J Bacteriol, 1997 Feb, 179(4), 987 - 97 Cloning and characterization of the Helicobacter pylori flbA gene, which codes for a membrane protein involved in coordinated expression of flagellar genes; Schmitz A et al.; Flagellar motility has been shown to be an essential requirement for the ability of Helicobacter pylori to colonize the gastric mucosa . While some flagellar structural components have been studied in molecular detail, nothing was known about factors that play a role in the regulation of flagellar biogenesis . We have cloned and characterized an H . pylori homolog (named flbA) of the lcrD/flbF family of genes . Many proteins encoded by these genes are known to be involved in flagellar biogenesis or secretion of virulence-associated proteins via type III secretion systems . The H . pylori flbA gene (2,196 bp) is capable of coding for a predicted 732-amino-acid, 80.9-kDa protein that has marked sequence similarity with other known members of the LcrD/FlbF protein family . An isogenic strain with a mutation in the flbA gene was constructed by disruption of the gene with a kanamycin resistance cassette and electroporation-mediated allelic exchange mutagenesis . The mutant strain expressed neither the FlaA nor the FlaB flagellin protein . The expression of the FlgE hook protein was reduced in comparison with the wild-type strain, and the extent of this reduction was growth phase dependent . The flbA gene disruption was shown to downregulate the expression of these flagellar genes on the transcriptional level . The flbA mutants were aflagellate and completely nonmotile . Occasionally, assembled hook structures could be observed, indicating that export of axial flagellar filament components was still possible in the absence of the flbA gene product . The hydrophilic part of the FlbA protein was expressed in Escherichia coli, purified, and used to raise a polyclonal rabbit antiserum against the FlbA protein . Western blot experiments with this antiserum indicated that the FlbA protein is predominantly associated with the cytoplasmic membrane in H . pylori . The antiserum cross-reacted with two other proteins (97 and 43 kDa) whose expression was not affected by the flbA gene disruption and which might represent further H . pylori homologs of the LcrD/FlbF protein family. Curr Genet, 1997 Feb, 31(2), 133 - 8 Isolation and characterisation of a gene encoding protein disulphide isomerase, pdiA, from Aspergillus niger; Ngiam C et al.; Current strategies to improve the secretion of heterologous proteins from Aspergillus niger include the manipulation of chaperones and foldases specific to the endoplasmic reticulum (ER) . Here we report the isolation of a gene, pdiA, encoding a putative protein disulphide isomerase (PDI) from A . niger using the Saccharomyces cerevisiae PDI gene as a probe . Sequencing of a genomic clone and RT-PCR products predict a 515-aa protein comprising a 20-aa ER-translocation signal sequence and a 495-aa mature protein (Mr = 54.3 kDa) . The predicted protein also contains two thiol oxidoreductase active sites with a -CGHC- motif and a carboxy terminal -HDEL ER-retention signal . Three introns were identified within the pdiA gene and Southern- and dot-blot analysis indicates that the gene is present in a single copy . Northern-blot analysis shows a transcript of the predicted size . Sequence homology to a motif associated with protein trafficking and the induction of chaperones has been identified in the pdiA promoter . Transcription of pdiA is induced 3-4-fold after treatment with tunicamycin, an inhibitor of N-linked glycosylation . The kinetics of induction suggest that pdiA expression is not part of the primary stress response. J Gen Virol, 1997 Feb, 78 ( Pt 2), 343 - 51 Monoclonal antibodies specific for Semliki Forest virus replicase protein nsP2; Kujala P et al.; A panel of monoclonal antibodies (MAbs) was raised against Semliki Forest virus (SFV) nonstructural protein nsP2, which is a protease, an NTPase, a putative RNA helicase, and a regulator of the synthesis of the subgenomic 26S mRNA encoding the structural proteins . nsP2, used for immunization, was expressed as a histidine fusion protein in Escherichia coli and purified by metal affinity chromatography . Dot-blot assay, using a membrane fraction from SFV-infected cells as antigen, gave 33 positive clones . Of these, 30 MAbs recognized nsP2 in Western immunoblotting, and 25 showed positive indirect immunofluorescence (IFAT) in SFV-infected