by its capacity to facilitate branching growth of mouse mammary epithelial cells in type I collagen gels. J Med Microbiol, 1998 Apr, 47(4), 283 - 93 The 18th C.L . Oakley Lecture . Pathogenicity of enteropathogenic Escherichia coli; Baldwin TJ; Enteropathogenic Escherichia coli (EPEC) remain an important world-wide cause of diarrhoeal disease and mortality of infants and young children . Research programmes around the world have, in recent times, made enormous strides towards a better understanding of EPEC pathogenesis, yielding unique insights into the molecular intercourse between host and pathogen . Recombinant DNA and cell biology techniques have provided powerful tools, giving the first intriguing glimpses of a wealth of bacterial products mediating complex host:pathogen interactions involving the subversion of normal host signalling processes . Much has been discovered since 1945, when E . coli was first implicated as a cause of diarrhoea . However, many questions remain unanswered and many more remain unasked . Much remains to be discovered, especially in the area of molecular interactions between host and pathogen and how they relate to the manifestation of disease in the patient. Protein Sci, 1998 Apr, 7(4), 1057 - 60 Crystallization and preliminary X-ray analysis of a 1:1 complex between a designed monomeric interferon-gamma and its soluble receptor; Randal M et al.; A variant of human interferon-gamma (IFN-gamma) has been created in which the two chains of the homodimeric cytokine were linked N- to C-terminus by an eight residue polypeptide linker . The sequence of this linker was derived from a loop in bira bifunctional protein, and was determined from a structural database search . This "single-chain" variant was used to create an IFN-gamma molecule that binds only a single copy of the alpha-chain receptor, rather than the 2 alpha-chain receptor: 1 IFN-gamma binding stoichiometry observed for the native hormone . Crystals have been grown of a 1:1 complex between this single-chain molecule and the extracellular domain of its alpha-chain receptor . These crystals diffract beyond 2.0 A, significantly better than the 2.9 A observed for the native 2:1 complex . Density calculations suggest these crystals contain two complexes in the asymmetric unit; a self-rotation function confirms this conclusion. Protein Sci, 1998 Apr, 7(4), 961 - 5 A differential scanning calorimetric study of the thermal unfolding of apo- and holo-cytochrome b562; Robinson CR et al.; Cytochrome b562 is a four-helix-bundle protein containing a non-covalently bound b-type heme prosthetic group . In the absence of heme, cytochrome b562 remains highly structured under native conditions . Here we report thermodynamic data for the thermal denaturation of the holo- and apoproteins as determined by differential scanning calorimetry . Thermal denaturation of holocytochrome b562 is a highly reversible process, and unexpectedly does not involve dissociation of the heme prosthetic group . Thermal denaturation of the corresponding apoprotein, with the heme group chemically removed, remains a cooperative, reversible process . Apocytochrome b562 is substantially destabilized relative to the holoprotein: the t1/2 is more than ten degrees lower, and enthalpy and heat capacity changes are about one-half of the holoprotein values . However, the energetic parameters of apocytochrome b562 denaturation are within the range of observed values for small proteins. Mutagenesis, 1998 Mar, 13(2), 127 - 32 Mutation frequency decline in MMS-treated Escherichia coli K-12 mutS strains; Grzesiuk E et al.; It has been shown that the decline in mutant frequency (MFD) (argE3(ochre)-->Arg+) which occurs in MMS-treated and then transiently starved AB1157 Escherichia coli K-12 cells concerns revertants which arose by supL suppressor formation in a process which is umuDC dependent . Here we have examined whether MMS-induced Arg+ revertants are susceptible to decline when bacteria are deficient in mismatch repair . We show that there is an absence of MFD in MMS-treated M1 (mutS) and in EC2416 (mutS delta umuDC) cells defective in mismatch repair which is associated with a change in the spectrum of MMS-induced mutations formed . In contrast to AB1157, transformation of M1 (mutS) bacteria with plasmids harbouring various combinations of umuD(D')C genes does not enhance the level of MMS-induced mutations but may influence the proportion of supL mutations . These supL mutations show MFD . Repair processes under MFD conditions were confirmed by analysis of plasmid DNA isolated from MMS-treated bacteria at different stages of their starvation and digestion with Fpg protein. Mutagenesis, 1998 Mar, 13(2), 109 - 14 Spontaneous mutation in lacI transgenic mice: a comparison of tissues; de Boer JG et al.; The nature of spontaneous mutations in the lacI transgene of Big Blue mice was determined in selected tissues . The mutant frequencies ranged from 2.5 x 10(-5) to 7.1 x 10(-5) for liver, spleen, bladder, stomach, kidney, bone marrow, lung and skin . We also determined the DNA sequence alterations in the mutants recovered from these tissues . In all tissues the predominant class of mutations was G:C-->A:T transitions, most of which occurred at 5'-CpG-3' dinucleotide sequences . Bladder, kidney and skin display the highest contribution of G:C-->A:T transitions . The second most common class of mutations was G:C-->T:A transversions . All other base substitution classes contributed < 10% each . Of the non-substitution events, the loss of a single base pair was the most frequently occurring event (< 10%) . The similarity of mutational spectra (in terms of kinds of mutations detected by the lacI transgenic system) in all tissues examined supports the idea that similar mutational pathways function in these tissues in the absence of chemical or physical stimulus. Br Poult Sci, 1998 Mar, 39(1), 152 - 5 Changes in plasma alpha 1-acid glycoprotein concentration and selected immune response in broiler chickens injected with Escherichia coli lipopolysaccharide; Takahashi K et al.; 1 . Changes in plasma alpha 1-acid glycoprotein (AGP) concentration and immune responses following Escherichia coli lipopolysaccharide (LPS) injection were studied in broiler chickens . 