EMBO J, 2000 Nov 15, 19(22), 6249 - 58 A case for sliding SeqA tracts at anchored replication forks during Escherichia coli chromosome replication and segregation; Brendler T et al.; SeqA is an Escherichia coli DNA-binding protein that acts at replication origins and controls DNA replication . However, binding is not exclusive to origins . Many fragments containing two or more hemi-methylated GATC sequences bind efficiently . Binding was optimal when two such sequences were closely apposed or up to 31 bases apart on the same face of the DNA helix . Binding studies suggest that neighboring bound proteins contact each other to form a complex with the intervening DNA looped out . There are many potential binding sites distributed around the E.coli chromosome . As replication produces a transient wave of hemi-methylation, tracts of SeqA binding are likely to associate with each fork as replication progresses . The number and positions of green fluorescent protein-SeqA foci seen in living cells suggest that they correspond to these tracts, and that the forks are tethered to planes of cell division . SeqA may help to tether the forks or to organize newly replicated DNA into a structure that aids DNA to segregate away from the replication machinery. EMBO J, 2000 Nov 15, 19(22), 6240 - 8 The eclipse period of Escherichia coli; von Freiesleben U et al.; The minimal time between successive initiations on the same origin (the eclipse) in Escherichia coli was determined to be approximately 25-30 min . An inverse relationship was found between the length of the eclipse and the amount of Dam methyltransferase in the cell, indicating that the eclipse corresponds to the period of origin hemimethylation . The SeqA protein was absolutely required for the eclipse, and DnaA titration studies suggested that the SeqA protein prevented the binding of multiple DnaA molecules on oriC (initial complex formation) . No correlation between the amount of SeqA and eclipse length was revealed, but increased SeqA levels affected chromosome partitioning and/or cell division . This was corroborated further by an aberrant nucleoid distribution in SeqA-deficient cells . We suggest that the SeqA protein's role in maintaining the eclipse is tied to a function in chromosome organization. EMBO J, 2000 Nov 15, 19(22), 6230 - 9 Euplotes telomerase contains an La motif protein produced by apparent translational frameshifting; Aigner S et al.; Telomerase is the ribonucleoprotein enzyme responsible for the replication of chromosome ends in most eukaryotes . In the ciliate Euplotes aediculatus, the protein p43 biochemically co-purifies with active telomerase and appears to be stoichiometric with both the RNA and the catalytic protein subunit of this telomerase complex . Here we describe cloning of the gene for p43 and present evidence that it is an authentic component of the telomerase holoenzyme . Comparison of the nucleotide sequence of the cloned gene with peptide sequences of the protein suggests that production of full-length p43 relies on a programmed ribosomal frameshift, an extremely rare translational mechanism . Anti-p43 antibodies immunodeplete telomerase RNA and telomerase activity from E.aediculatus nuclear extracts, indicating that the vast majority of mature telomerase complexes in the cell are associated with p43 . The sequence of p43 reveals similarity to the La autoantigen, an RNA-binding protein involved in maturation of RNA polymerase III transcripts, and recombinant p43 binds telomerase RNA in vitro . By analogy to other La proteins, p43 may function in chaperoning the assembly and/or facilitating nuclear retention of telomerase. EMBO J, 2000 Nov 15, 19(22), 5980 - 8 Protein folding in the periplasm in the absence of primary oxidant DsbA: modulation of redox potential in periplasmic space via OmpL porin; Dartigalongue C et al.; Disulfide bond formation in Escherichia coli is a catalyzed reaction accomplished by DsbA . We found that null mutations in a new porin gene, ompL, allowed a total bypass of the DsbA requirement for protein oxidation . These mutations acted as extragenic null suppressors for dsbA, and restored normal folding of alkaline phosphatase and relieved sensitivity to dithiothreitol . ompL dsbA double mutants were completely like wild-type mutants in terms of motility and lack of mucoidy . This suppression was not dependent on DsbC and DsbG, since the oxidation status of these proteins was unaltered in ompL dsbA strains . Purified OmpL allowed diffusion of small solutes, including sugars, but the suppression was not dependent on the carbon sources used . Suppression by ompL null mutations required DsbB, leading us to propose a hypothesis that DsbB oxidizes yet unidentified, low-molecular-weight redox agents in the periplasm . These oxidized agents accumulate and substitute for DsbA if their leakage into the medium is prevented by the absence of OmpL, presumed to form a specific channel for their diffusion. Cancer J, 2000 Sep-Oct, 6(5), 287 - 93 Antitumor interaction of short-course endostatin and ionizing radiation; Hanna NN et al.; PURPOSE: The purpose of this study was to evaluate whether endostatin, an antiangiogenic cleavage fragment of collagen XVIII, enhances the antitumor effects of ionizing radiation (IR) . Endostatin was injected to coincide with fractionated radiotherapy . METHODS: Xenografts of radioresistant SQ-20B tumor cells were established in athymic nude mice . Lewis lung carcinoma cells were injected into C57BI/6 mice . Mice bearing SQ-20B xenografts were injected intraperitoneally with 2.5 mg/kg/day of murine recombinant endostatin 5 times per week for 2 weeks 3 hours before IR treatment (50 Gy total dose) . Mice bearing Lewis lung carcinoma tumors were injected intraperitoneally with endostatin (2.5 mg/kg/day) four times; the first injection was given 24 hours before the first IR dose (15 Gy) and then 3 hours before IR (15 Gy/day) for 3 consecutive days . Microvascular density was assessed on tumor tissue sections by use of CD31 immunohistochemistry and light microscopy . Endothelial cell survival analyses were employed to evaluate endostatin effects on human aortic endothelial cells and human umbilical vein endothelial cells . Endothelial cell apoptosis was examined by use of FACS analysis and DAPI microscopy . RESULTS: In SQ-20B xenografts, combined treatment with endostatin and IR produced tumor growth inhibition that was most pronounced at the nadir of regression (day 21) . By day 35, tumors receiving combined treatment with endostatin and IR were 47% smaller than tumors treated with endostatin alone . Interactive cytotoxic treatment effects between endostatin and IR were also demonstrated in mice bearing Lewis lung carcinoma tumors . Significant tumor growth inhibition was observed in the endostatin/IR group at days 11 and 13 compared with IR alone . Histologic analyses demonstrated a reduction in microvascular density after combined treatment with endostatin and IR compared with endostatin treatment alone . Survival analyses confirmed interactive cytotoxicity between endostatin and IR in both human aortic endothelial cells and human umbilical vein endothelial cells but not in SQ-20B tumor cells . Combined treatment with endostatin and IR produced an increase in cow pulmonary artery endothelial apoptosis compared with either treatment alone . DISCUSSION: The tumor regression observed after combined treatment with endostatin and IR suggests additive antitumor effects in both human and murine tumors . Importantly, the concentrations of endostatin employed produced little tumor regression when endostatin was employed as a single agent . The results from the clonogenic and apoptosis assays support the hypothesis that the endothelial compartment is the target for the endostatin/IR interaction. FEBS Lett, 2000 Nov 10, 484(3), 265 - 70 The natural osmolyte trimethylamine N-oxide (TMAO) restores the ability of mutant tau to promote microtubule assembly; Smith MJ et al.; Coding region and intronic mutations in the gene for microtubule-associated protein tau cause frontotemporal dementia and Parkinsonism linked to chromosome 17 (FTDP-17) . Most coding region mutations effect a reduced ability of tau protein to interact with microtubules and lead to the formation of a filamentous pathology made of hyperphosphorylated tau . Here we show that trimethylamine N-oxide (TMAO) restores the ability of tau with FTDP-17 mutations to promote microtubule assembly . To mimic phosphorylation, serine and threonine residues in tau were singly or multiply mutated to glutamic acid, resulting in a reduced ability of tau to promote microtubule assembly . With the exception of the most heavily substituted protein (27 glutamic acid residues), TMAO increased the ability of mutant tau to promote microtubule assembly . However, it had no significant effect on heparin-induced assembly of tau into filaments. FEBS Lett, 2000 Nov 10, 484(3), 224 - 8 The roles of Glu93 and Tyr149 in astacin-like zinc peptidases; Yiallouros I et al.; The catalytic zinc of astacin, a prototype of the astacin family and the metzincin superfamily of metalloproteinases is coordinated by three histidines, a glutamate bound water and a tyrosine . In order to assess the roles of active site key residues, two mutants, Glu93Ala-astacin and Tyr149Phe-astacin, were expressed in Escherichia coli, affinity-purified and renatured . While the Glu93Ala mutant was inactive, the Tyr149Phe mutant retained about 2 . 5% residual activity toward Dns-Pro-Lys-Arg*Ala-Pro-Trp-Val, based on the k(cat)/K(m) value for recombinant wild-type astacin . These results support a model in which Glu93 is the general base in substrate hydrolysis, whereas Tyr149 contributes to transition state binding. Int J Mol Med, 2000 Dec, 6(6), 635 - 43 Design, expression, and renaturation of a lesion-targeted recombinant epidermal growth factor-von Willebrand factor fusion protein: efficacy in an animal model of experimental colitis; Hall FL et al.; In the present study, the mature epidermal growth factor (EGF) protein was engineered to incorporate a high affinity collagen-binding domain (CBD) derived from co-agulation von Willebrand factor, to specifically target EGF to colonic lesions . The fusion protein was expressed in an E . coli bacterial expression system, purified by metal chelate chromatography, and renatured by oxidative refolding into a soluble biologically active growth factor . The EGF-CBD fusion protein bound tightly to collagen matrices under conditions in which native non-targeted EGF was washed away . In biologic assays, the EGF-CBD fusion protein stimulated NIH3T3 cell proliferation with near wild-type biological activity . In vivo binding studies showed that the collagen-targeted EGF, but not the non-targeted EGF, accumulated at areas of exposed collagen on the luminal surface of the inflamed colon . Finally, a single colonic instillation of the collagen-targeted EGF-induced a more rapid regeneration of intestinal crypts 24 h after treatment (no . of crypts = 89.2+/-8.1) compared to the non-targeted EGF (no . of crypts = 52.2+/-29.8; p=0.027), and the PBS control (no . of crypts = 24 . 0+/-22.9; p=0.001) . Taken together, these findings indicate that intracolonic delivery of collagen-targeted EGF represents a potentially effective therapeutic strategy for acute or chronic inflammatory bowel disease. Plasmid, 2000 Nov, 44(3), 231 - 8 Green fluorescent protein and epitope tag fusion vectors for Dictyostelium discoideum; Levi S et al.; We have constructed expression vectors for Dictyostelium discoideum which encode a green fluorescent protein (GFP) sequence upstream of a multicloning site for introduction of sequences of interest . Insertion of cDNAs into the multicloning site results in expression of fusion protein bearing an amino- or carboxyl-terminal GFP tag which can be used for fluorescent localization studies in Dictyostelium cells . A parallel construct fuses a FLAG epitope tag at the amino terminus of expressed protein . Each fusion cartridge was placed either in a G418-resistance vector allowing transactivated Ddp2-based extrachromosomal replication or in a vector allowing autonomous Ddp1-based replication . Distinct differences in expression stability were observed in the two vector types . When GFP-expressing cells were analyzed by fluorescence microscopy, significant cell-to-cell variability in expression level was observed when expression was based on the Ddp2 vector, while less cell-to-cell variation in expression level was observed when the Ddp1 backbone was used for expression . J Biol Chem, 2001 Mar 9, 276(10), 7266 - 71 Epub 2000 Nov 16. Co-crystal of Escherichia coli RNase HI with Mn2+ ions reveals two divalent metals bound in the active site; Goedken ER et al.; Ribonuclease H (RNase H) selectively degrades the RNA strand of RNA.DNA hybrids in a divalent cation-dependent manner . Previous structural studies revealed a single Mg(2+) ion-binding site in Escherichia coli RNase HI . In the crystal structure of the related RNase H domain of human immunodeficiency virus reverse transcriptase, however, two Mn(2+) ions were observed suggesting a different mode of metal binding . E . coli RNase HI shows catalytic activity in the presence of Mg(2+) or Mn(2+) ions, but these two metals show strikingly different optimal concentrations . Mg(2+) ions are required in millimolar concentrations, but Mn(2+) ions are only required in micromolar quantities . Based upon the metal dependence of E . coli RNase HI activity, we proposed an activation/attenuation model in which one metal is required for catalysis, and binding of a second metal is inhibitory . We have now solved the co-crystal structure of E . coli RNase HI with Mn(2+) ions at 1.9-A resolution . Two octahedrally coordinated Mn(2+) ions are seen to bind to the enzyme-active site . Residues Asp-10, Glu-48, and Asp-70 make direct (inner sphere) coordination contacts to the first (activating) metal, whereas residues Asp-10 and Asp-134 make direct contacts to the second (attenuating) metal . This structure is consistent with biochemical evidence suggesting that two metal ions may bind RNase H but liganding a second ion inhibits RNase H activity. J Biol Chem, 2001 Feb 9, 276(6), 4441 - 6 Epub 2000 Nov 14. tau binds and organizes Escherichia coli replication proteins through distinct domains . Domain IV, located within the unique C terminus of tau, binds the replication fork, helicase, DnaB; Gao D et al.; Interaction between the tau subunit of the DNA polymerase III holoenzyme and the DnaB helicase is critical for coupling the replicase and the primosomal apparatus at the replication fork (Kim, S., Dallmann, H . G., McHenry, C . S., and Marians, K . J . (1996) Cell 84, 643-650) . In the preceding manuscript, we reported the identification of five putative structural domains within the tau subunit (Gao, D., and McHenry, C . (2000) J . Biol . Chem . 