cells; 15 MAbs stained the cytoplasmic vacuoles (CPVI), which are the sites of viral RNA synthesis in alphavirus-infected cells . MAb 3B5 recognized only CPVIs, as shown by double immunofluorescence staining with polyclonal anti-nsP3 antiserum . Most of the MAbs (20/33) recognized the nuclear form of nsP2, which may be associated with SFV neurovirulence . Immunoprecipitation with MAbs revealed that the SFV nonstructural proteins are associated with each other . None of the MAbs recognized Sindbis virus nsP2 in immunoblotting, indicating that they were directed to non-conserved sequences specific for SFV . Interestingly, these epitopes were located mostly within the N-terminal half of nsP2 . Unexpectedly, the anti-nsP2 MAb 1E9 cross-reacted strongly with a host protein of 78 kDa from uninfected human, murine, avian and insect cells . This protein was identified as the immunoglobulin binding protein, BiP, by 2-D gel mapping and reaction with anti-BiP antiserum. J Gen Virol, 1997 Feb, 78 ( Pt 2), 337 - 41 Internal proteolysis of the NS3 protein specified by dengue virus 2; Teo KF et al.; The NS3 protein of flaviviruses is a multifunctional polypeptide required for virus replication . Enzymic activities that have been demonstrated or predicted from the presence of sequence motifs include protease, NTPase, helicase and RNA triphosphatase . Both full-length and truncated forms of NS3 have been identified in infected cells . To examine internal cleavage of the NS3 protein of dengue virus 2 (DEN-2), infected cells or COS cells transfected with cDNA encoding NS2B/3 were radiolabelled and immunoprecipitated with antiserum against NS3 or hyperimmune mouse ascitic fluid . The polypeptides detected were NS2B/3 (Mr 83000), NS3 (Mr 69000), NS3' (Mr 50000) and NS3" (Mr 19000) . The latter polypeptide has not been previously identified . For DEN-2, it has been proposed that NS3' results from cleavage at the site ...R457R / GR460.. . within an RNA helicase sequence motif of NS3 . Our results demonstrated that cleavage occurred at this site, and that prior cleavage between NS2B/NS3 was not necessary. Biophys J, 1997 Feb, 72(2 Pt 1), 936 - 41 pH-dependent conformational changes in Escherichia coli dihydrofolate reductase revealed by Raman difference spectroscopy; Chen YQ et al.; The catalytic site of all dihydrofolate reductases contains an invariant carboxylic acid, equivalent to Asp-27 in Escherichia coli dihydrofolate reductase (ecDHFR) . It has been found that various kinetic and ligand binding properties of ecDHFR show a pH profile with a pKa of about 6.5 . The group responsible for this pKa is often assumed to be carboxyl group of Asp-27 . To determine the ionization state of this carboxyl and its pKa, we have employed a novel method, based on Raman difference spectroscopy, to obtain its vibrational spectrum in situ . The method is general for the study of protein carboxyl groups, which are often significantly implicated in protein function and structure; this study establishes the method's limits and problems . The Raman difference spectrum between wild-type ecDHFR and the Asp-27 to serine mutant (D27S) in the pH range 5.6-9.0 has been taken . No protonation of the carboxyl group was detected, implying that its pKa is probably less than 5.0 . We did, however, detect a pH dependence in the intensity of Raman bands in the difference spectrum with a pKa of 6.3, indicating that the apo enzyme undergoes a pH-dependent conformational change . Because the carboxyl group of Asp-27 at the active site is the only ionizable group in the binding site, other groups, away from the catalytic site, must be responsible for the pH behavior of ecDHFR. Nucleic Acids Res, 1997 Feb 1, 25(3), 682 - 4 High efficiency of site-directed mutagenesis mediated by a single PCR product; Chen X et al.; We describe a highly efficient procedure for site-specific mutagenesis of double-stranded plasmids . The method relies on a single PCR primer which incorporates both the mutations at the selection site and the desired single base substitutions at the mutant site . This primer is annealed to the denatured plasmid and directs the synthesis of the mutant strand . After digestion with selection enzyme, the plasmid DNA is amplified into Escherichia coli strain BMH71-18 and subjected to a second digestion and amplification into the bacterial strain DH5alpha . A mutagenesis efficiency >80% was consistently achieved in the case of two unrelated plasmids. Nucleic Acids Res, 1997 