2 . Higher plasma AGP concentrations were observed from 12 to 48 h after a single injection of LPS . 3 . The highest concentration of plasma AGP was observed on day 2 followed by a gradual decrease in chicks injected with 150 micrograms/kg body weight of LPS every day for 13 d . 4 . Plasma AGP concentration in chicks injected daily with LPS at 900 micrograms/kg body weight for 13 d increased on day 2, and decreased on day 4 to the concentration found before the injection . The concentration increased again on day 10 . 5 . Changes in plasma interleukin-1 (IL-1) like activity were similar to those in plasma AGP concentration when LPS was injected daily at 900 micrograms/kg body weight for 3 d . 6 . Responses of blood mononuclear cell (MNC) proliferation to mitogen or concanavalin A, (Con A), and pokeweed mitogen (PWM) were positively correlated with changes in plasma AGP concentration . 7 . The results suggest that plasma AGP concentration could be used as a positive indicator of changes in blood MNC proliferation to a mitogen and in plasma IL-1 like activity. Virology, 1998 Apr 10, 243(2), 482 - 91 DNA packaging by L1 and L2 capsid proteins of bovine papillomavirus type 1; Zhao KN et al.; Encapsidation of circular DNA by papillomavirus capsid protein was investigated in Cos-1 cells . Plasmids carrying both an SV40 origin of replication (ori) and an E . coli ori were introduced into Cos-1 cells by DNA transfection PV capsid proteins were supplied in trans by recombinant vaccinia viruses . Pseudovirions were purified from infected cells and their packaged DNA was extracted and used to transform E . coli as an indication of packaging efficacy . VLPs assembled from BPV-1 L1 alone packaged little plasmid DNA, whereas VLPs assembled from BPV-1 L1 + L2 packaged plasmid DNA at least 50 times more effectively . BPV-1 L1 + L2 VLPs packaged a plasmid containing BPV-1 sequence 8.2 +/- 3.1 times more effectively than a plasmid without BPV sequences . Using a series of plasmid constructs comprising a core BPV-1 sequence and spacer DNA it was demonstrated that BPV VLPs could accommodate a maximum of about 10.2 kb of plasmid DNA, and that longer closed circular DNA was truncated to produce less dense virions with shorter plasmid sequences . The present study suggests that packaging of genome within PV virions involves interaction of L2 protein with specific DNA sequences, and demonstrates that PV pseudovirions have the potential to be used as DNA delivery vectors for plasmids of up to 10.2 kb. Virology, 1998 Apr 10, 243(2), 423 - 31 Expression of human papillomavirus type 16 L1 protein in Escherichia coli: denaturation, renaturation, and self-assembly of virus-like particles in vitro; Zhang W et al.; Major capsid protein L1 of HPV16 was produced in a fused form in Escherichia coli using an inducible expression system . The protein formed insoluble aggregations (inclusion bodies) and the yield was more than 10% of total cell proteins . The inclusion bodies were isolated and solubilised with 8 M urea and the L1 proteins were purified by chromatographic separation . Following removal of the urea by gradual dialysis, the denatured L1 proteins spontaneously renatured and subsequently assembled into polymorphologic aggregations in vitro . Electron microscopy showed that the assembled material included structures resembling native empty capsids as well as incompletely formed capsids . After separation from the pool of polymorphologic structures by sucrose gradient sedimentation, the correctly formed virus-like particles (VLE . coliPs) were recognised by a HPV16 type-specific, conformational-dependent monoclonal antibody in an ELISA . This system offers not only a model for investigation of the intrinsic interactions that occur during L1 assembly, but also a potential route for convenient manufacture of highly purified VLP vaccines. Virology, 1998 Apr 10, 243(2), 275 - 82 Type specific and genotype cross reactive B epitopes of the L1 protein of HPV16 defined by a panel of monoclonal antibodies; Kulski JK et al.; Mouse monoclonal antibodies (mAbs) were raised against the major capsid protein, L1, of human papillomavirus type 16 (HPV16), produced in Escherichia coli with the expression plasmid pTrcL1 . Epitope specificity could be assigned to 11 of these 12 antibodies using a series of linear peptides and fusion proteins from HPV16 . One mAb (MC53) recognized a novel linear epitope that appears to be unique to the HPV16 genotype . A further 11 mAbs were characterized as recognizing novel and previously defined linear and conformational epitopes shared among more than one HPV genotype . The apparently genotype specific mAb could be useful for the development of diagnostic tests for vegetative virus infection in clinical specimens. Eur Surg Res, 1998, 30(2), 79 - 94 Treatment with growth hormone and insulin-like growth factor-1 in septicemia: effects on carbohydrate metabolism; Balteskard L et al.; Growth hormone (GH) and insulin-like growth factor-1 (IGF-1) may be beneficial against the protein catabolism seen in injury and septicemia . Further understanding of their effects on carbohydrate metabolism is needed . In a septic porcine model receiving total parenteral nutrition, pretreatment with GH or IGF-1 (or no treatment in controls) was followed by an infusion of live Escherichia coli bacteria . Endogenous glucose production, carbohydrate oxidation, glucose and lactate fluxes over the liver, gastrointestinal organs, kidney, and hindleg were determined . Endogenous glucose production increased during septicemia in the GH group . The metabolic acidosis induced by septicemia was augmented by GH, but attenuated by IGF-1 . The alanine and lactate levels were significantly higher in the GH- than in the IGF-1 treated animals during septicemia . IGF-1 pretreatment appeared to induce favorable effects while GH pretreatment might produce unfavorable effects on carbohydrate metabolism in septic piglets. Mol Cell Biol, 1998 May, 18(5), 3081 - 8 Selective disruption of genes transiently induced in differentiating mouse embryonic stem cells by using gene trap mutagenesis and site-specific recombination; Thorey IS et al.; A strategy employing gene trap mutagenesis and site-specific recombination (Cre/loxP) has been used to identify genes that are transiently expressed during early mouse development . Embryonic stem cells expressing a reporter plasmid that codes for neomycin phosphotransferase and Escherichia coli LacZ were infected with a retroviral gene trap vector (U3Cre) carrying coding sequences for Cre recombinase (Cre) in the U3 region . Activation of Cre expression from integrations into active genes resulted in a permanent switching between the two selectable marker genes and consequently the expression of beta-galactosidase (beta-Gal) . As a result, clones in which U3Cre had disrupted genes that were only transiently expressed could be selected . Moreover, U3Cre-activating cells acquired a cell autonomous marker that could be traced to cells and tissues of the developing embryo . Thus, when two of the clones with inducible U3Cre integrations were passaged in the germ line, they generated spatial patterns of beta-Gal expression. Mol Cell Biol, 1998 May, 18(5), 2721 - 8 Fission yeast rad12+ regulates cell cycle checkpoint control and is homologous to the Bloom's syndrome disease gene; Davey S et al.; The human BLM gene is a member of the Escherichia coli recQ helicase family, which includes the Saccharomyces cerevisiae SGS1 and human WRN genes . Defects in BLM are responsible for the human disease Bloom's syndrome, which is characterized in part by genomic instability and a high incidence of cancer . Here we describe the cloning of rad12+, which is the fission yeast homolog of BLM and is identical to the recently reported rhq1+ gene . We showed that rad12 null cells are sensitive to DNA damage induced by UV light and gamma radiation, as well as to the DNA synthesis inhibitor hydroxyurea . Overexpression of the wild-type rad12+ gene also leads to sensitivity to these agents and to defects associated with the loss of the S-phase and G2-phase checkpoint control . We showed genetically and biochemically that rad12+ acts upstream from rad9+, one of the fission yeast G2 checkpoint control genes, in regulating exit from the S-phase checkpoint . The physical chromosome segregation defects seen in rad12 null cells combined with the checkpoint regulation defect seen in the rad12+ overproducer implicate rad12+ as a key coupler of chromosomal integrity with cell cycle progression. Clin Exp Immunol, 1998 Apr, 112(1), 84 - 91 Recombinant human IL-16 inhibits HIV-1 replication and protects against activation-induced cell death (AICD); Idziorek T et al.; The chemoattractant cytokine IL- 16 has been reported to suppress lymphocyte activation and to inhibit HIV-1 replication in acutely infected T cells . We have cloned and expressed human IL-16 in Escherichia coli and investigated whether the recombinant protein could regulate the level of lymphocyte apoptosis from HIV-1-infected subjects . After purification and refolding, only 2-10% of the recombinant cytokine was present in a biologically active homotetrameric form . This could explain the need for high concentrations of the bacterially derived IL- 16 to induce significant inhibition of HIV-1 replication . Addition of IL-16 to unstimulated peripheral blood mononuclear cell (PBMC) cultures from HIV-1-infected subjects did not modify the observed level of spontaneous lymphocyte apoptosis . In contrast, IL-16 added to PBMC cultures stimulated with anti-CD3, anti-CD95 or dexamethasone reduced significantly the percentage of lymphocytes undergoing AICD . This effect was found to correlate with the ability of the cytokine to decrease CD95 expression on activated CD4+ T cells . Comparative studies on PBMC from healthy individuals indicated that the regulation of apoptosis levels by IL-16 is a complex phenomenon and could depend on the nature of the activator used and/or the immune status of lymphocytes tested . The outcome of CD4 cross-linking on T cells by various ligands is discussed in the context of the observed beneficial activities of IL- 16 and its potential role in the treatment of HIV disease. Chem Biol Interact, 1998 Feb 20, 109(1-3), 153 - 67 Redefining reductive sulfate assimilation in higher plants: a role for APS reductase, a new member of the thioredoxin superfamily? Wray JL, Campbell EI, Roberts MA, Gutierrez-Marcos JF. The reaction steps leading from the intermediate adenosine 5'-phosphosulfate (APS) to sulfide within the higher plant reductive sulfate