275, 4433-4440) . As part of our systematic effort to assign functions to each of these domains, we expressed a series of truncated, biotin-tagged tau fusion proteins and determined their ability to bind DnaB by surface plasmon resonance on streptavidin-coated surfaces . Only tau fusion proteins containing domain IV bound DnaB . The DnaB-binding region was further limited to a highly basic 66-amino acid residue stretch within domain IV . Unlike the binding of immobilized tau(4) to the DnaB hexamer, the binding of monomeric domain IV to DnaB(6) was dependent upon the density of immobilized domain IV, indicating that DnaB(6) is bound by more than one tau protomer . This observation implies that both the leading and lagging strand polymerases are tethered to the DnaB helicase via dimeric tau . These double tethers of the leading and lagging strand polymerases proceeding through the tau-tau link and an additional tau-DnaB link are likely important for the dynamic activities of the replication fork. J Biol Chem, 2001 Feb 9, 276(6), 4433 - 40 Epub 2000 Nov 14. tau binds and organizes Escherichia coli replication through distinct domains . Partial proteolysis of terminally tagged tau to determine candidate domains and to assign domain V as the alpha binding domain; Gao D et al.; The tau subunit dimerizes Escherichia coli DNA polymerase III core through interactions with the alpha subunit . In addition to playing critical roles in the structural organization of the holoenzyme, tau mediates intersubunit communications required for efficient replication fork function . We identified potential structural domains of this multifunctional subunit by limited proteolysis of C-terminal biotin-tagged tau proteins . The cleavage sites of each of eight different proteases were found to be clustered within four regions of the tau subunit . The second susceptible region corresponds to the hinge between domain II and III of the highly homologous delta' subunit, and the third region is near the C-terminal end of the tau-delta' alignment (Guenther, B., Onrust, R., Sali, A., O'Donnell, M., and Kuriyan, J . (1997) Cell 91, 335-345) . We propose a five-domain structure for the tau protein . Domains I and II are based on the crystallographic structure of delta' by Guenther and colleagues . Domains III-V are based on our protease cleavage results . Using this information, we expressed biotin-tagged tau proteins lacking specific protease-resistant domains and analyzed their binding to the alpha subunit by surface plasmon resonance . Results from these studies indicated that the alpha binding site of tau lies within its C-terminal 147 residues (domain V). J Biol Chem, 2001 Feb 9, 276(6), 4447 - 53 Epub 2000 Nov 14. Tau binds and organizes Escherichia coli replication proteins through distinct domains . Domain III, shared by gamma and tau, binds delta delta ' and chi psi; Gao D et al.; The DnaX complex of the DNA polymerase holoenzyme assembles the beta(2) processivity factor onto the primed template enabling highly processive replication . The key ATPases within this complex are tau and gamma, alternative frameshift products of the dnaX gene . Of the five domains of tau, I-III are shared with gamma In vivo, gamma binds the auxiliary subunits deltadelta' and chipsi (Glover, B . P., and McHenry, C . S . (2000) J . Biol . Chem . 275, 3017-3020) . To localize deltadelta' and chipsi binding domains within gamma domains I-III, we measured the binding of purified biotin-tagged DnaX proteins lacking specific domains to deltadelta' and chipsi by surface plasmon resonance . Fusion proteins containing either DnaX domains I-III or domains III-V bound deltadelta' and chipsi subunits . A DnaX protein only containing domains I and II did not bind deltadelta' or chipsi . The binding affinity of chipsi for DnaX domains I-III and domains III-V was the same as that of chipsi for full-length tau, indicating that domain III contained all structural elements required for chipsi binding . Domain III of tau also contained deltadelta' binding sites, although the interaction between deltadelta' and domains III-V of tau was 10-fold weaker than the interaction between deltadelta' and full length tau . The presence of both delta and chipsi strengthened the delta'-C(0)tau interaction by at least 15-fold . Domain III was the only domain common to all of tau fusion proteins whose interaction with delta' was enhanced in the presence of delta and chipsi . Thus, domain III of the DnaX proteins not only contains the deltadelta' and chipsi binding sites but also contains the elements required for the positive cooperative assembly of the DnaX complex. J Biol Chem, 2001 Feb 9, 276(6), 4245 - 50 Epub 2000 Nov 14. The transport of group 2 capsular polysaccharides across the periplasmic space in Escherichia coli . Roles for the KpsE and KpsD proteins; Arrecubieta C et al.; The cell surface expression of group 2 capsular polysaccharides involves the translocation of the polysaccharide from its site of synthesis on the inner face of the cytoplasmic membrane onto the cell surface . The transport process is independent of the repeat structure of the polysaccharide, and translocation across the periplasm requires the cytoplasmic membrane-anchored protein KpsE and the periplasmic protein KpsD . In this paper we establish the topology of the KpsE protein and demonstrate that the C terminus interacts with the periplasmic face of the cytoplasmic membrane . By chemical cross-linking we show that KpsE is likely to exist as a dimer and that dimerization is independent of the other Kps proteins or the synthesis of capsular polysaccharide . No interaction between KpsD and KpsE could be demonstrated by chemical cross-linking, although in the presence of both KpsE and Lpp, KpsD could be cross-linked to a 7-kDa protein of unknown identity . In addition, we demonstrate that KpsD is present not only within the periplasm but is also in both the cytoplasmic and outer membrane fractions and that the correct membrane association of KpsD was dependent on KpsE, Lpp, and the secreted polysaccharide molecule . Both KpsD and KpsE showed increased proteinase K sensitivity in the different mutant backgrounds, reflecting conformational changes in the KpsD and KpsE proteins as a result of the disruption of the transport process . Collectively the data suggest that the trans-periplasmic export involves KpsD acting as the link between the cytoplasmic membrane transporter and the outer membrane with KpsE acting to facilitate this transport process. Proc Natl Acad Sci U S A, 2000 Nov 21, 97(24), 12997 - 3002 A dual-specificity aminoacyl-tRNA synthetase in the deep-rooted eukaryote Giardia lamblia; Bunjun S et al.; Cysteinyl-tRNA (Cys-tRNA) is essential for protein synthesis . In most organisms the enzyme responsible for the formation of Cys-tRNA is cysteinyl-tRNA synthetase (CysRS) . The only known exceptions are the euryarchaea Methanococcus jannaschii and Methanobacterium thermoautotrophicum, which do not encode a CysRS . Deviating from the accepted concept of one aminoacyl-tRNA synthetase per amino acid, these organisms employ prolyl-tRNA synthetase as the enzyme that carries out Cys-tRNA formation . To date this dual-specificity prolyl-cysteinyl-tRNA synthetase (ProCysRS) is only known to exist in archaea . Analysis of the preliminary genomic sequence of the primitive eukaryote Giardia lamblia indicated the presence of an archaeal prolyl-tRNA synthetase (ProRS) . Its proS gene was cloned and the gene product overexpressed in Escherichia coli . By using G . lamblia, M . jannaschii, or E . coli tRNA as substrate, this ProRS was able to form Cys-tRNA and Pro-tRNA in vitro . Cys-AMP formation, but not Pro-AMP synthesis, was tRNA-dependent . The in vitro data were confirmed in vivo, as the cloned G . lamblia proS gene was able to complement a temperature-sensitive E . coli cysS strain . Inhibition studies of CysRS activity with proline analogs (thiaproline and 5'-O-{N-(l-prolyl)-sulfamoyl}adenosine) in a Giardia S-100 extract predicted that the organism also contains a canonical CysRS . This prediction was confirmed by cloning and analysis of the corresponding cysS gene . Like a number of archaea, Giardia contains two enzymes, ProCysRS and CysRS, for Cys-tRNA formation . In contrast, the purified Saccharomyces cerevisiae and E . coli ProRS enzymes were unable to form Cys-tRNA under these conditions . Thus, the dual specificity is restricted to the archaeal genre of ProRS . G . lamblia's archaeal-type prolyl- and alanyl-tRNA synthetases refine our understanding of the evolution and interaction of archaeal and eukaryal translation systems. J Cardiovasc Pharmacol, 2000 Nov, 36(5 Suppl 1), S19 - 21 Relationship between soluble intracellular endothelin-converting enzyme and endothelin-1 synthesis: effect of inhibitors of the secretory pathway; Corder R et al.; The relationship between soluble and membrane-bound endothelin-converting enzyme (ECE) activity with the level of endothelin-1 (ET-1) synthesis was investigated in cultured endothelial cells . Escherichia coli lipopolysaccharide (LPS) was used to stimulate ET-1 synthesis, and brefeldin A, monensin, colchicine or cytochalasin B, which disrupt peptide biosynthetic pathways in a variety of ways, were tested for their ability to modify changes in ET-1 synthesis and ECE levels . LPS increased ET-1 secretion by more than twofold . Levels of soluble ECE activity, but not those of membrane-bound ECE activity, correlated with ET-1 synthesis . These results suggest the soluble ECE activity is likely to play a role in ET-1 biosynthesis. Bioorg Med Chem Lett, 2000 Nov 6, 10(21), 2441 - 4 Synthesis of glutaminyl adenylate analogues that are inhibitors of glutaminyl-tRNA synthetase; Bernier S et al.; Glutaminol adenylate 5 is a competitive inhibitor of glutaminyl-tRNA synthetase with respect to glutamine (Ki = 280 nM) and to ATP (Ki = 860 nM) . The corresponding methyl phosphate ester 4 is a weaker inhibitor (Ki approximately 10 microM) with respect to glutamine. J Dent Res, 2000 Oct, 79(10), 1752 - 7 Antibodies against mycobacterial antigens in the synovial fluid of patients with temporomandibular disorders; Adachi N et al.; In the absence of active pulmonary disease, mycobacterial infection frequently causes arthritis and can be considered to initiate autoimmune diseases such as rheumatoid arthritis . Temporomandibular disorder (TMD) is a disease in which pain and impaired mandibular movement appear to arise directly from degenerative or inflammatory changes within the temporomandibular joint, but its precise pathogeny has not been elucidated . Here we examined whether mycobacterial infection is related to the pathology of TMD . The antibody levels against mycobacterial antigen in the synovial fluid (SF) of patients with TMD were assessed by enzyme-linked immunosorbent assay (ELISA) . Six of 17 TMD patients (35%) were found to possess mycobacterial antigen-specific immunoglobulin (Ig) G but not IgM, while the six healthy volunteers possessed neither . Western-blot analysis was used to isolate the reacted antigen, and the IgG reacted strongly to 44-kDa antigen . The first 14 N-terminal amino acid sequences were determined, and computer analysis revealed that it was homologous to translational elongation factor Tu (EF-Tu) of Mycobacterium tuberculosis, which was a major target antigen for these antibodies . The 44-kDa protein of Mycobacterium bovis BCG (BCG) was identical with the EF-Tu of M . tuberculosis . We cloned the gene encoding the EF-Tu of BCG by using the synthesized oligonucleotide primers by means of polymerase chain-reaction . The gene was expressed in Escherichia coli . The protein was purified, and the antibody levels against this recombinant protein in the SF of TMD patients were assessed by ELISA . Our findings suggest that some cases of TMD are concerned with the synovial IgG against the EF-Tu of M . tuberculosis, and that the existence of the antibody is a clinical indication of TMD. Genome Res, 2000 Nov, 10(11), 1788 - 95 DNA cloning using in vitro site-specific recombination; Hartley JL et al.; As a result of numerous genome sequencing projects, large numbers of candidate open reading frames are being identified, many of which have no known function . Analysis of these genes typically involves the transfer of DNA segments into a variety of vector backgrounds for protein expression and functional analysis . We describe a method called recombinational cloning that uses in vitro site-specific recombination to accomplish the directional cloning of PCR products and the subsequent automatic subcloning of the DNA segment into new vector backbones at high efficiency . Numerous DNA segments can be transferred in parallel into many different vector backgrounds, providing an approach to high-throughput, in-depth functional analysis of genes and rapid optimization of protein expression . The resulting subclones maintain orientation and reading frame register, allowing amino- and carboxy-terminal translation fusions to be generated . In this paper, we outline the concepts of this approach and provide several examples that highlight some of its potential. Biochemistry, 2000 Nov 14, 39(45), 13939 - 44 Catalysis by Escherichia coli ribonuclease HI is facilitated by a phosphate group of the substrate; Haruki M et al.; To investigate the role of the phosphate group 3' to the scissile phosphodiester bond of the substrate in the catalytic mechanism of Escherichia coli ribonuclease HI (RNase HI), we have used modified RNA-DNA hybrid substrates carrying a phosphorothioate substitution at this position or lacking this phosphate group for the cleavage reaction . Kinetic parameters of the H124A mutant enzyme, in which His(124) was substituted with Ala, as well as those of the wild-type RNase HI, were determined . Substitution of the pro-R(p)-oxygen of the phosphate group 3' to the scissile phosphodiester bond of the substrate with sulfur reduced the k(cat) value of the wild-type RNase HI by 6.9-fold and that of the H124A mutant enzyme by only 1 . 