Feb 1, 25(3), 597 - 603 Replication bypass and mutagenic effect of alpha-deoxyadenosine site-specifically incorporated into single-stranded vectors; Shimizu H et al.; alpha-2'-Deoxyadenosine (alpha) is a major adenine lesion produced by gamma-ray irradiation of DNA under anoxic conditions . In this study, single-stranded recombinant M13 vectors containing alpha were constructed and transfected into Escherichia coli to assess lethal and mutagenic effects of this lesion . The data for alpha were further compared with those obtained with M13 vectors containing normal A or a model abasic site (F) at the same site . The transfection assay revealed that alpha constituted a moderate block to DNA replication . The in vivo replication capacity to pass through alpha was approximately 20% relative to normal A, but 20-fold higher than that of F constituting an almost absolute replication block . Similar data were obtained by in vitro replication of oligonucleotide templates containing alpha or F by E.coli DNA polymerase I . The mutagenic consequence of replicating M13 DNA containing alpha was analyzed by direct DNA sequencing of progeny phage . Mutagenesis was totally targeted at the site of alpha introduced into the vector . Mutation was exclusively a single nucleotide deletion and no base substitutions were detected . The deletion frequency associated alpha was dependent on the 3'-nearest neighbor base: with the 3'-nearest neighbor base T mutation (deletion) frequency was 26%, whereas 1% with the 3'-nearest neighbor base G . A possible mechanism of the single nucleotide deletion associated with alpha is discussed on the basis of the misinsertion-strand slippage model. Nucleic Acids Res, 1997 Feb 1, 25(3), 537 - 45 Characterization of DbpA, an Escherichia coli DEAD box protein with ATP independent RNA unwinding activity; Boddeker N et al.; DbpA is a putative Escherichia coli ATP dependent RNA helicase belonging to the family of DEAD box proteins . It hydrolyzes ATP in the presence of 23S ribosomal RNA and 93 bases in the peptidyl transferase center of 23S rRNA are sufficient to trigger 100% of the ATPase activity of DbpA . In the present study we characterized the ATPase and RNA unwinding activities of DbpA in more detail . We report that-in contrast to eIF-4A, the prototype of the DEAD box protein family-the ATPase and the helicase activities of DbpA are not coupled . Moreover, the RNA unwinding activity of DbpA is not specific for 23S rRNA, since DbpA is also able to unwind 16S rRNA hybrids . Furthermore, we determined that the ATPase activity of DbpA is triggered to a significant extent not only by the 93 bases of the 23S rRNA previously reported but also by other regions of the 23S rRNA molecule . Since all these regions of 23S rRNA are either part of the 'functional core' of the 50S ribosomal subunit or involved in the 50S assembly, DbpA may play an important role in the ribosomal assembly process. Nucleic Acids Res, 1997 Feb 1, 25(3), 491 - 6 Selective recognition of a cisplatin-DNA adduct by human mismatch repair proteins; Yamada M et al.; The antitumor agent cis-diamminedichloroplatinum(II) (cisplatin) introduces cytotoxic DNA damage predominantly in the form of intrastrand crosslinks between adjacent purines . Binding assays using a series of duplex oligonucleotides containing a single 1,2 diguanyl intrastrand crosslink indicate that human cell extracts contain factors that preferentially recognise this type of damage when the complementary strand contains T opposite the 3', and C opposite the 5'guanine in the crosslink . Under the conditions of the band-shift assay used, little binding is observed if the positions of the T and C are reversed in the complementary strand . Similarly, duplexes containing CC or TT opposite the crosslink are recognised relatively poorly . The binding activity is absent from extracts of the colorectal carcinoma cell lines LoVo and DLD-1 in which the hMutSalpha mismatch recognition complex is inactivated by mutation . Extensively purified human hMutSalpha exhibits the same substrate preference and binds to the mismatched platinated DNA at least as well as to an identical unplatinated duplex containing a single G.T mismatch . It is likely, therefore, that human mismatch repair may be triggered by 1,2 diguanyl intrastrand crosslinks that have undergone replicative bypass. Nucleic Acids Res, 1997 Feb 1, 25(3), 480 - 91 Differential human nucleotide excision repair of paired and mispaired cisplatin-DNA