assimilation pathway are the subject of controversy . Two pathways have been proposed: a 'bound intermediate' pathway in which the sulfo group of APS is first transferred by APS sulfotransferase to a carrier molecule to form a bound sulfite intermediate and is then further reduced by thiosulfonate reductase to bound sulfide; and a 'free intermediate' pathway in which APS is further activated to 3'-phosphoadenosine 5'-phosphosulfate (PAPS) by APS kinase followed by reduction of the sulfo group to free sulfite by PAPS reductase . Sulfite is then reduced to free sulfide by sulfite reductase . Sulfide, either free or bound, is then incorporated into organic form (as cysteine) by the enzyme O-acetylserine (thiol) lyase . In order to better characterize the pathway we attempted to clone PAPS reductase cDNAs by functional complementation of an Escherichia coli cysH mutant to prototrophy . We found no evidence for PAPS reductase cDNAs but did identify cDNAs that encode a small family of novel, chloroplast-localized proteins with APS reductase activity that are new members of the thioredoxin superfamily . We show here that the thioredoxin domain of these proteins is functional . We speculate that rather than proceeding via either of the pathways proposed above, reductive sulfate assimilation proceeds via the reduction of APS to sulfite by APS reductase and the subsequent reduction of sulfite to sulfide by sulfite reductase . In this scheme the product of the APS kinase reaction, PAPS, is not a direct intermediate in the pathway but rather acts as a substrate for sulfotransferase action and perhaps as a store of activated sulfate that can be returned to the pathway as APS via phosphohydrolase action on PAPS . Interactions between enzyme isoforms within the chloroplast stroma may bring about substrate channeling of APS and contribute to the partitioning of APS between sulfotransferase reactions on the one hand and the synthesis of cysteine and related metabolites via the reductive sulfate assimilation pathway on the other. Chem Biol Interact, 1998 Feb 20, 109(1-3), 129 - 35 Effects of 3'-phosphoadenosine 5'-phosphate on the activity and folding of phenol sulfotransferase; Yang YS et al.; Known spectroscopic and kinetic data are used to formulate pathways of the physiological and transfer reactions and the substrate inhibition of phenol sulfotransferase . Kinetic mechanisms indicate that release of PAP from enzyme complex is required for the physiological reaction but not for the transfer reaction . The pathways explain rate difference between the physiological and transfer reactions since the release of PAP is the rate-limiting step of the former reaction . Two enzyme species of phenol sulfotransferase which distinguish the physiological and transfer reaction were found to involve the binding of PAP . Differences between two forms of phenol sulfotransferase, alpha and beta, indicate that they assemble through different folding process . It is demonstrated that only alpha enzyme renatures in the presence of PAP and beta enzyme renatures only in the absence of PAP in vitro . In the over-expressed system, formation of alpha and beta phenol sulfotransferase is also dependent on the availability of PAP in Escherichia coli . It is concluded that folding of phenol sulfotransferase is assisted by PAP to form alpha enzyme . In the absence of PAP, beta form of phenol sulfotransferase is produced. Chem Biol Interact, 1998 Feb 20, 109(1-3), 43 - 52 Novel sulfotransferases cloned by RT-PCR: real proteins or PCR artifacts? Gaedigk A, Lekas P, Berchuk M, Grant DM. During studies designed to subclone human phenol sulfotransferase (STP and STM) sequences for use in heterologous E . coli-based expression systems, we designed two oligonucleotide primers that would allow for the simultaneous PCR amplification of expression cassettes containing the coding regions of the STP1, STP2 and STM cDNAs . Following total RNA isolation from human liver, reverse transcription of cDNA, PCR amplification under standard conditions, plasmid subcloning and restriction analysis to select for suitable ST recombinants, we recovered plasmids containing inserts corresponding to STP1, STP2 and STM . However, ten additional, closely related but apparently novel ST sequences were also isolated . Alignments of the three known ST sequences (and one published allelic variant) with these new clones revealed that each one appears to be a PCR-generated modular chimera possessing a combination of DNA segments derived from STP1, STP2 and STM . This observation should serve as an alert to the potential pitfalls of using PCR techniques for the cloning of highly related genes and their cDNA products, especially when PCR primer design allows for the amplification of multiple products in a single reaction. Mol Biochem Parasitol, 1998 Mar 15, 91(2), 237 - 49 Identification and heterologous expression of a new dense granule protein (GRA7) from Toxoplasma gondii; Jacobs D et al.; Immunoscreening of an expression library constructed with Toxoplasma gondii tachyzoite mRNA with sera from toxoplasmosis-positive humans has led to the identification of a new parasite antigen . Sequence analysis of the gene encoding this antigen allowed the calculation of the theoretical molecular mass (25,857 Da) and showed that the protein contains a putative signal sequence . The C-terminal region contains two hydrophobic regions, the last of which has the characteristics of a membrane-spanning domain . When the protein was heterologously expressed in E . coli and tested by Western blot, it reacted with the human sera originally used for