9-fold . In contrast, substitution of the pro-S(p)-oxygen of the phosphate group at this position with sulfur had little effect on the k(cat) value of the wild-type and H124A mutant enzymes . The results obtained for the substrate lacking this phosphate group were consistent with those obtained for the substrates with the phosphorothioate substitutions . In addition, a severalfold increase in the K(m) value was observed by the substitution of the pro-R(p)-oxygen of the substrate with sulfur or by the substitution of His(124) of the enzyme with Ala, suggesting that a hydrogen bond is formed between the pro-R(p)-oxygen and His(124) . These results allow us to propose that the pro-R(p)-oxygen contributes to orient His(124) to the best position for the catalytic function through the formation of a hydrogen bond. Biochemistry, 2000 Nov 14, 39(45), 13800 - 9 Electrogenic reactions of cytochrome bd; Jasaitis A et al.; Cytochrome bd is one of the two terminal quinol oxidases in the respiratory chain of Escherichia coli . The enzyme catalyzes charge separation across the bacterial membrane during the oxidation of quinols by dioxygen but does not pump protons . In this work, the reaction of cytochrome bd with O(2) and related reactions has been studied by time-resolved spectrophotometric and electrometric methods . Oxidation of the fully reduced enzyme by oxygen is accompanied by rapid generation of membrane potential (delta psi, negative inside the vesicles) that can be described by a two-step sequence of (i) an initial oxygen concentration-dependent, electrically silent, process (lag phase) corresponding to the formation of a ferrous oxy compound of heme d and (ii) a subsequent monoexponential electrogenic phase with a time constant <60 mus that matches the formation of ferryl-oxo heme d, the product of the reaction of O(2) with the 3-electron reduced enzyme . No evidence for generation of an intermediate analogous to the "peroxy" species of heme-copper oxidases could be obtained in either electrometric or spectrophotometric measurements of cytochrome bd oxidation or in a spectrophotometric study of the reaction of H(2)O(2) with the oxidized enzyme . Backflow of electrons upon flash photolysis of the singly reduced CO complex of cytochrome bd leads to transient generation of a delta psi of the opposite polarity (positive inside the vesicles) concurrent with electron flow from heme d to heme b(558) and backward . The amplitude of the delta psi produced by the backflow process, when normalized to the reaction yield, is close to that observed in the direct reaction during the reaction of fully reduced cytochrome bd with O(2) and is apparently associated with full transmembrane translocation of approximately one charge. Biochemistry, 2000 Nov 14, 39(45), 13719 - 29 Novel recombinant hemoglobin, rHb (beta N108Q), with low oxygen affinity, high cooperativity, and stability against autoxidation; Tsai CH et al.; Using our Escherichia coli expression system, we have constructed rHb (beta N108Q), a new recombinant hemoglobin (rHb), with the amino acid substitution located in the alpha(1)beta(1) subunit interface and in the central cavity of the Hb molecule . rHb (beta N108Q) exhibits low oxygen affinity, high cooperativity, enhanced Bohr effect, and slower rate of autoxidation of the heme iron atoms from the Fe(2+) to the Fe(3+) state than other low-oxygen-affinity rHbs developed in our laboratory, e.g., rHb (alpha V96W) and rHb (alpha V96W, beta N108K) . It has been reported by Olson and co-workers {Carver et al . (1992) J . Biol . Chem . 267, 14443-14450; Brantley et al . (1993) J . Biol . Chem . 268, 6995-7010} that the substitution of phenylalanine for leucine at position 29 of myoglobin can inhibit autoxidation in myoglobin and at position 29 of the alpha-chain of hemoglobin can lower NO reaction in both the deoxy and the oxy forms of human normal adult hemoglobin . Hence, we have further introduced this mutation, alpha L29F, into beta N108Q . rHb (alpha L29F, beta N108Q) is stabilized against auto- and NO-induced oxidation as compared to rHb (beta N108Q), but exhibits lower oxygen affinity at pH below 7.4 and good cooperativity as compared to Hb A . Proton nuclear magnetic resonance (NMR) studies show that rHb (beta N108Q) has similar tertiary structure around the heme pockets and quaternary structure in the alpha(1)beta(1) and alpha(1)beta(2) subunit interfaces as compared to those of Hb A . The tertiary structure of rHb (alpha L29F, beta N108Q) as measured by (1)H NMR, especially the alpha-chain heme pocket region (both proximal and distal histidyl residues), is different from that of CO- and deoxy-Hb A, due to the amino acid substitution at alpha L29F . (1)H NMR studies also demonstrate that rHb (beta N108Q) can switch from the R quaternary structure to the T quaternary structure without changing ligation state upon adding an allosteric effector, inositol hexaphosphate, and reducing the temperature . On the basis of its low oxygen affinity, high cooperativity, and stability against autoxidation, rHb (beta N108Q) is considered a potential candidate for the Hb-based oxygen carrier in a blood substitute system. Biochemistry, 2000 Nov 14, 39(45), 13641 - 50 Selective inhibition of human Molt-4 leukemia type II inosine 5'-monophosphate dehydrogenase by the 1,5-diazabicyclo{3.1.0}hexane-2,4-diones; Barnes BJ et al.; Inosine 5'-monophosphate dehydrogenase (IMPDH) is the rate-limiting enzyme in de novo purine biosynthesis . IMPDH activity results from expression of two isoforms . Type I is constitutively expressed and predominates in normal resting cells, while Type II is selectively up-regulated in neoplastic and replicating cells . Inhibitors of IMPDH activity selectively targeting the Type II isoform have great potential as cancer chemotherapeutic agents . For this study, an expression system was developed which yields 35-50 mg of soluble, purified recombinant Type I and II protein from 1 L of bacteria . In addition, three 1,5-diazabicyclo{3.1.0}hexane-2,4-diones were synthesized and shown to act as specific inhibitors of human recombinant Type II IMPDH . The agents are competitive inhibitors with respect to the endogenous substrate IMP and K(i) values range from 5 to 44 microM but were inactive as inhibitors of the Type I isoform at concentrations ranging from 0.5 to 500 microM . IC(50) values for recombinant Type II inhibition were determined and compared to IC(50) values obtained from Molt-4 cell extracts of IMPDH . Cytotoxicity assays revealed that the compounds inhibited Molt-4 leukemia growth with ED(50) values of 3.2-7.6 microM . Computational docking studies predict that the compounds bind to IMPDH in the IMP-binding site, although interactions with residues differ from those previously determined to interact with bound IMP . While all residues predicted to interact directly with the bound compounds are conserved in the Type I and Type II isoforms, sequence divergence within a helix adjacent to the active site may contribute to the observed selectivity for the human Type II isoform . These compounds represent the first class of selective IMPDH Type II inhibitors which may serve as lead compounds for the development of isoform-selective cancer chemotherapy. Biochemistry, 2000 Nov 14, 39(45), 13633 - 40 Characterization of enzymatic processes by rapid mix-quench mass spectrometry: the case of dTDP-glucose 4,6-dehydratase; Gross JW et al.; The single-turnover kinetic mechanism for the reaction catalyzed by dTDP-glucose 4,6-dehydratase (4,6-dehydratase) has been determined by rapid mix-chemical quench mass spectrometry . Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was employed to analyze quenched samples . The results were compatible with the postulated reaction mechanism, in which NAD(+) initially oxidizes glucosyl C4 of dTDP-glucose to NADH and dTDP-4-ketoglucose . Next, water is eliminated between C5 and C6 of dTDP-4-ketoglucose to form dTDP-4-ketoglucose-5,6-ene . Hydride transfer from NADH to C6 of dTDP-4-ketoglucose-5,6-ene regenerates NAD(+) and produces the product dTDP-4-keto-6-deoxyglucose . The single-turnover reaction was quenched at various times on the millisecond scale with a mixture of 6 M guanidine hydrochloride and sodium borohydride, which stopped the reaction and reductively stabilized the intermediates and product . Quantitative MALDI-TOF MS analysis of the quenched samples allowed the simultaneous observation of the disappearance of substrate, transient appearance and disappearance of dTDP-hexopyranose-5,6-ene (the reductively stabilized dTDP-4-ketoglucose-5,6-ene), and the appearance of product . Kinetic modeling of the process allowed rate constants for most of the steps of the reaction of dTDP-glucose-d(7) to be evaluated . The transient formation and reaction of dTDP-4-ketoglucose could not be observed, because this intermediate did not accumulate to detectable concentrations. Biol Chem, 2000 Sep-Oct, 381(9-10), 877 - 85 Molecular basis for interactions of the DnaK chaperone with substrates; Mayer MP et al.; Hsp70 chaperones assist a large variety of protein folding processes in the cell by transient association with short peptide segments of proteins . The substrate binding and release cycle is driven by the switching between the low affinity ATP bound state and the high affinity ADP bound state of Hsp70 . Considerable progress has been made recently by the identification of in vivo substrates for the Escherichia coli homolog, DnaK, and the molecular mechanisms which govern the DnaK-substrate interactions . Here we review the processes that generate DnaK substrates in vivo and the properties of these substrates, and we describe insights gained from structural and kinetic analysis of DnaK-substrate interaction. J Mol Microbiol Biotechnol, 2000 Oct, 2(4), 543 - 50 Characterization and expression of the arginine biosynthesis gene cluster of Streptomyces clavuligerus; Rodriguez-Garcia A et al.; A cluster of genes argCJBDRGH containing most of the arginine biosynthesis genes has been found in Streptomyces clavuligerus after sequencing a 8.3 kb DNA region containing overlapping sequences of two DNA fragments known to contain arginine biosynthesis genes . Subcloning, complementation of E . coli arginine auxotrophic strains and enzymatic assays confirmed the identity of each gene . S1 nuclease mapping studies and Northern hybridization analysis revealed the formation of two large transcripts corresponding to argCJBDR and argGH . The amount of each of these mRNAs is 10 to 44 times higher in a S . clavuligerus argR-disrupted mutant than in the wild type confirming the existence of an ArgR-mediated control of arginine biosynthesis gene expression . A low level constitutive monocistronic transcript of argR was observed in S . clavuligerus cells . Most of the argGH transcript initiating at an adenine 29 nt upstream of the argG initiation codon appears to stop at a termination stem and loop structure present downstream of the argG gene. J Mol Microbiol Biotechnol, 2000 Oct, 2(4), 455 - 62 Technological advances in the molecular biology of Leptospira; Zuerner R et al.; Pathogenic members of the genus Leptospira have been refractory to genetic study due to lack of known mechanisms of genetic exchange . To bypass this limitation, several techniques have been useful for Leptospira gene discovery, including heterologous complementation of Escherichia coli mutants, screening of DNA libraries with probes, and random sequence analysis . Construction of combined physical and genetic maps revealed the presence of two circular chromosomal replicons . The organization of the L . interrogans genome is quite variable, with genetically similar strains differentiated by many rearrangements . These rearrangements likely occur through recombination between repetitive DNA elements found scattered throughout the genome . Analysis of intervening sequences and genes encoding LPS biosynthetic enzymes provide evidence of lateral transfer of DNA between Leptospira spp . We have also gained insight into the biology of these bacteria by analyzing genes encoding LPS and outer membrane proteins (OMPs) . Some of these OMPs are differentially expressed . Characterization of mechanisms governing the expression of the OMP genes should provide insight into host-parasite interactions . Furthermore, recent advances in heterologous expression of leptospiral OMP genes are opening new avenues of vaccine development. Methods Enzymol, 2000, 328, 478 - 96 Protein engineering by expressed protein ligation; Blaschke UK et al.; By allowing the controlled assembly of synthetic peptides and recombinant polypeptides, expressed protein ligation permits unnatural amino acids, biochemical probes, and biophysical probes to be specifically incorporated into semisynthetic proteins . A powerful feature of the method is its modularity; once the reactive recombinant pieces are in hand and the optimal ligation conditions have been developed, it is possible to quickly generate an array of semisynthetic analogs by simply attaching different synthetic peptide cassettes--in most cases the synthetic peptides will be small and easy to make . From a practical perspective, the rate-determining step in the process is usually not the ligation step (it is based on a simple and efficient chemical reaction), but rather the generation of the reactive polypeptide building blocks . In particular, optimizing the yields of recombinant polypeptide