adducts; Moggs JG et al.; In order to understand the action of the chemotherapeutic drug cisplatin, it is necessary to determine why some types of cisplatin-DNA intrastrand crosslinks are repaired better than others . Using cell extracts and circular duplex DNA, we compared nucleotide excision repair of uniquely placed 1,2-GG, 1,2-AG, and 1,3-GTG cisplatin-crosslinks, and a 2-acetylaminofluorene lesion . The 1,3 crosslink and the acetylaminofluorene lesion were repaired by normal cell extracts approximately 15-20 fold better than the 1,2 crosslinks . No evidence was found for selective shielding of 1,2 cisplatin crosslinks from repair by cellular proteins . Fractionation of cell extracts to remove putative shielding proteins did not improve repair of the 1,2-GG crosslink, and cell extracts did not selectively inhibit access of UvrABC incision nuclease to 1,2-GG crosslinks . The poorer repair of 1,2 crosslinks in comparison to the 1,3 crosslink is more likely a consequence of different structural alterations of the DNA helix . In support of this, a 1,2-GG-cisplatin crosslink was much better repaired when it was opposite one or two non-complementary thymines . Extracts from cells defective in the hMutSalpha mismatch binding activity also showed preferential repair of the 1,3 crosslink over the 1,2 crosslink, and increased repair of the 1,2 adduct when opposite thymines, showing that hMutSalphais not involved in the differential NER of these substrates in vitro . Mismatched cisplatin adducts could arise by translesion DNA synthesis, and improved repair of such adducts could promote cisplatin-induced mutagenesis in some cases. Nucleic Acids Res, 1997 Feb 1, 25(3), 474 - 9 Kinetics of excision of purine lesions from DNA by Escherichia coli Fpg protein; Karakaya A et al.; The kinetics of excision of damaged purine bases from oxidatively damaged DNA by Escherichia coli Fpg protein were investigated . DNA substrates, prepared by treatment with H2O2/Fe(III)-EDTA or by gamma-irradiation under N2O or air, were incubated with Fpg protein, followed by precipitation of DNA . Precipitated DNA and supernatant fractions were analyzed by gas chromatography/isotope-dilution mass spectrometry . Kinetic studies revealed efficient excision of 8-hydroxyguanine (8-OH-Gua), 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua) and 4, 6-diamino-5-formamidopyrimidine (FapyAde) . Thirteen other modified bases in the oxidized DNA substrates, including 5-hydroxycytosine and 5-hydroxyuracil, were not excised . Excision was measured as a function of enzyme concentration, substrate concentration, time and temperature . The rate of release of modified purine bases from the three damaged DNA substrates varied significantly even though each DNA substrate contained similar levels of oxidative damage . Specificity constants (kcat/KM) for the excision reaction indicated similar preferences of Fpg protein for excision of 8-OH-Gua, FapyGua and FapyAde from each DNA substrate . These findings suggest that, in addition to 8-OH-Gua, FapyGua and FapyAde may be primary substrates for this enzyme in cells. Arch Biochem Biophys, 1997 Feb 1, 338(1), 104 - 10 Catalytic and structural importance of Gly-454, Tyr-455, and Leu-456 in the carboxy-terminal region of Escherichia coli F1-ATPase alpha subunit; Yabuki M et al.; Monoclonal antibody alpha110 recognizes Leu-456 in the alpha subunit of the Escherichia coli F1-ATPase . Binding of this antibody to the alpha subunit or mutation of this residue to Pro caused enhancement of the ATPase activity, suggesting that this residue is involved in the catalytic mechanism of this molecule (H . Kanazawa et al . (1995) Arch . Biochem . Biophys . 317, 348-356) . Leu-456 together with Gly-454 and Tyr-455 are the only residues in the carboxy-terminal 75 amino acids conserved among various species, suggesting that these three residues play important roles in catalysis by the ATPase . Here, we introduced site-directed mutations into these residues . Not only L456P but also G454L, Y455K, Y455L, and L456N mutations caused enhancement of the ATPase activity . Surprisingly, Y455V, L456H, and L456S caused assembly defects of F1 subunits on the membrane . Reconstitution of the alpha betagamma complex from the wild-type beta and gamma subunits with the mutant alpha subunit (L4gamma6P) exhibited enhanced ATPase activity . Addition of delta or epsilon fused to glutathione S-transferase which are functionally similar to the delta and epsilon