screening . The new antigen also reacted with a monoclonal antibody raised against the entire parasite . Ultrastructural analysis showed that the protein is localized in the dense granules . After host cell invasion, the protein is secreted into the vacuolar network, the parasitophorous vacuole membrane, and into extensions protruding in the cytoplasm . Therefore, it is suggested to designate this new dense granule protein GRA7, following the established nomenclature for this protein family. Mol Biochem Parasitol, 1998 Mar 15, 91(2), 221 - 35 Thioredoxin peroxidase from Onchocerca volvulus: a major hydrogen peroxide detoxifying enzyme in filarial parasites; Lu W et al.; Random screening of an Onchocerca volvulus third-stage (L3) cDNA library identified a highly abundant cDNA encoding a newly discovered antioxidant enzyme, thioredoxin peroxidase (TPx), a member of the peroxidoxin superfamily . This TPx cDNA (Ov-tpx-2) encodes a polypeptide of 199 amino acid residues with a calculated molecular weight of 21,890 Da . The Ov-tpx-2 cDNA represents roughly 2.5% of the total cDNAs from the L3 cDNA library . The gene was expressed in Escherichia coli and the protein product was shown to have antioxidant activity . Antiserum raised against Ov-TPX-2 recognized a native protein from extracts of both the L3 and adult-stages with a molecular weight of 22 kD . The localization and stage-specificity of Ov-TPX-2 protein was analyzed by immunocytochemistry and immunoelectron microscopy using monospecific antibodies . Expression was detected in late first-stage larvae during development in the vector and increased in intensity during differentiation to the infective L3-stage . The antigen was also detected in post-infective larvae and adult worms . In larvae, Ov-TPX-2 protein was predominantly localized to the hypodermis and cuticle, with additional sites in the hypodermal chords and multivesicular bodies . In adult worms, the primary sites of expression were the uterine epithelium and intestine, with additional labeling of the body wall and cuticle . Developing embryos and microfilariae in utero were bathed in Ov-TPX-2 protein discharged from epithelial cells . These results suggest that Ov-TPX-2 may protect the parasites from being damaged by host-generated oxidative stress and that Ov-TPX-2 protein provides the H2O2-detoxifying activity predicted but not previously identified in filarial parasites . Its highly upregulated expression in infective larvae may aid in parasite establishment following transmission to the definitive host. J Biomol NMR, 1998 Jan, 11(1), 97 - 102 An efficient and cost-effective isotope labeling protocol for proteins expressed in Escherichia coli; Cai M et al.; A cost-effective protocol for uniform 15N and/or 13C isotope labeling of bacterially expressed proteins is presented . Unlike most standard protocols, cells are initially grown in a medium containing nutrients at natural abundance and isotopically labeled nutrients are only supplied at the later stages of growth and during protein expression . This permits the accumulation of a large cell mass without the need to employ expensive isotopically labeled nutrients . The abrupt decrease in oxygen consumption that occurs upon complete exhaustion of essential nutrients is used to precisely time the switch between unlabeled and labeled nutrients . Application of the protocol is demonstrated for wild-type and a mutant of the N-terminal zinc-binding domain of HIV-1 integrase. J Biomol NMR, 1998 Jan, 11(1), 17 - 29 A NOESY-HSQC simulation program, SPIRIT; Zhu L et al.; A program SPIRIT (Simulation Program considering Incomplete Recovery of z magnetization and INEPT Transfer efficiency) has been developed to simulate three-dimensional NOESY-HSQC spectra . This program takes into account (1) different transfer efficiency during INEPT and reverse INEPT durations due to differential relaxation rates and 1J coupling constants; (2) the different effect of the sensitivity-enhancement scheme on CH, CH2 and CH3 systems; and (3) incomplete recovery of longitudinal magnetization between scans . The simulation program incorporates anisotropic tumbling mode for symmetric tops, and allows for differential external relaxation rates for protons . Some well-defined internal motions, such as the fast rotation of methyl groups, are taken into account . The simulation program also allows for input of multiple conformations and their relative populations to calculate the average relaxation matrix to account for slow internal motions . With the SPIRIT program, the sensitivity-enhanced NOESY-HSQC experiment can be used directly in the evaluation of the accuracy of structures, which can potentially be improved by direct refinement against the primary data. Prikl Biokhim Mikrobiol, 1998 Jan-Feb, 34(1), 120 - 6 {Preparation of lymphotoxin and properties of it}; Lebedev LR et al.; The process of isolation of highly purified human lymphotoxin was studied and optimized . A set of methods providing an increase in the content of the target product in the biomass (plasmid DNA amplification and selection of clones of transformed cells) was applied at the stage of cultivation . A two-step purification scheme (ion-exchange chromatography on DEAE-Biotone A1 and hydroxylapatite) was developed . A number of physical characteristics were studied. Mem Inst Oswaldo Cruz, 1997 Sep-Oct, 92(5), 637 - 41 Expression of recombinant antigens in Escherichia coli: application on immunochemical studies of Schistosoma mansoni tegumental antigens; Abath FG et al.; Sm15 and Sm13 are recognized by antibodies from mice protectively vaccinated with tegumental