building blocks can require some initial effort . However, it should be noted that the initial investment in time required to optimize the production of the recombinant fragment is offset by the ease and speed with which one can produce the material thereafter . In the example described in this chapter, the yield of soluble intein fusion protein was slightly better using the GyrA intein than for the VMA intein, although in both cases significant amounts of fusion protein were present in the cell pellet . Studies are currently underway to identify optimal refolding conditions for GyrA fusion proteins solubilized from inclusion bodies. Methods Enzymol, 2000, 328, 103 - 10 Use of a yeast three-hybrid system to clone bridging proteins; Zhang J; The three-hybrid system described here should facilitate screening cDNA libraries to clone "bridging" proteins in ternary complex . The pDela constructs were initially designed to be compatible with the Gal4-based two-hybrid system, such as pGAD424/pGBT9 . The Ura selection offered by pDela makes it easy to introduce the "bridging" protein into the yeast containing the other two hybrids, the BD fusion protein and the AD fusion protein . Because many of the widely used two-hybrid systems employ Trp-, Leu-, His-, or ziocin selection in the shuttle vectors, the Ura(+)-based pDela constructs can be used directly in those systems. J Biol Chem, 2001 Feb 9, 276(6), 3872 - 8 Epub 2000 Nov 13. Analysis of the cooperativity of human beta-cell glucokinase through the stimulatory effect of glucose on fructose phosphorylation; Moukil MA et al.; Using overexpressed Escherichia coli sorbitol-6-phosphate dehydrogenase to monitor fructose 6-phosphate formation, we found that the stimulation of fructose phosphorylation by glucose was reduced in two human beta-cell glucokinase mutants with a low Hill coefficient or when the activity of wild type glucokinase was decreased by replacing ATP with poorer nucleotide substrates . Mutation of two other residues, neighboring glucose-binding residues in the catalytic site, also reduced the affinity for glucose as a stimulator of fructose phosphorylation . Among a series of glucose analogs, only 3, all substrates of glucokinase, stimulated fructose phosphorylation; other analogs were either inactive or inhibited glucokinase . Glucose increased the apparent affinity for inhibitors that are glucose analogs but not for the glucokinase regulatory protein or palmitoyl-CoA . These data indicate that the stimulatory effect of glucose on fructose phosphorylation reflects the positive cooperativity for glucose and is mediated by binding of glucose to the catalytic site . They support models involving the existence of two slowly interconverting conformations of glucokinase that differ through their affinity for glucose and for glucose analogs . We show by computer simulation that such a model can account for the kinetic properties of glucokinase, including the differential ability of mannoheptulose and N-acetylglucosamine to suppress cooperativity (Agius, L., and Stubbs, M . (2000) Biochem . J . 346, 413-421). J Bacteriol, 2000 Dec, 182(23), 6842 - 4 Hydrogen peroxide activates the SoxRS regulon in vivo; Manchado M et al.; By multiplex reverse transcription-PCR, we demonstrate that the SoxRS response, which protects cells against superoxide toxicity, is triggered also by hydrogen peroxide . SoxR-dependent inductions of 7 . 3-, 7.6-, 4.6-, 2.2-, and 2.6-fold were quantified for soxS, micF, sodA, inaA, and fpr transcripts, respectively . This finding suggests an extensive and tight connectivity between different regulatory pathways in the Escherichia coli response to oxidative stress. J Bacteriol, 2000 Dec, 182(23), 6831 - 3 Interspecies complementation of Escherichia coli ccm mutants: CcmE (CycJ) from Bradyrhizobium japonicum acts as a heme chaperone during cytochrome c maturation; Schulz H et al.; Biogenesis of c-type cytochromes in alpha- and gamma-proteobacteria requires the function of a set of orthologous genes (ccm genes) that encode specific maturation factors . The Escherichia coli CcmE protein is a periplasmic heme chaperone . The membrane protein CcmC is required for loading CcmE with heme . By expressing CcmE (CycJ) from Bradyrhizobium japonicum in E . coli we demonstrated that heme is bound covalently to this protein at a strictly conserved histidine residue . The B . japonicum homologue can transfer heme to apocytochrome c in E . coli, suggesting that it functions as a heme chaperone . CcmC (CycZ) from B . japonicum expressed in E . coli was capable of inserting heme into CcmE. J Bacteriol, 2000 Dec, 182(23), 6827 - 30 Comparison of the UDP-N-acetylmuramate:L-alanine ligase enzymes from Mycobacterium tuberculosis and Mycobacterium leprae; Mahapatra S et al.; In the peptidoglycan of Mycobacterium leprae, L-alanine of the side chain is replaced by glycine . When expressed in Escherichia coli, MurC (UDP-N-acetyl-muramate:L-alanine ligase) of M . leprae showed K(m) and V(max) for L-alanine and glycine similar to those of Mycobacterium tuberculosis MurC, suggesting that another explanation should be sought for the presence of glycine. J Bacteriol, 2000 Dec, 182(23), 6774 - 82 Interdependence of activation at rhaSR by cyclic AMP receptor protein, the RNA polymerase alpha subunit C-terminal domain, and rhaR; Holcroft CC et al.; The Escherichia coli rhaSR operon encodes two AraC family transcription activators, RhaS and RhaR, and is activated by RhaR in the presence of L-rhamnose . beta-Galactosidase assays of various rhaS-lacZ promoter fusions combined with mobility shift assays indicated that a cyclic AMP receptor protein (CRP) site located at -111.5 is also required for full activation of rhaSR expression . To address the mechanisms of activation by CRP and the RNA polymerase alpha-subunit C-terminal domain (alpha-CTD) at rhaSR, we tested the effects of alanine substitutions in CRP activating regions 1 and 2, overexpression of a truncated version of alpha (alpha-Delta235), and alanine substitutions throughout alpha-CTD . We found that DNA-contacting residues in alpha-CTD are required for full activation, and for simplicity, we discuss alpha-CTD as a third activator of rhaSR . CRP and RhaR could each partially activate transcription in the absence of the other two activators, and alpha-CTD was not capable of activation alone . In the case of CRP, this suggests that this activation involves neither an alpha-CTD interaction nor cooperative binding with RhaR, while in the case of RhaR, this suggests the likelihood of direct interactions with core RNA polymerase . We also found that CRP, RhaR, and alpha-CTD each have synergistic effects on activation by the others, suggesting direct or indirect interactions among all three . We have some evidence that the alpha-CTD-CRP and alpha-CTD-RhaR interactions might be direct . The magnitude of the synergistic effects was usually greater with just two activators than with all three, suggesting possible redundancies in the mechanisms of activation by CRP, alpha-CTD, and RhaR. J Bacteriol, 2000 Dec, 182(23), 6751 - 61 Maturation of IncP pilin precursors resembles the catalytic Dyad-like mechanism of leader peptidases; Eisenbrandt R et al.; The pilus subunit, the pilin, of conjugative IncP pili is encoded by the trbC gene . IncP pilin is composed of 78 amino acids forming a ring structure (R . Eisenbrandt, M . Kalkum, E.-M . Lai, C . I . Kado, and E . Lanka, J . Biol . Chem . 274:22548-22555, 1999) . Three enzymes are involved in maturation of the pilin: LepB of Escherichia coli for signal peptide removal and a yet-unidentified protease for removal of 27 C-terminal residues . Both enzymes are chromosome encoded . Finally, the inner membrane-associated IncP TraF replaces a four-amino-acid C-terminal peptide with the truncated N terminus, yielding the cyclic polypeptide . We refer to the latter process as "prepilin cyclization." We have used site-directed mutagenesis of trbC and traF to unravel the pilin maturation process . Each of the mutants was analyzed for its phenotypes of prepilin cyclization, pilus formation, donor-specific phage adsorption, and conjugative DNA transfer abilities . Effective prepilin cyclization was determined by matrix-assisted laser desorption-ionization-mass spectrometry using an optimized sample preparation technique of whole cells and trans-3-indolyl acrylic acid as a matrix . We found that several amino acid exchanges in the TrbC core sequence allow prepilin cyclization but disable the succeeding pilus assembly . We propose a mechanism explaining how the signal peptidase homologue TraF attacks a C-terminal section of the TrbC core sequence via an activated serine residue . Rather than cleaving and releasing hydrolyzed peptides, TraF presumably reacts as a peptidyl transferase, involving the N terminus of TrbC in the aminolysis of a postulated TraF-acetyl-TrbC intermediate . Under formal loss of a C-terminal tetrapeptide, a new peptide bond is formed in a concerted action, connecting serine 37 with glycine 114 of TrbC. J Bacteriol, 2000 Dec, 182(23), 6742 - 50 Role of the dinB gene product in spontaneous mutation in Escherichia coli with an impaired replicative polymerase; Strauss BS et al.; We isolated several new mutator mutations of the Escherichia coli replicative polymerase dnaE subunit alpha and used them and a previously reported dnaE mutation to study spontaneous frameshift and base substitution mutations . Two of these dnaE strains produce many more mutants when grown on rich (Luria-Bertani) than on minimal medium . A differential effect of the medium was not observed when these dnaE mutations were combined with a mismatch repair mutation . The selection scheme for the dnaE mutations required that they be able to complement a temperature-sensitive strain . However, the ability to complement is not related to the mutator effect for at least one of the mutants . Comparison of the mutation rates for frameshift and base substitution mutations in mutS and dnaE mutS strains suggests that the mismatch repair proteins respond differently to the two types of change . Deletion of dinB from both chromosome and plasmid resulted in a four- to fivefold decrease in the rate of frameshift and base substitution mutations in a dnaE mutS double mutant background . This reduction indicates that most mistakes in replication occur as a result of the action of the auxiliary rather than the replicative polymerase in this dnaE mutant . Deletion of dinB from strains carrying a wild-type dnaE had a measurable effect, suggesting that a fraction of spontaneous mutations occur as a result of dinB polymerase action even in cells with a normal replicative polymerase. J Bacteriol, 2000 Dec, 182(23), 6679 - 86 Catalase-peroxidases of Legionella pneumophila: cloning of the katA gene and studies of KatA function; Bandyopadhyay P et al.; Legionella pneumophila, the causative organism of Legionnaires' pneumonia, contains two enzymes with catalatic and peroxidatic activity, KatA and KatB . To address the issue of redundant, overlapping, or discrete in vivo functions of highly homologous catalase-peroxidases, the gene for katA was cloned and its function was studied in L . pneumophila and Escherichia coli and compared with prior studies of katB in this laboratory . katA is induced during exponential growth and is the predominant peroxidase in stationary phase . When katA is inactivated, L . pneumophila is more sensitive to exogenous hydrogen peroxide and less virulent in the THP-1 macrophage cell line, similar to katB . Catalatic-peroxidatic activity with different peroxidatic cosubstrates is comparable for KatA and KatB, but KatA is five times more active towards dianisidine . In contrast with these examples of redundant or overlapping function, stationary-phase survival is decreased by 100- to 10,000-fold when katA is inactivated, while no change from wild type is seen for the katB null . The principal clue for understanding this discrete in vivo function was the demonstration that KatA is periplasmic and KatB is cytosolic . This stationary-phase phenotype suggests that targets sensitive to hydrogen peroxide are present outside the cytosol in stationary phase or that the peroxidatic activity of KatA is critical for stationary-phase redox reactions in the periplasm, perhaps disulfide bond formation . Since starvation-induced stationary phase is a prerequisite to acquisition of virulence by L . pneumophila, further studies on the function and regulation of katA in stationary phase may give insights on the mechanisms of infectivity of this pathogen. J Bacteriol, 2000 Dec, 182(23), 6667 - 72 S-Adenosylmethionine decarboxylase from the archaeon Methanococcus jannaschii: identification of a novel family of pyruvoyl enzymes; Kim AD et al.; Polyamines are present in high concentrations in archaea, yet little is known about their synthesis, except by extrapolation from bacterial and eucaryal systems . S-Adenosylmethionine (AdoMet) decarboxylase, a pyruvoyl group-containing enzyme that is required for spermidine biosynthesis, has been previously identified in eucarya and Escherichia coli . Despite spermidine concentrations in the Methanococcales that are several times higher than in E . coli, no AdoMet decarboxylase gene was recognized in the complete genome sequence of Methanococcus jannaschii . The gene encoding AdoMet decarboxylase in this archaeon is identified herein as a highly diverged homolog of the E . coli speD gene (less than 11% identity) . The M . jannaschii enzyme has been expressed in E . coli and purified to homogeneity . Mass spectrometry showed that the enzyme is composed of two subunits of 61 and 63 residues that are derived from a common proenzyme; these proteins associate in an (alphabeta)(2) complex . The pyruvoyl-containing subunit is less than one-half the size of that in previously reported AdoMet decarboxylases, but the holoenzyme has enzymatic activity comparable to that of other AdoMet decarboxylases . The sequence of the M . jannaschii enzyme is a prototype of a class of AdoMet decarboxylases that includes homologs in other archaea and diverse bacteria . The broad phylogenetic distribution of this group suggests that the canonical SpeD-type decarboxylase was derived from an archaeal enzyme within the gamma proteobacterial lineage . Both SpeD-type and archaeal-type enzymes have diverged widely in sequence and size from analogous eucaryal enzymes. J Bacteriol, 2000 Dec, 182(23), 6630 - 7 Expression of different-size transcripts from the clpP-clpX operon of Escherichia coli during carbon deprivation; Li C et al.; Transcription of the clpP-clpX operon of Escherichia coli leads to the production of two different sizes of transcripts . In log phase, the level of the longer transcript is higher than the level of the shorter transcript . Soon after the onset of carbon starvation, the level of the shorter transcript increases significantly, and the level of the longer transcript decreases . The longer transcript consists of the entire clpP-clpX operon, whereas the shorter transcript contains the entire clpP gene but none of the clpX coding sequence . The RpoH protein is required for the increase in the level of the shorter transcript during carbon starvation . Primer extension experiments suggest that there is increased usage of the sigma(32)-dependent promoter of the clpP-clpX operon within 15 min after the start of carbon starvation . Expression of the clpP-clpX operon from the promoters upstream of the clpP gene decreases to a very low level by 20 min after the onset of carbon starvation . Various pieces of evidence suggest, though they do not conclusively prove, that production of the shorter transcript may involve premature termination of the longer transcript . The half-life of the shorter transcript is much less than that of the longer transcript during carbon starvation . E . coli rpoB mutations that affect transcription termination efficiency alter the ratio of the shorter clpP-clpX transcript to the longer transcript . The E . coli rpoB3595 mutant, with an RNA polymerase that terminates transcription with lower efficiency than the wild type, accumulates a lower percentage of the shorter transcript during carbon starvation than does the isogenic wild-type strain . In contrast, the rpoB8 mutant, with an RNA polymerase that terminates transcription with higher efficiency than the wild type, produces a higher percentage of the shorter clpP-clpX transcript when E . coli is in log phase . These and other data are consistent with the hypothesis that the shorter transcript results from premature transcription termination during production of the longer transcript. J Bacteriol, 2000 Dec, 182(23), 6598 - 604 Escherichia coli responses to a single DNA adduct; Pandya GA et al.; To study the mechanisms by which Escherichia coli modulates the genotoxic effects of DNA damage, a novel system has been developed which permits quantitative measurements of various E . coli pathways involved in mutagenesis and DNA repair . Events measured include fidelity and efficiency of translesion DNA synthesis, excision repair, and recombination repair . Our strategy involves heteroduplex plasmid DNA bearing a single site-specific DNA adduct and several mismatched regions . The plasmid replicates in a mismatch repair-deficient host with the mismatches serving as strand-specific markers . Analysis of progeny plasmid DNA for linkage of the strand-specific markers identifies the pathway from which the plasmid is derived . Using this approach, a single 1, N(6)-ethenodeoxyadenosine adduct was shown to be repaired inefficiently by excision repair, to inhibit DNA synthesis by approximately 80 to 90%, and to direct the incorporation of correct dTMP opposite this adduct . This approach is especially useful in analyzing the damage avoidance-tolerance mechanisms . Our results also show that (i) progeny derived from the damage avoidance-tolerance pathway(s) accounts for more than 15% of all progeny; (ii) this pathway(s) requires functional recA, recF, recO, and recR genes, suggesting the mechanism to be daughter strand gap repair; (iii) the ruvABC genes or the recG gene is also required; and (iv) the RecG pathway appears to be more active than the RuvABC pathway . Based on these results, the mechanism of the damage avoidance-tolerance pathway is discussed. J Bacteriol, 2000 Dec, 182(23), 6592 - 7 The unphosphorylated receiver domain of PhoB silences the activity of its output domain; Ellison DW et al.; PhoB is the response regulator of the Pho regulon . It is composed of two distinct domains, an N-terminal receiver domain and a C-terminal output domain that binds DNA and interacts with sigma(70) to activate transcription of the Pho regulon . Phosphorylation of the receiver domain is required for activation of the protein . The mechanism of activation by phosphorylation has not yet been determined . To better understand the function of the receiver domain in controlling the activity of the output domain, a direct comparison was made between unphosphorylated PhoB and its solitary DNA-binding domain (PhoB(DBD)) for DNA binding and transcriptional activation . Using fluorescence anisotropy, it was found that PhoB(DBD) bound to the pho box with an affinity seven times greater than that of unphosphorylated PhoB . It was also found that PhoB(DBD) was better able to activate transcription than the full-length, unmodified protein . We conclude that the unphosphorylated receiver domain of PhoB silences the activity of its output domain . These results suggest that upon phosphorylation of the receiver domain of PhoB, the inhibition placed upon the output domain is relieved by a conformational change that alters interactions between the unphosphorylated receiver domain and the output domain. J Bacteriol, 2000 Dec, 182(23), 6570 - 6 Vanadate-induced trapping of nucleotides by purified maltose transport complex requires ATP hydrolysis; Sharma S et al.; The maltose transport system in Escherichia coli is a member of the ATP-binding cassette superfamily of transporters that is defined by the presence of two nucleotide-binding domains or subunits and two transmembrane regions . The bacterial import systems are unique in that they require a periplasmic substrate-binding protein to stimulate the ATPase activity of the transport complex and initiate the transport process . Upon stimulation by maltose-binding protein, the intact MalFGK(2) transport complex hydrolyzes ATP with positive cooperativity, suggesting that the two nucleotide-binding MalK subunits interact to couple ATP hydrolysis to transport . The ATPase activity of the intact transport complex is inhibited by vanadate . In this study, we investigated the mechanism of inhibition by vanadate and found that incubation of the transport complex with MgATP and vanadate results in the formation of a stably inhibited species containing tightly bound ADP that persists after free vanadate and nucleotide are removed from the solution . The inhibited species does not form in the absence of MgCl(2) or of maltose-binding protein, and ADP or another nonhydrolyzable analogue does not substitute for ATP . Taken together, these data conclusively show that ATP hydrolysis must precede the formation of the vanadate-inhibited species in this system and implicate a role for a high-energy, ADP-bound intermediate in the transport cycle . Transport complexes containing a mutation in a single MalK subunit are still inhibited by vanadate during steady-state hydrolysis; however, a stably inhibited species does not form . ATP hydrolysis is therefore necessary, but not sufficient, for vanadate-induced nucleotide trapping. Plant Sci, 2000 Nov 6, 159(2), 281 - 288 Effects of overexpression of Escherichia coli katE and bet genes on the tolerance for salt stress in a freshwater cyanobacterium Synechococcus sp . PCC 7942; Kaku N et al.; To explore the potential role of catalase and glycine betaine in the protection of cyanobacteria from damage due to salt stress, we transformed a freshwater cyanobacterium Synechococcus sp . PCC 7942 with shuttle vectors that contained the Escherichia coli katE, bet, and katE plus bet (katE/bet) genes . The catalase activity in the cells overexpressing katE and katE/bet genes was about 1.4-1.8-fold higher than that in the control cells . The control and transformant cells had a similar growth rate in the medium with a low salinity . However, under a high-salinity condition, the cells transformed with katE grew faster than the control cells, and the cells expressing katE/bet genes grew faster than those expressing either the katE or bet gene alone . These results indicate that high-salinity caused oxidative stress and the coexpression of katE and bet genes in Synechococcus cells were superior to the expression of either katE or bet alone for the protection of the cells from the damage due to high-salinity. J Biol Chem, 2001 Mar 2, 276(9), 6243 - 52 Epub 2000 Nov 09. Sequence recognition, cooperative interaction, and dimerization of the initiator protein DnaA of Streptomyces; Majka J et al.; Using a combined PCR-gel retardation assay, the preferred recognition sequence of the Streptomyces initiator protein DnaA was determined . The protein showed a preference toward DNA containing two Escherichia coli-like DnaA boxes in a head-to-head arrangement (consensus sequence TTATCCACA, whereas the consensus sequence of the DnaA boxes found in the Streptomyces oriC region is TTGTCCACA) . In quantitative band shift experiments, the kinetics of the Streptomyces DnaA-DnaA box interaction was characterized . The DnaA protein can form dimers while binding to a single DnaA box; dimer formation is mediated by the domain III of the protein, and the dissociation constant of this process was between 35 and 115 nm . Streptomyces initiator protein DnaA interacts in a cooperative manner with DNA containing multiple binding sites . For the cooperativity effect, which seems to be independent of the distance separating the DnaA boxes, domain I (or I and II) is responsible . The cooperativity constant is moderate and is in the range of 20-110. J Biol Chem, 2001 Mar 2, 276(9), 6499 - 505 Epub 2000 Nov 09. Reconstitution of mitochondrial processing peptidase from the core proteins (subunits I and II) of bovine heart mitochondrial cytochrome bc(1) complex; Deng K et al.; Mature core I and core II proteins of the bovine heart mitochondrial cytochrome bc(1) complex were individually overexpressed in Escherichia coli as soluble proteins using the expression vector pET-I and pET-II, respectively . Purified recombinant core I and core II alone show no mitochondrial processing peptidase (MPP) activity . When these two proteins are mixed together, MPP activity is observed . Maximum activity is obtained when the molar ratio of these two core proteins reaches 1 . This indicates that only the two core subunits of thebc(1) complex are needed for MPP activity . The properties of reconstituted MPP are similar to those of Triton X-100-activated MPP in the bovine bc(1) complex . When Rieske iron-sulfur protein precursor is used as substrate for reconstituted MPP, the processing activity stops when the amount of product formation (subunit IX) equals the amount of reconstituted MPP used in the system . Addition of Triton X-100 to the product-inhibited reaction mixture restores MPP activity, indicating that Triton X-100 dissociates bound subunit IX from the active site of reconstituted MPP . The aromatic group, rather than the hydroxyl group, at Tyr(57) of core I is essential for reconstitutive activity. J Biol Chem, 2001 Apr 13, 276(15), 11545 - 51 Epub 2000 Oct 30. Electron transfer and binding of the c-type cytochrome TorC to the trimethylamine N-oxide reductase in Escherichia coli; Gon S et al.; Reduction of trimethylamine N-oxide (E'(0(TMAO/TMA)) = +130 mV) in Escherichia coli is carried out by the Tor system, an electron transfer chain encoded by the torCAD operon and made up of the periplasmic terminal reductase TorA and the membrane-anchored pentahemic c-type cytochrome TorC . Although the role of TorA in the reduction of trimethylamine N-oxide (TMAO) has been clearly established, no direct evidence for TorC involvement has been presented . TorC belongs to the NirT/NapC c-type cytochrome family based on homologies of its N-terminal tetrahemic domain (TorC(N)) to the cytochromes of this family, but TorC contains a C-terminal extension (TorC(C)) with an additional heme-binding site . In this study, we show that both domains are required for the anaerobic bacterial growth with TMAO . The intact TorC protein and its two domains, TorC(N) and TorC(C), were produced independently and purified for a biochemical characterization . The reduced form of TorC exhibited visible absorption maxima at 552, 523, and 417 nm . Mediated redox potentiometry of the heme centers of the purified components identified two negative midpoint potentials (-177 and -98 mV) localized in the tetrahemic TorC(N) and one positive midpoint potential (+120 mV) in the monohemic TorC(C) . In agreement with these values, the in vitro reconstitution of electron transfer between TorC, TorC(N), or TorC(C) and TorA showed that only TorC and TorC(C) were capable of electron transfer to TorA . Surprisingly, interaction studies revealed that only TorC and TorC(N) strongly bind TorA . Therefore, TorC(C) directly transfers electrons to TorA, whereas TorC(N), which probably receives electrons from the menaquinone pool, is involved in both the electron transfer to TorC(C) and the binding to TorA. Mol Vis, 2000 Nov 08, 6, 232 - 6 Inability of chaperones to fold mutant zeta crystallin, an aggregation-prone eye lens protein; Goenka S et al.; PURPOSE: zeta-crystallin is a quinone oxido-reductase, recruited in the eye lens of hystricomorphic rodents and camels . A deletion mutation constituting the NADPH-binding domain causes congenital cataract in a strain of guinea pigs . The presence of large quantities of a-crystallin, a molecular chaperone, does not provide any protection against this . In order to investigate whether the underlying reason for the lack of protection is the formation of a folding-incompetent protein, we have expressed the mutant protein in a heterologous system along with other known chaperones . METHODS: We expressed the mutant zeta-crystallin in E . coli along with other chaperones such as GroEL/ES and DnaK/DnaJ/GrpE and then analyzed whether these chaperones could increase the amount of protein partitioning into the soluble fraction of E . coli cells . RESULTS: These chaperones were unable to rescue the mutant protein from partitioning into inclusion bodies, although they could increase the yield of soluble wild-type zeta-crystallin . CONCLUSIONS: The deletion of 34 amino acids, constituting the NADPH-binding domain of zeta-crystallin, makes the protein incompetent to fold correctly and thus form insoluble aggregates . It perhaps suggests why the mutant strain of guinea pigs have cataract at birth even though their lenses contain high amounts of alpha-crystallin . This study also shows that certain mutations can render proteins incompetent to fold into soluble molecules despite abundant assistance. RNA, 2000 Oct, 6(10), 1403 - 12 NMR and biochemical characterization of recombinant human tRNA(Lys)3 expressed in Escherichia coli: identification of posttranscriptional nucleotide modifications required for efficient initiation of HIV-1 reverse transcription; Tisne C et al.; Reverse transcription of HIV-1 viral RNA uses human tRNA(Lys)3 as a primer . Some of the modified nucleotides carried by this tRNA must play a key role in the initiation of this process, because unmodified tRNA produced in vitro is only marginally active as primer . To provide a better understanding of the contribution of base modifications in the initiation complex, we have designed a recombinant system that allows tRNA(Lys)3 expression in Escherichia coli . Because of their high level of overexpression, some modifications are incorporated at substoichiometric levels . We have purified the two major recombinant tRNA(Lys)3 subspecies, and their modified nucleotide contents have been characterized by a combination of NMR and biochemical techniques . Both species carry psis, Ds, T, t6A, and m7G . Differences are observed at position 34, within the anticodon . One fraction lacks the 5-methylaminomethyl group, whereas the other lacks the 2-thio group . Although the s2U34-containing recombinant tRNA is a less efficient primer, it presents most of the characteristics of the mammalian tRNA . On the other hand, the mnm5U34-containing tRNA has a strongly reduced activity . Our results demonstrate that the modifications that are absent in E . coli (m2G10, psi27, m5C48, m5C49, and m1A58) as well as the mnm5 group at position 34 are dispensable for initiation of reverse transcription . In contrast, the 2-thio group at position 34 seems to play an important part in this process. RNA, 2000 Oct, 6(10), 1393 - 402 Translation during cold adaptation does not involve mRNA-rRNA base pairing through the downstream box; La Teana A et al.; The downstream box (DB) has been proposed to enhance translation of several mRNAs and to be a key element controlling the expression of cold-shocked mRNAs . However, the proposal that the DB exerts its effects through a base pairing interaction with the complementary anti-downstream box (antiDB) sequence (nt 1469-1483) located in the penultimate stem (helix 44) of 16S rRNA remains controversial . The existence of this interaction during initiation of protein synthesis under cold-shock conditions has been investigated in the present work using an Escherichia coli strain whose ribosomes lack the potential to base pair with mRNA because of a 12 bp inversion of the antiDB sequence in helix 44 . Our results show that this strain is capable of cold acclimation, withstands cold shock, and its ribosomes translate mRNAs that contain or lack DB sequences with similar efficiency, comparable to that of the wild type . The structure of helix 44 in 30S ribosomal subunits from cells grown at 37 degrees C and from cells subjected to cold shock was also analyzed by binding a 32P-labeled oligonucleotide complementary to the antiDB region and by chemical probing with DMS and kethoxal . Both approaches clearly indicate that this region is in a double-stranded conformation and therefore not available for base pairing with mRNA. RNA, 2000 Oct, 6(10), 1356 - 79 Search for characteristic structural features of mammalian mitochondrial tRNAs; Helm M et al.; A number of mitochondrial (mt) tRNAs have strong structural deviations from the classical tRNA cloverleaf secondary structure and from the conventional L-shaped tertiary structure . As a consequence, there is a general trend to consider all mitochondrial tRNAs as "bizarre" tRNAs . Here, a large sequence comparison of the 22 tRNA genes within 31 fully sequenced mammalian mt genomes has been performed to define the structural characteristics of this specific group of tRNAs . Vertical alignments define the degree of conservation/variability of primary sequences and secondary structures and search for potential tertiary interactions within each of the 22 families . Further horizontal alignments ascertain that, with the exception of serine-specific tRNAs, mammalian mt tRNAs do fold into cloverleaf structures with mostly classical features . However, deviations exist and concern large variations in size of the D- and T-loops . The predominant absence of the conserved nucleotides G18G19 and T54T55C56, respectively in these loops, suggests that classical tertiary interactions between both domains do not take place . Classification of the tRNA sequences according to their genomic origin (G-rich or G-poor DNA strand) highlight specific features such as richness/poorness in mismatches or G-T pairs in stems and extremely low G-content or C-content in the D- and T-loops . The resulting 22 "typical" mammalian mitochondrial sequences built up a phylogenetic basis for experimental structural and functional investigations . Moreover, they are expected to help in the evaluation of the possible impacts of those point mutations detected in human mitochondrial tRNA genes and correlated with pathologies. J Leukoc Biol, 2000 Nov, 68(5), 627 - 32 Dynamics of rab5 activation in endocytosis and phagocytosis; Roberts RL et al.; Fluid-phase endocytosis is stimulated by H-ras-linked growth factor receptors and this stimulation requires activation of rab5 . We utilized a GFP-rab5a:wt fusion protein to monitor GFP-rab5a:wt activation in living fibroblasts and in J774 macrophages . Control CHO cells that expressed GFP-rab5a:wt were cultured in serum-free conditions and showed GFP-rab5a:wt localized to endosomal vesicles with a mean diameter of 0.3 +/- 0.1 microm . Endosome fusion, membrane ruffling, and pinosome formation were rarely detected in these cells . Coexpression of H-ras:G12V, a constitutively active H-ras mutant that activates rab5a, in cells resulted in marked enlargement of labeled endosomes (mean diameter 0.7 +/- 0.2 microm) and large numbers of giant GFP-rab5a:wt-positive endosomes were present . Time-lapse recordings showed abundant fusion among giant labeled endosomes, and membrane ruffling and pinosome formation were commonly observed . Alterations in GFP-rab5a:wt endosome structure and activity in cells expressing H-ras:G12V were linked to rab5a activation because these changes were identical to those found in cells expressing GFP-rab5a:Q79L, a constitutively activated rab5a mutant . Furthermore, cells co-expressing H-ras:G12V and GFP-rab5a:S34N, an inactive rab5a mutant, exhibited no evidence of H-ras:G12V-induced endosome enlargement . To observe changes in endosome structure and activity that directly followed activation of GFP-rab5a:wt, we performed time-lapse recordings of cells cultured overnight in serum-free media after addition of EGF . EGF caused a rapid increase in endosome fusion and in membrane ruffling activity . Membrane ruffling was often associated with GFP-rab5a:wt-positive vesicle (pinosome) formation at the base of membrane ruffles . Endosome and pinosome fusion were common in EGF-stimulated cells . Phagocytosis is also regulated by GFP-rab5a:wt . J774 macrophages that expressed GFP-rab5a:wt showed transiently activation and recruitment of GFP-rab5a:wt to newly formed phagosomes that contained rhodamine-labeled Escherichia coli . These studies show that GFP-rab5a:wt activation results in dynamic alterations in the structure and activity of the early endosomal and early phagosomal elements. J Vet Med Sci, 2000 Oct, 62(10), 1101 - 4 Expression of porcine interleukin-2 in Escherichia coli; Iwata H et al.; A mature form of porcine interleukin-2 (IL-2) protein without signal peptides was expressed as glutathione S-transferase (GST) fusion proteins in Escherichia coli using pGEX vector . Since most of GST-IL-2 fusion protein was detected in an insoluble fraction on SDS-PAGE analysis, the insoluble fusion protein was solubilized by refolding procedure using urea . The recombinant IL-2 (rIL-2) was purified by a batch method using Glutathione Sepharose 4B and factor Xa digestion and used for preparation of antisera in mice . The antisera reacted with rIL-2 expressed in baculovirus system on immunoblot analysis . In addition, the purified rIL-2 showed a high biological activity on CTLL-2 proliferative response. Biochim Biophys Acta, 2000 Nov 15, 1494(1-2), 129 - 36 Sheep cytosolic branched-chain amino acid aminotransferase: cDNA cloning, primary structure and molecular modelling and its unique expression in muscles; Bonfils J et al.; This paper presents the cloning and the molecular modelling of the cytosolic branched-chain amino acid aminotransferase (BCATc) from sheep brain . The sheep BCATc cDNA (3 kb) encodes a mature polypeptide of 385 amino acids with a calculated molecular mass of 43072.93 Da . The sequence of the sheep BCATc cDNA is more similar to other mammalian BCATc cDNAs (53-87% identical) than to the sheep mitochondrial branched-chain amino acid aminotransferase (52%) . Sheep BCATc belongs to the IV Folding class of pyridoxal-5'-phosphate-depending enzymes . Based on the known structure of the branched-chain amino acid aminotransferase (BCAT) from Escherichia coli, a molecular model of sheep BCATc (amino acid residues 62-385) was built . This is the first three-dimensional model of any mammalian BCAT . It suggests that the enzymatic mechanism of sheep BCATc and likely of all mammalian BCAT is very similar to the mechanism of the E . coli BCAT and confirms the hypotheses regarding to the substrate binding sites of E . coli BCAT . Sheep skeletal muscle, which is the main in vivo site for transamination of branched-chain amino acids, exhibits an unique expression of BCATc. Biochim Biophys Acta, 2000 Nov 15, 1494(1-2), 43 - 53 The LipB protein is a negative regulator of dam gene expression in Escherichia coli; Vaisvila R et al.; Transcription initiation of the major promoter (P2) of the Escherichia coli dam gene increases with growth rate . The presence of three partially palindromic motifs, (TTCAGT(N(20))TGAG), designated G (growth)-boxes, within the -52 to +31 region of the promoter, may be related to growth rate control . Deletion of two of these repeats, downstream of the transcription initiation point, result in constitutive high activity of the promoter . The unlinked cde-4::miniTn10 insertion also results in severalfold higher activity of the dam P2 promoter, suggesting that this mutation resulted in the loss of a putative dam P2 repressor . The cde-4 mutation was mapped to the lipB (lipoic acid) gene, which we show encodes a 24 kDa protein initiating at a TTG codon . LipB is a highly conserved protein in animal and plant species, other bacteria, Archaea, and yeast . Plasmids expressing the native or hexahistidine-tagged LipB complement the phenotype of the cde-4 mutant strain . The level of LipB in vivo was higher in exponentially growing cells than those in the stationary phase . Three G-box motifs were also found in the lipB region . Models for the regulation of expression of the two genes are discussed. Nucleic Acids Res . 2000 Nov 15;28(22):E97. A rapid method for efficient gene replacement in the filamentous fungus Aspergillus nidulans; Chaveroche MK et al.; The construction of mutant fungal strains is often limited by the poor efficiency of homologous recombination in these organisms . Higher recombination efficiencies can be obtained by increasing the length of homologous DNA flanking the transformation marker, although this is a tedious process when standard molecular biology techniques are used for the construction of gene replacement cassettes . Here, we present a two-step technology which takes advantage of an Escherichia coli strain expressing the phage lambda Red(gam, bet, exo) functions and involves (i) the construction in this strain of a recombinant cosmid by in vivo recombination between a cosmid carrying a genomic region of interest and a PCR-generated transformation marker flanked by 50 bp regions of homology with the target DNA and (ii) genetic exchange in the fungus itself between the chromosomal locus and the circular or linearized recombinant cosmid . This strategy enables the rapid establishment of mutant strains carrying gene knock-outs with efficiencies >50% . It should also be appropriate for the construction of fungal strains with gene fusions or promoter replacements. Nucleic Acids Res, 2000 Nov 15, 28(22), 4566 - 72 Biochemical characterization of I-CmoeI reveals that this H-N-H homing endonuclease shares functional similarities with H-N-H colicins; Drouin M et al.; Endonuclease assays of the H-N-H proteins encoded by two group I introns in the Chlamydomonas moewusii chloroplast psbA gene revealed that the CmpsbA.1 intron specifies a site-specific DNA endonuclease, designated I-CMOE:I . Like most previously reported intron-encoded endonucleases, I-CMOE:I generates a double-strand break near the insertion site of its encoding intron, leaving 3' extensions of 4 nt . This enzyme was purified from Escherichia coli as a fusion protein with a His tag at its N-terminus . The recombinant protein (rI-CMOE:I) requires a divalent alkaline earth cation for DNA cleavage (Mg(2+) > Ca(2+) > Sr(2+) > Ba(2+)) . It also requires a metal cofactor for DNA binding, a property shared with H-N-H colicins but