membranes, suggesting a potential role in protective immunity . In order to raise antibodies for immunochemical investigations, the genes for these antigens were expressed in pGEX and pMal vectors so that comparisons could be made among different expression systems and different genes . The fusion proteins corresponding to several parts of the gene for the precursor of Sm15 failed in producing antibodies recognizing the parasite counterpart . On the other hand, antibodies raised against Sm13 MBP-fusion proteins recognized the 13 kDa tegumental protein . Thus the peculiarities of the gene of interest are important and the choice of the expression system must sometimes be decided on an empirical basis. J Mol Biol, 1998 Mar 13, 276(5), 913 - 25 Molecular genetic analysis of transposase-end DNA sequence recognition: cooperativity of three adjacent base-pairs in specific interaction with a mutant Tn5 transposase; Zhou M et al.; Transposition of Tn5 and IS50 requires the specific binding of transposase (Tnp) to the end inverted repeats, the outside end (OE) and the inside end (IE) . OE and IE have 12 identical base-pairs and seven non-identical base-pairs . Previously we described the isolation of a Tnp mutant, EK54, that shows an altered preference for OE versus IE compared to wild-type (wt) Tnp . EK54 enhances OE recognition and decreases IE recognition both in DNA binding and in overall transposition . Here we report that base-pairs 10, 11 and 12 of the OE are critical for the specific recognition by EK54 Tnp . These three adjacent base-pairs act cooperatively; all three must be present in order for EK54 Tnp to interact very favorably with the end DNA . The existence of only one or two of these three base-pairs decreases binding of EK54 Tnp . The combined use of EK54 Tnp and a new OE/IE mosaic end sequence containing the OE base-pairs 10, 11 and 12 gives rise to an extraordinarily high transposition frequency . Just as the Tnp is a multifunctional protein, the nucleotides in the 19 bp Tn5 ends also affect other functions besides Tnp binding . Furthermore, the fact that we were able to isolate end sequence variants that transpose at a higher frequency than the natural ends (OE and IE) with wt Tnp reveals yet another way in which the wt transposition frequency is depressed, i.e . by keeping the end sequences suboptimal. J Mol Biol, 1998 Mar 13, 276(5), 861 - 75 On the mechanism of inhibition of phage T7 RNA polymerase by lac repressor; Lopez PJ et al.; We study here the effect on phage T7 RNA polymerase activity of lac repressor bound downstream of the T7 promoter . When repressor binds in vitro at an operator centered at +13 or +15 with respect to transcription start, it does not prevent initiation, though the transcript yield is reduced . However, the processivity of the polymerase is depressed and transcript extension is blocked at positions +4 and +6, respectively . These results indicate that repressor and polymerase do not simply exclude each other from the promoter . Rather, they would come into steric conflict and compete for establishment or retention of interactions with the same segment of DNA, without this leading to the immediate displacement of either polymerase or repressor . The resulting destabilization of the transcription complex would depress both initiation rate and enzyme processivity . In contrast to the above results, little reduction in runoff transcription is observed when operator is centered at +47 . The decreased sensitivity of polymerase to repressor bound at +47 versus +13 or +15 is likely to be due to the higher stability of the elongation complex during the transcription of downstream regions in comparison with the first transcribed nucleotides . We also show that under conditions of leaky repression and with operator centered at +13, a mutant T7 RNA polymerase showing normal promoter affinity but a slower elongation rate is more sensitive to repression than the wild-type enzyme, both in vitro and in vivo . In vitro, this higher sensitivity is largely due to a reduced ability of the mutant to overcome the elongation block at position +4 . The parallel between the in vitro and in vivo data suggests that in vivo the repressor also does not prevent polymerase from binding to promoter, but interferes with subsequent steps in initiation and transcript extension, in this case presumably largely extension beyond +4. Fold Des, 1998, 3(2), 87 - 93 Coupling protein stability and protein function in Escherichia coli CspA; Hillier BJ et al.; BACKGROUND: CspA is a small protein that binds single-stranded RNA and DNA . The binding site of CspA consists of a cluster of aromatic amino acids, which form an unusually large nonpolar patch on the surface of the protein . Because nonpolar residues are generally found in the interiors of proteins, this cluster may have evolved to bind nucleic acids at the expense of protein stability . RESULTS: Three neighboring phenylalanines have been mutated singly and in combination to leucine and to serine . All mutations adversely affect DNA binding . Surprisingly, all mutations, and especially those to serine, are destabilizing . CONCLUSIONS: The aromatic cluster in CspA is required not only for protein function but also for protein stability . This result is pertinent to the design of beta-sheet proteins and single-stranded nucleic acid binding proteins, whose binding mode is proposed to be of aromatic-aromatic intercalation. J Chromatogr A, 1998 Mar 6, 798(1-2), 117 - 23 High-performance liquid chromatographic peak identification of 2,4-dinitrophenylhydrazine derivatives of lipid peroxidation aldehydes