not with the homing endonucleases characterized to date . rI-CMOE:I binds its recognition sequence as a monomer, as revealed by gel retardation assays . K:(m) and k(cat) values of 100 +/- 40 pM and 0.26 +/- 0.04 min(-1), respectively, were determined . Replacement of the first histidine of the H-N-H motif by an alanine residue abolishes both rI-CMOE:I activity and binding to its substrate . We propose that this conserved histidine residue plays a role in binding the metal cofactor and that such binding induces a structural modification of the enzyme which is required for DNA recognition. Nucleic Acids Res, 2000 Nov 15, 28(22), 4544 - 51 Biochemical characterization of the hjc holliday junction resolvase of Pyrococcus furiosus; Komori K et al.; The Hjc protein of Pyrococcus furiosus is an endonuclease that resolves Holliday junctions, the intermediates in homologous recombination . The amino acid sequence of Hjc is conserved in Archaea, however, it is not similar to any of the well-characterized Holliday junction resolvases . In order to investigate the similarity and diversity of the enzymatic properties of Hjc as a Holliday junction resolvase, highly purified Hjc produced in recombinant Escherichia coli was used for detailed biochemical characterizations . Hjc has specific binding activity to the Holliday-structured DNA, with an apparent dissociation constant (K:(d)) of 60 nM . The dimeric form of Hjc binds to the substrate DNA . The optimal reaction conditions were determined using a synthetic Holliday junction as substrate . Hjc required a divalent cation for cleavage activity and Mg(2+) at 5-10 mM was optimal . Mn(2+) could substitute for Mg(2+), but it was much less efficient than Mg(2+) as the cofactor . The cleavage reaction was stimulated by alkaline pH and KCl at approximately 200 mM . In addition to the high specific activity, Hjc was found to be extremely heat stable . In contrast to the case of SULFOLOBUS:, the Holliday junction resolving activity detected in P . furiosus cell extract thus far is only derived from Hjc. Biochem Biophys Res Commun, 2000 Nov 11, 278(1), 72 - 8 Elongation factor-1 alpha is a novel substrate of rho-associated kinase; Izawa T et al.; Rho-associated kinase (Rho-kinase), which is activated by the Rho small GTPase, phosphorylates the myosin-binding subunit (MBS) of myosin phosphatase, myosin light chain (MLC), the ERM family proteins, and adducin, thereby regulating the formation of stress fibers, focal adhesions, microvillus formation, and cell motility . Here, to further understand the role of Rho-kinase in the regulation of the numerous cellular processes by Rho, we purified a novel substrate of Rho-kinase having a molecular mass of 48 kDa (p48) from a rat liver cytosol extract . Mass spectral analysis revealed p48 to be elongation factor-1 alpha (EF-1 alpha), which is known as an actin-binding protein besides a cofactor of polypeptide elongation . Rho-kinase directly phosphorylated recombinant EF-1alpha in vitro . A high- speed cosedimentation assay revealed that phosphorylation of EF-1 alpha by Rho-kinase decreased the binding activity of EF-1 alpha to filamentous actin (F-actin) . A low-speed sedimentation assay revealed that phosphorylation of EF-1 alpha by Rho-kinase decreased the F-actin-bundling activity . In addition, EF-1 alpha bound to MBS of myosin phosphatase, suggesting that both Rho-kinase and myosin phosphatase regulate the phosphorylation state of EF-1 alpha downstream of Rho as other substrates of Rho-kinase, i.e., MLC, adducin, and the ERM family . These results suggest that the Rho/Rho-kinase pathway regulates the organization of actin cytoskeleton via the phosphorylation of EF-1 alpha . J Mol Biol, 2000 Nov 17, 304(1), 43 - 53 Potentiating AZT activation: structures of wild-type and mutant human thymidylate kinase suggest reasons for the mutants' improved kinetics with the HIV prodrug metabolite AZTMP; Ostermann N et al.; The 60-fold reduced phosphorylation rate of azidothymidine (AZT) monophosphate (AZTMP), the partially activated AZT metabolite, by human thymidylate kinase (TMPK) severely limits the efficacy of this anti-HIV prodrug . Crystal structures of different TMPK nucleotide complexes indicate that steric hindrance by the azido group of AZTMP prevents formation of the catalytically active closed conformation of the P-loop of TMPK . The F105Y mutant and a chimeric mutant that contains sequences of the human and Escherichia coli enzyme phosphorylate AZTMP 20-fold faster than the wild-type enzyme . The structural basis of the increased activity is assigned to stabilization of the closed P-loop conformation . J Mol Biol, 2000 Nov 17, 304(1), 11 - 20 Deletion analysis defines distinct functional domains for protein-protein and nucleic acid interactions in the ORF1 protein of mouse LINE-1; Martin SL et al.; LINE-1, or L1, is a non-LTR retrotransposon in mammals . Retrotransposition of L1 requires the action of two element-encoded proteins, ORF1p and ORF2p . ORF2p provides essential enzymatic activities for the reverse transcription and integration of a newly transposed copy of L1, whereas the exact role of ORF1p is less well understood . The 43 kDa ORF1p copurifies as a large complex with L1 RNA in extracts of human and mouse cells . Mouse ORF1p purified from Escherichia coli binds RNA and single-stranded DNA in vitro, exhibits nucleic acid chaperone activity, and is capable of protein-protein interaction . In this study we create a series of deletions in the ORF1 sequence, express the truncated proteins and examine their activities to delineate the region of ORF1p responsible for these different functions . By both yeast two-hybrid analysis and GST pull-down assay, the protein-protein interaction domain is defined as a coiled-coil domain that encompasses about one third of the protein near its N terminus . Based on data obtained with UV-cross-linking, electrophoretic mobility-shift assay and an annealing assay, the C-terminal one third of ORF1p is both necessary and sufficient for nucleic acid binding and to promote annealing of complementary oligonucleotides . Separation of these activities into different domains of ORF1p will facilitate detailed biochemical analyses of the structure and function of this protein and understanding of its role during L1 retrotransposition . Genome Inform Ser Workshop Genome Inform, 1997, 8, 125 - 134 Clustering Molecular Sequences with Their Components; Suharnan S et al.; Motivation: Several methods in genetic information have recently been developed to estimate classification of protein sequences through their sequence similarity . These methods are essential for understanding the function of predicted open reading frames (ORFs) and their molecular evolutionary processes . However, since many protein sequences consist of a number of independently evolved structural units (we refer to these units as components), the combinatorial nature of the components makes it difficult to classify the sequences . Results: This paper presents a new method for classifying uncharacterized protein sequences . As the measure of sequence similarity, we use similarity score computed by a method based on the Smith-Waterman local alignment algorithm . Here we introduce how this method cope when sequences have multi-component structure . This method was applied to predicted ORFs on the Escherichia coli genome and we discuss the algorithm and experimental results. J Biol Chem, 2001 Feb 23, 276(8), 5975 - 84 Epub 2000 Nov 08. The regulation of the Escherichia coli mazEF promoter involves an unusual alternating palindrome; Marianovsky I et al.; The Escherichia coli mazEF system is a chromosomal "addiction module" that, under starvation conditions in which guanosine-3',5'-bispyrophosphate (ppGpp) is produced, is responsible for programmed cell death . This module specifies for the toxic stable protein MazF and the labile antitoxic protein MazE . Upstream from the mazEF module are two promoters, P(2) and P(3) that are strongly negatively autoregulated by MazE and MazF . We show that the expression of this module is positively regulated by the factor for inversion stimulation . What seems to be responsible for the negative autoregulation of mazEF is an unusual DNA structure, which we have called an "alternating palindrome." The middle part, "a," of this structure may complement either the downstream fragment, "b," or the upstream fragment, "c" . When the MazE.MazF complex binds either of these arms of the alternating palindrome, strong negative autoregulation results . We suggest that the combined presence of the two promoters, the alternating palindrome structure and the factor for inversion stimulation-binding site, all permit the expression of the mazEF module to be sensitively regulated under various growth conditions. J Biol Chem, 2001 Feb 9, 276(6), 4182 - 9 Epub 2000 Nov 08. RNA passes through the hole of the protein hexamer in the complex with the Escherichia coli Rho factor; Burgess BR et al.; Escherichia coli transcription termination factor Rho is a ring-shaped hexameric protein that uses the energy derived from ATP hydrolysis to dissociate RNA transcripts from the ternary elongation complex . To test a current model for the interaction of Rho with RNA, three derivatives of Rho were made containing single cysteine residues and modified with a photo-activable cross-linker . The positions for the cysteines were: 1) in part of the primary RNA-binding site in the N terminus (Cys-82 Rho); 2) in a connecting polypeptide proposed to be on the outside of the hexamer (Cys-153 Rho); and 3) near the proposed secondary RNA-binding site in the ATP-binding domain (Cys-325 Rho) . Results from the cross-linking of the modified Rho proteins to a series of lambda cro RNA derivatives showed that Cys-82 Rho formed cross-links with all transcripts containing the Rho utilization (rut) site, that Cys-325 Rho formed cross-links to transcripts that had the rut site and 10 or more residues 3' of the rut site, and that Cys-153 did not form cross-links with any of the transcripts . From a model of the quaternary structure of Rho, which is largely based on homology to the F(1)-ATPase, amino acid 82 is located near the top of the hexamer, and amino acid 325 is located on a solvent-accessible loop in the center of the hexamer . These data are consistent with binding of the rut region of RNA around the crown, with its 3'-segment passing through the center of the Rho hexamer. Mol Biochem Parasitol, 2000 Oct, 110(2), 283 - 91 Gene organization of a Plasmodium falciparum serine hydroxymethyltransferase and its functional expression in Escherichia coli; Alfadhli S et al.; The global emergence of drug-resistant malarial parasites necessitates identification and characterization of novel drug targets . Three reactions are involved in methylenetetrahydrofolate recycling: Thymidylate synthase (TS), dihydrofolate reductase (DHFR), and serine hydroxymethyltransferase (SHMT) . Malarial bifunctional DHFR-TS is a well-studied, important target of established drugs such as pyrimethamine and cycloguanil . In sharp contrast, malarial SHMT remains largely uncharacterized . In the present study, a Plasmodium falciparum SHMT coding region was characterized . It had 1603 bp including two introns near the 5'-end of the gene: one 118 bp intron immediately after the start methionine and a 159 bp intron after an additional 34 amino acids . The three exons together coded for a 442 amino acid protein with 38-47% identity to SHMT sequences from other species . Expression of malarial SHMT coding sequence (minus the introns) into glyA mutants of Escherichia coli relieved glycine auxotrophy and permitted direct assay of SHMT catalytic activity in bacterial cell lysates . This is the first SHMT cloned and expressed from a protozoan parasite . The molecular tools developed in this study will be useful for developing potential antimalarials directed at SHMT. Mol Biochem Parasitol, 2000 Oct, 110(2), 207 - 21 PPB1, a putative spliced leader RNA gene transcription factor in Trypanosoma cruzi; Wen LM et al.; In trypanosomatids, the spliced leader RNA, or SL RNA, donates its 5' 39 nucleotides to mature nuclear mRNAs in a process termed trans-splicing . We have previously characterized the SL RNA gene from Trypanosoma cruzi and identified its transcription promoter, including a 14 nt proximal sequence element, or PSE, that binds a putative transcription factor and activates transcription of the gene . Herein, we describe establishment of a yeast one-hybrid system using the 14 nt PSE as bait, and use this system to select T . cruzi cDNAs encoding a putative transcription factor that activates transcription of the SL RNA gene . The cDNA was selected from a normalized library and encodes an approximately 45 kDa putative PSE promoter-binding protein, PPB1 . PPB1 in vitro translated or overexpressed in and isolated from transformed E . coli, showed PSE-specific binding activity by electrophoretic mobility shift assays . Finally, overexpression of PPB1 in T . cruzi led to increased expression of the SL RNA gene as well as reporter genes in episomal constructs under the control of the SL RNA gene promoter . These observations suggest that PPB1 is a transcription factor that plays an important role in SL RNA gene expression. Mol Biol Evol, 2000 Nov, 17(11), 1610 - 9 Evolution of sequence recognition by restriction-modification enzymes: selective pressure for specificity decrease; Chinen A et al.; Several type II restriction-modification (RM) gene complexes kill host bacterial cells that have lost them, through attack on the chromosomal recognition sites of these cells . Two RM gene complexes recognizing the same sequence cannot simultaneously enjoy such stabilization through postsegregational host killing, because one will defend chromosomal sites from attack by the other . In the present work, we analyzed intrahost competition between two RM gene complexes when the recognition sequence of one was included in that of the other . When the EcoRII gene complex, recognizing 5'-CCWGG (W = A, T), is lost from the host, the SsoII gene complex, which recognizes 