by photodiode array detection; Cordis GA et al.; Malonaldehyde (MDA), a product of lipid peroxidation, is a presumptive marker for the development of oxidative stress in tissues and plasmas . In this study we report the photodiode array detection of the 2,4-dinitrophenylhydrazine (DNPH) derivatives of MDA using HPLC . Oxidative stress was produced by injecting (i.p.) bacterial lipopolysaccharide (LPS) into rats at a dose of 100 micrograms/kg, or i.v . into rabbits (1 microgram/kg), or added to freshly drawn human blood (200 ng/ml) . Blood was collected at several time points up to 5 h, centrifuged, and equal volumes of 20% TCA were used to precipitate proteins from the plasma . The supernatants were derivatized with DNPH, and the aldehyde-DNPHs were extracted with pentane . After evaporation, aliquots of 10 microliters in acetonitrile were injected onto a Beckman Ultrasphere C18 (3 microns) column, chromatographed with an acetonitrile-water-acetic acid gradient mobile phase and scanned using Waters 996 photodiode array detector . Peak identification and homogeneity was determined by comparing the experimental peaks and UV scans with those of authentic standards . A significant increase in the DNPH derivative of malonaldehyde (MDA-DNPH), but not of the other aldehyde-DNPH derivatives of formaldehyde (FDA), acetaldehyde (ADA), acetone and propionaldehyde (PDA) was seen over the first hour after LPS administration in anesthetized rats, while in conscious rabbits this trend lasted up to 3 h . The retention times as well as the UV scans of the derivatized aldehydes matched the authentic standards . Thus, photodiode array detection has proved valuable in establishing this HPLC method for estimating oxidative stress . This technique could accurately measure pmol amounts of MDA-DNPH indicating the usefulness of photodiode array detection method for estimating small changes in the oxidative stress. J Chromatogr A, 1998 Mar 6, 798(1-2), 73 - 82 Accelerated recombinant protein purification process development automated, robotics-based integration of chromatographic purification and analysis; Londo T et al.; Recovery of recombinant proteins from endogenous, host molecules can be an experimentally intensive and time-consuming task . Often the time to analyze material during development of recovery procedures is the rate-limiting step . Nowadays, modern techniques and equipment are being specifically engineered to make this effort much more efficient . We present a case study to illustrate how a new, automation tool, designed for easy, systematic methods development, can be used for very rapid process and analytical optimization . This tool uses robotics to integrate process development with rapid LC-based analysis requiring no user intervention . The methods and procedures described can be generalized to any recombinant protein recovery campaign. Science, 1998 Apr 10, 280(5361), 286 - 9 Ribosome-catalyzed peptide-bond formation with an A-site substrate covalently linked to 23S ribosomal RNA; Green R et al.; In the ribosome, the aminoacyl-transfer RNA (tRNA) analog 4-thio-dT-p-C-p-puromycin crosslinks photochemically with G2553 of 23S ribosomal RNA (rRNA) . This covalently linked substrate reacts with a peptidyl-tRNA analog to form a peptide bond in a peptidyl transferase-catalyzed reaction . This result places the conserved 2555 loop of 23S rRNA at the peptidyl transferase A site and suggests that peptide bond formation can occur uncoupled from movement of the A-site tRNA . Crosslink formation depends on occupancy of the P site by a tRNA carrying an intact CCA acceptor end, indicating that peptidyl-tRNA, directly or indirectly, helps to create the peptidyl transferase A site. Biochemistry, 1998 Mar 24, 37(12), 4160 - 8 Reaction of Escherichia coli cytochrome bo3 with substoichiometric ubiquinol-2: a freeze-quench electron paramagnetic resonance investigation; Schultz BE et al.; The reaction of the quinol oxidase cytochrome bo3 from Escherichia coli with ubiquinol-2 (UQ2H2) was carried out using substoichiometric (0.5 equiv) amounts of substrate . Reactions were monitored through the use of freeze-quench EPR spectroscopy . Under 1 atm of argon, semiquinone was formed at the QB site of the enzyme with a formation rate constant of 140 s-1; the QB semiquinone EPR signal decayed with a rate constant of about 5 s-1 . Heme b and CuB were reduced within the 10-ms dead time of the freeze-quench experiment and remained at a constant level of reduction over the 1-s time course of the experiment . Quantitation of the reduction levels of QB and heme b during this reaction yielded a reduction potential of 30-60 mV for heme b . Under a dioxygen atmosphere, the rates of semiquinone formation and its subsequent decay were not altered significantly . However, accurate quantitation of the EPR signals for heme b and heme o3 could not be made, due to interference from dioxygen . In the reaction between the QB-depleted enzyme and UQ2H2 under substoichiometric conditions, there was no observable change in the EPR spectra of the enzyme over the time course of the reaction, suggesting an electron transfer from heme b to the binuclear site in the absence of QB which occurs within the dead time of the freeze-quench apparatus . Analysis of the thermodynamics and kinetics of electron transfers in this enzyme suggests that a Q-cycle mechanism for proton translocation is more likely than a cytochrome c oxidase-type ion-pump mechanism. Biochemistry, 1998 Mar 24, 37(12), 4148 - 59 Localization of histidine residues responsible for heme axial ligation in cytochrome b556 of complex II (succinate:ubiquinone oxidoreductase) in Escherichia coli; Vibat CR et al.; Complex II (succinate:ubiquinone oxidoreductase) from Escherichia coli contains four different subunits . Two of the subunits (SDHC and SDHD) are hydrophobic and anchor the two more hydrophilic (flavin and iron-sulfur) subunits (SDHA and SDHB) to the cytoplasmic membrane . Previous studies have shown that the complex of SDHC/SDHD is required to maintain the heme B component of the enzyme and that the heme B is ligated to the protein by two histidine ligands . In the current work, the histidines within SDHC and SDHD have been systematically mutated . SDHC-His91 and SDHD-His14 were eliminated as potential ligands by these studies . SDHC-His84 and SDHD-His71 have been identified as the most likely heme axial ligands in the E . coli enzyme, suggesting that the heme bridges these two subunits in the membrane . Furthermore, the results show that the four-subunit Complex II assembles and retains function despite the absence of the heme B prosthetic group in the membrane . The results do not rule out completely SDHC-His30 as a candidate for heme ligation, but do show that mutation at this position prevents assembly of Complex II in the membrane. Biochemistry, 1998 Mar 24, 37(12), 4071 - 9 Structural basis for different inhibitory specificities of human cystatins C and D; Hall A et al.; Human cystatins C and D share almost identical primary structures of two out of the three segments proposed to be of importance for enzyme interactions but have markedly different profiles for inhibition of the target cysteine peptidases, cathepsins B, H, L, and S . To investigate if the N-terminal binding regions of the inhibitors are responsible for the different inhibition profiles, and thereby confer biological selectivity, two hybrid cystatins were produced in Escherichia coli expression systems . In one hybrid, the N-terminal segment of cystatin C was placed on the framework of cystatin D, and the second was engineered with the N-terminal segment of cystatin D on the cystatin C scaffold . Truncated cystatin C and D variants, devoid of their N-terminal segments, were obtained by incubation with glycyl endopeptidase and isolated, in a second approach to assess the importance of the N-terminal binding regions for cystatin function and specificity . The affinities of the four cystatin variants for cathepsins B, H, L, and S were measured . By comparison with corresponding results for wild-type cystatins C and D, it was concluded (1) that both the N-terminal and framework part of the molecules significantly contribute to the observed differences in inhibitory activities of cystatins C and D and (2) that the N-terminal segment of cystatin C increases the inhibitory activity of cystatin D against cathepsin S and cathepsin L but results in decreased activity against cathepsin H . These differences in specificity were explained by the residues interacting with the S2 subsite of peptidases (Val- and Ala-10 in cystatin C and D, respectively) . Also, removal of the N-terminal segment results in total loss of enzyme affinity for cystatin D but not for cystatin C . Therefore, structural differences in the framework parts, as well as in the N-terminal segments, are critical for both inhibitory specificity and potency . Homology modeling was used to identify residues likely responsible for the generally reduced inhibitory potency of cystatin D. EMBO J, 1998 Mar 16, 17(6), 1838 - 45 Functional interactions between the holliday junction resolvase and the branch migration motor of Escherichia coli; van Gool AJ et al.; Homologous recombination generates genetic diversity and provides an important cellular pathway for the repair of double-stranded DNA breaks . Two key steps in this process are the branch migration of Holliday junctions followed by their resolution into mature recombination products . In E.coli, branch migration is catalysed by the RuvB protein, a hexameric DNA helicase that is loaded onto the junction by RuvA, whereas resolution is promoted by the RuvC endonuclease . Here we provide direct evidence for functional interactions between RuvB and RuvC that link these biochemically distinct processes . Using synthetic Holliday junctions, RuvB was found to stabilize the binding of RuvC to a junction and to stimulate its resolvase activity . Conversely, RuvC facilitated interactions between RuvB and the junction such that RuvBC complexes catalysed branch migration . The observed synergy between RuvB and RuvC provides new insight into the structure and function of a RuvABC complex that is capable of facilitating branch migration and resolution of Holliday junctions via a concerted enzymatic mechanism. EMBO J, 1998 Mar 16, 17(6), 1829 - 37 Stationary phase induction of dnaN and recF, two genes of Escherichia coli involved in DNA replication and repair; Villarroya M et al.; The beta subunit of DNA polymerase III holoenzyme, the Escherichia coli chromosomal replicase, is a sliding DNA clamp responsible for tethering the polymerase to DNA and endowing it with high processivity . The gene encoding beta, dnaN, maps between dnaA and recF, which are involved in initiation of DNA replication at oriC and resumption of DNA replication at disrupted replication forks, respectively . In exponentially growing cells, dnaN and recF are expressed predominantly from the dnaA promoters . However, we have found that stationary phase induction of the dnaN promoters drastically changes the expression pattern of the dnaA operon genes . As a striking consequence, synthesis of the beta subunit and RecF protein increases when cell metabolism is