5'-CCNGG (N = A, T, G, C), will prevent host death by protecting CCWGG sites on the chromosome . However, when the SsoII (CCNGG) gene complex is lost, the EcoRII (CCWGG) gene complex will be unable to prevent host death through attack by SsoII on 5'-CCSGG (S = C, G) sites . These predictions were verified in our experiments, in which we analyzed plasmid maintenance, cell growth, cell shape, and chromosomal DNA . Our results demonstrate the presence of selective pressure for decrease in the specificity of recognition sequence of RM systems in the absence of invading DNA. J Virol, 2000 Dec, 74(23), 11254 - 61 Characterization of promoter function and cell-type-specific expression from viral vectors in the nervous system; Smith RL et al.; Viral vectors have become important tools to effectively transfer genes into terminally differentiated cells, including neurons . However, the rational for selection of the promoter for use in viral vectors remains poorly understood . Comparison of promoters has been complicated by the use of different viral backgrounds, transgenes, and target tissues . Adenoviral vectors were constructed in the same vector background to directly compare three viral promoters, the human cytomegalovirus (CMV) immediate-early promoter, the Rous sarcoma virus (RSV) long terminal repeat, and the adenoviral E1A promoter, driving expression of the Escherichia coli lacZ gene or the gene for the enhanced green fluorescent protein . The temporal patterns, levels of expression, and cytotoxicity from the vectors were analyzed . In sensory neuronal cultures, the CMV promoter produced the highest levels of expression, the RSV promoter produced lower levels, and the E1A promoter produced limited expression . There was no evidence of cytotoxicity produced by the viral vectors . In vivo analyses following stereotaxic injection of the vector into the rat hippocampus demonstrated differences in the cell-type-specific expression from the CMV promoter versus the RSV promoter . In acutely prepared hippocampal brain slices, marked differences in the cell type specificity of expression from the promoters were confirmed . The CMV promoter produced expression in hilar regions and pyramidal neurons, with minimal expression in the dentate gyrus . The RSV promoter produced expression in dentate gyrus neurons . These results demonstrate that the selection of the promoter is critical for the success of the viral vector to express a transgene in specific cell types. Am J Respir Crit Care Med, 2000 Nov, 162(5), 1656 - 61 Endotoxin-induced lung maturation in preterm lambs is not mediated by cortisol; Jobe AH et al.; Antenatal exposure to glucocorticoids, amnionitis, intraamniotic interleukin (IL)-1alpha, or endotoxin can improve postnatal lung function after preterm delivery . The relationship between early lung maturation and the dose and duration of a proinflammatory stimulus has not been evaluated . The effects of proinflammatory stimuli on fetal plasma cortisol also have not been evaluated . We hypothesized that intraamniotic endotoxin would induce early lung maturation in fetal sheep without increasing fetal cortisol . Intraamniotic injections of 1, 4, 20, or 100 mg of Escherichia coli 055:beta5 endotoxin caused 2-fold increases in compliance, 4- to 5-fold increases in lung gas volumes, and 20-fold increases in alveolar saturated phosphatidylcholine (Sat PC) when given 7 d before preterm delivery at 125 d gestation . Animals treated with 20 mg endotoxin for treatment to delivery intervals of 5 h to 15 d had no significant elevations in cord plasma cortisol levels . Increases in Sat PC in lung tissue and alveolar washes were detected 2 d after endotoxin treatment and lung function improved 4 d after endotoxin treatment . Two doses of endotoxin given 3 and 7 d or 7 and 15 d before treatment resulted in lung maturation responses equivalent to single dose comparison groups without elevations in cortisol . Early lung maturation induced by intraamniotic endotoxin in fetal sheep occurred without an increase in fetal plasma cortisol, indicating that endotoxin promoted lung maturation by a mechanism independent of cortisol. Mol Microbiol, 2000 Nov, 38(3), 639 - 49 A 76-residue polypeptide of colicin E9 confers receptor specificity and inhibits the growth of vitamin B12-dependent Escherichia coli 113/3 cells; Penfold CN et al.; The mechanism by which E colicins recognize and then bind to BtuB receptors in the outer membrane of Escherichia coli cells is a poorly understood first step in the process that results in cell killing . Using N- and C-terminal deletions of the N-terminal 448 residues of colicin E9, we demonstrated that the smallest polypeptide encoded by one of these constructs that retained receptor-binding activity consisted of residues 343-418 . The results of the in vivo receptor-binding assay were supported by an alternative competition assay that we developed using a fusion protein consisting of residues 1-497 of colicin E9 fused to the green fluorescent protein as a fluorescent probe of binding to BtuB in E . coli cells . Using this improved assay, we demonstrated competitive inhibition of the binding of the fluorescent fusion protein by the minimal receptor-binding domain of colicin E9 and by vitamin B12 . Mutations located in the minimum R domain that abolished or reduced the biological activity of colicin E9 similarly affected the competitive binding of the mutant colicin protein to BtuB . The sequence of the 76-residue R domain in colicin E9 is identical to that found in colicin E3, an RNase type E colicin . Comparative sequence analysis of colicin E3 and cloacin DF13, which is also an RNase-type colicin but uses the IutA receptor to bind to E . coli cells, revealed significant sequence homology throughout the two proteins, with the exception of a region of 92 residues that included the minimum R domain . We constructed two chimeras between cloacin DF13 and colicin E9 in which (i) the DNase domain of colicin E9 was fused onto the T+R domains of cloacin DF13; and (ii) the R domain and DNase domain of colicin E9 were fused onto the T domain of cloacin DF13 . The killing activities of these two chimeric colicins against indicator strains expressing BtuB or IutA receptors support the conclusion that the 76 residues of colicin E9 confer receptor specificity . The minimum receptor-binding domain polypeptide inhibited the growth of the vitamin B12-dependent E . coli 113/3 mutant cells, demonstrating that vitamin B12 and colicin E9 binding is mutually exclusive. Mol Microbiol, 2000 Nov, 38(3), 575 - 87 Both transmembrane domains of SecG contribute to signal sequence recognition by the Escherichia coli protein export machinery; Bost S et al.; A chimeric protein containing the uncleaved signal sequence of plasminogen activators inhibitor-2 (PAI2) fused to alkaline phosphatase (AP) interferes with Escherichia coli protein export and arrests growth . Suppressors of this toxicity include secG mutations that define the Thr-41-Leu-42-Phe-43 (TLF) domain of SecG . These mutations slow down the export of PAI2-AP . Another construct encoding a truncated PAI2 signal sequence (hB-AP) is also toxic . Most suppressors exert their effect on both chimeric proteins . We describe here five secG suppressors that only suppress the toxicity of hB-AP and selectively slow down its export . These mutations do not alter the TLF domain: three encode truncated SecG, whereas two introduce Arg residues in the transmembrane domains of SecG . The shortest truncated protein only contains 13 residues of SecG, suggesting that the mutation is equivalent to a null allele . Indeed, a secG disruption selectively suppresses the toxicity of hB-AP . However, the missense mutations are not null alleles . They allow SecG binding to SecYE, although with reduced affinity . Furthermore, these mutated SecG are functional, as they facilitate the export of endogenous proteins . Thus, SecG participates in signal sequence recognition, and both transmembrane domains of SecG contribute to ensure normal signal sequence recognition by the translocase. Mol Microbiol, 2000 Nov, 38(3), 565 - 74 RuvABC-dependent double-strand breaks in dnaBts mutants require recA; Seigneur M et al.; Replication fork arrest can cause DNA double-strand breaks (DSBs) . These DSBs are caused by the action of the Holliday junction resolvase RuvABC, indicating that they are made by resolution of Holliday junctions formed at blocked forks . In this work, we study the homologous recombination functions required for RuvABC-mediated breakage in cells deficient for the accessory replicative helicase Rep or deficient for the main Escherichia coli replicative helicase DnaB . We show that, in the rep mutant, RuvABC-mediated breakage occurs in the absence of the homologous recombination protein RecA . In contrast, in dnaBts mutants, most of the RuvABC-mediated breakage depends on the presence of RecA, which suggests that RecA participates in the formation of Holliday junctions at forks blocked by the inactivation of DnaB . This action of RecA does not involve the induction of the SOS response and does not require any of the recombination proteins essential for the presynaptic step of homologous recombination, RecBCD, RecF or RecO . Consequently, our observations suggest a new function for RecA at blocked replication forks, and we propose that RecA acts by promoting homologous recombination without the assistance of known presynaptic proteins. Mol Microbiol, 2000 Nov, 38(3), 526 - 34 Acquisition of double-stranded DNA-binding ability in a hybrid protein between Escherichia coli CspA and the cold shock domain of human YB-1; Wang N et al.; Escherichia coli CspA, a major cold shock protein, is dramatically induced upon temperature downshift . As it binds co-operatively to single-stranded DNA (ssDNA) and RNA without apparent sequence specificity, it has been proposed that CspA acts as an RNA chaperone to facilitate transcription and translation at low temperature . CspA consists of a five-stranded beta-barrel structure containing two RNA-binding motifs, RNP1 and RNP2 . Eukaryotic Y-box proteins, such as human YB-1, are a family of nucleic acid-binding proteins that share a region of high homology with CspA (43% identity), termed the cold shock domain (CSD) . Their cellular functions are very diverse and are associated with growth-related processes . Here, we replaced the six-residue loop region of CspA between the beta3 and beta4 strands with the corresponding region of the CSD of human YB-1 protein . The resulting hybrid protein became capable of binding to double-stranded DNA (dsDNA) in addition to ssDNA and RNA . The dsDNA-binding ability of an RNP1 point mutant (F20L) of the hybrid was almost unchanged . On the other hand, the dsDNA-binding ability of the hybrid protein was abolished in high salt concentrations in contrast to its ssDNA-binding ability . These results indicate that the loop region between the beta3 and beta4 strands of Y-box proteins, which is a little longer and more basic than that of CspA, plays an important role in their binding to dsDNA. Mol Microbiol, 2000 Nov, 38(3), 493 - 505 Disruption of the F plasmid partition complex in vivo by partition protein SopA; Lemonnier M et al.; The SopA protein plays an essential, though so far undefined, role in partition of the mini-F plasmid but, when overproduced, it causes loss of mini-F from growing cells . Our investigation of this phenomenon has revealed that excess SopA protein reduces the linking number of mini-F . It appears to do so by disturbing the partition complex, in which SopB normally introduces local positive supercoiling upon binding to the sopC centromere, as it occurs only in plasmids carrying sopC and in the presence of SopB protein . SopA-induced reduction in linking number is not associated with altered sop promoter activity or levels of SopB protein and occurs in the absence of changes in overall supercoil density . SopA protein mutated in the ATPase nucleotide-binding site (K120Q) or lacking the presumed SopB interaction domain does not induce the reduction in linking number, suggesting that excess SopA disrupts the partition complex by interacting with SopB to remove positive supercoils in an ATP-dependent manner . Destabilization of mini-F also depends on sopC and SopB, but the K120Q mutant retains some capacity for destabilizing mini-F . SopA-induced destabilization thus appears to be complex and may involve more than one SopA activity . The results are interpreted in terms of a regulatory role for SopA in the oligomerization of SopB dimers bound to the centromere. Mol Microbiol, 2000 Oct, 38(2), 392 - 400 Role of the mukB gene in chromosome and plasmid partition in Escherichia coli; Weitao T et al.; The intracellular locations of oriC and oriR1, the replication origins of the chromosome and plasmid R1, respectively, were visualized by fluorescence in situ hybridization (FISH) in exponentially growing populations of Escherichia coli . The locations of oriC and oriR1 (from a Par+ R1 plasmid) were unique and different in the wild-type host . In a mukB mutant, the positions were perturbed for both origins . The position of oriR1 from a plasmid with active partition (Par+) in the mukB host was as randomized as that of oriR1 from the Par- plasmid in a wild-type host . However, this mukB-induced randomization did not result in unstable inheritance of the Par+ plasmid, as measured by the conventional segregation assay . This might result from the preferential association of the Par+ plasmid with the bigger, decondensed nucleoid-containing daughters during cell division of MukB- cells, whereas the Par- plasmids were distributed at random and were lost by frequently ending up in anucleate cells. Mol Microbiol, 2000 Oct, 38(2), 368 - 80 The Escherichia coli UVM response is accompanied by an SOS-independent error-prone DNA replication activity demonstrable in vitro; Al Mamun AA et al.; UVM is an SOS-independent inducible response characterized by elevated mutagenesis at a site-specific 3, N4-ethenocytosine (epsilonC) residue borne on M13 single-stranded DNA transfected into Escherichia coli<