subunits, respectively, to the reconstituted F1-ATPase did not cause significant enhancement of its activity . Decreased interaction between alpha and beta subunits with the L456P mutation was detected by the yeast two-hybrid system . According to the deduced three-dimensional structure of the bovine a subunit, Leu-456, Gly-454, and Tyr-455 are included in a small alpha helix . These results suggest that this alpha helix affects interaction of the alpha subunit with the beta subunit but not with delta or epsilon, which may be important for the catalytic mechanism and F1 assembly. Arch Biochem Biophys, 1997 Feb 1, 338(1), 83 - 9 Reversal of the nucleotide specificity of ketol acid reductoisomerase by site-directed mutagenesis identifies the NADPH binding site; Rane MJ et al.; Analysis of the published amino acid sequences of the enzyme ketol acid reductoisomerase (KARI) from seven organisms identified three regions with highly conserved sequences . One of these regions is predicted to be the dinucleotide fold where NADPH binds . In order to confirm that this region did include the NADPH binding site, we used oligonucleotide-mediated site-directed mutagenesis to study the function of specific amino acids in this region in terms of their interactions with NADPH . Four positively charged amino acids, R68, K69, K75, and R76, were mutated singly, in different combinations, and finally as a quartet in order to evaluate electrostatic interactions with NADPH . Mutation of each of the arginines singly to glutamine results in a 60- to 100-fold reduction in k(cat)/K(m) for NADPH . Mutation of each of the lysines singly does not significantly alter the steady state kinetic parameters associated with NADPH . None of these mutations significantly alters the affinity of the enzyme for NADH . After looking at double mutations of these four amino acids, we constructed the quadruplet mutant R68DK69LK75VR76D . This mutant has K(m) and k(cat) values of 19.3 microM and 5.3 min(-1) for NADH, which compares to 207 microM and 0.11 min(-1) for the wild-type enzyme . For the quadruplet mutant the corresponding values for NADPH are >200 microM for K(m) and 2 min(-1) for k(cat) compared to 7.3 microM and 7.2 min for the wild-type enzyme . By altering these four amino acids, the specificity constants for NADH and NADPH are almost exactly reversed in the mutant relative to the wild type. Arch Biochem Biophys, 1997 Feb 1, 338(1), 57 - 66 Mutation of conserved residues in the NADP(H)-binding domain of the proton translocating pyridine nucleotide transhydrogenase of Escherichia coli; Bragg PD et al.; Possible NADP(H)-binding sites of the beta subunit of the pyridine nucleotide transhydrogenase of Escherichia coli were examined by site-directed mutagenesis . The sequence of the beta subunit at positions 314-350 showed several features typical of NADP(H)-binding sites . Mutation of betaGly314, the first glycine residue of the GXGXXV motif, and of betaArg350, which probably interacts with the 2'-phosphate of the substrate NADP(H), resulted in drastic loss of enzyme activity . The loss of activity in the betaArg350 mutants was not due to loss of ability to bind NADP(H) . Several residues (betaVal319, betaGly337, betaHis345, and betaArg350) were mutated to make the sequence more similar to that of a NAD(H)-binding site . The introduction of multiple mutations resulted in improper assembly of the enzyme and decreased incorporation into the membrane . The GXGXXG motif, typical of beta alphabeta nucleotide-binding folds, in the sequence of the beta subunit at positions 274-279 was mutated without causing major changes in transhydrogenase activities . It is unlikely to be part of a nucleotide-binding domain . Deletion of the carboxy-terminal 32 amino acids of the beta subunit, a possible nucleotide-binding site, prevented assembly and incorporation of the truncated enzyme into the cytoplasmic membrane of E . coli. Arch Biochem Biophys, 1997 Feb 1, 338(1), 35 - 42 Cytochrome P450 2C11: Escherichia coli expression, purification, functional characterization, and mechanism-based inactivation of the enzyme; Licad-Coles E et al.; The male-specific P450 enzyme CYP 2C11, whose expression is developmentally and hormonally regulated, is the major steroid 16alpha-hydroxylase of the untreated rat liver . The enzyme metabolizes a host of substrates, including mechanism-based inactivators, such as 3,5-dicarbethoxy-2,6-dimethyl-4-ethyl-1,4-dihydropyridine (DDEP) and spironolactone (SPL) . Structural and functional characterization of the specific mode of such inactivation, however, requires sufficient quantities of the fully purified enzyme . Although several laboratories including our own have isolated and purified the enzyme from male rats, the yields are typically low and of the order of 1% . For these reasons, we chose to heterologously express the enzyme in Escherichia coli . The full-length cDNA was excised from the yeast vector pD2M1 and cloned into the plasmid vector pCW after appropriate modifications for optimal expression in E . coli . The enzyme was isolated and purified from E . coli membranes in relatively high yields (approximately 60%) and relatively high specific content (19 nmol/mg protein) . The purified recombinant enzyme had spectral and functional characteristics comparable to those reported for the native rat liver enzyme, including mechanism-based inactivation by DDEP and SPL . Studies with 14C-heme-labeled enzyme indicated that the major mode of DDEP inactivation was via heme-N-ethylation . On the other hand, studies with radiolabeled SPL-SH (the proximal inactivating deacetylated metabolite of SPL) revealed that although both {22-14C}SPL-SH and SPL-35SH inactivated the enzyme, only SPL-35SH was found to irreversibly radiolabel the 2C11 protein . The latter findings thus suggest that during mechanism-based inactivation of 2C11, the thiol moiety of SPL-SH is oxidatively activated to a species that attacks the 2C11 protein during or after cleavage from the thiosteroid . Thus, these modes of mechanism-based 2C11 inactivation by DDEP and SPL-SH considerably differ from the corresponding modes of P450 3A inactivation by these agents, wherein heme modification of the protein predominates. J Immunol, 1997 Feb 1, 158(3), 1293 - 301 A Ca2+-dependent autoregulation of lipopolysaccharide-induced IL-8 receptor expression in human polymorphonuclear neutrophils; Bhattacharya C et al.; IL-8, a potent neutrophil chemotactic agent, is known to be a key mediator in several inflammatory diseases . We found that 10 ng/ml of serum-activated LPS (Escherichia coli) efficiently up-regulated IL-8R on the surface of neutrophils within 30 min of LPS stimulation by 115 to 120% through de novo protein synthesis . After 30 min of LPS stimulation, reduction of IL-8R level was initiated and the normal level was restored within 2 h of LPS interaction . EDTA or EGTA and bestatin separately inhibited the receptor down-regulation by 98%, indicating the involvement of metalloprotease(s), more specifically an aminopeptidase in the process . Induction and subsequent reduction of IL-8 binding in serum-activated LPS-stimulated cells have been demonstrated in autoradiography . Intracellular Ca2+ level in these stimulated neutrophils was increased and decreased with alteration of IL-8R level . Although IL-8 binding was drastically reduced, the total IL-8R level, as detected by anti-IL-8R Ab measured by 125I-labeled anti-rabbit IgG, remained almost unaltered, indicating that minimal proteolysis occurred in IL-8R . Anti-IL-8R Ab and IL-8 itself could prevent this down-regulation significantly, suggesting that the susceptible epitope(s) might be in the IL-8 binding domain of the receptor . Under Ca2+-depleted conditions, the proteolysis was inhibited, which was accelerated upon addition of 1 mM of CaCl2 . The study demonstrates that LPS-induced up-regulation of IL-8R leads to amplified IL-8-mediated biologic responses of neutrophils that are restored to normal level by activation of a Ca2+-dependent aminopeptidase . This may be useful for understanding the regulation of LPS-mediated inflammatory responses of neutrophils during bacterial infection. Insect Mol Biol, 1997 Feb, 6(1), 97 - 104 The nonvitellogenic female protein of Musca domestica is an adult-specific hexamerin; Capurro Mde L et al.; During Musca domestica vitellogenesis a protein is preferentially synthesized by the female fat body and accumulates in the haemolymph but not in the ovaries . This protein, designated nonvitellogenic female protein (NVFP), was purified and shown to be a hexamer with an M(r) = 430 kDa, and subunits of M(r) = 70 kDa . The hexamer dissociates into subunits when the pH is elevated from 7.0 to 9.0 . Two cDNA clones, F0 and F2, were isolated and analysed . The 2.2 kb F2 clone has an open reading frame that encodes a conceptual translation product that has similarity to the Drosophila melanogaster LSP-2 hexamerin . Recombinant protein from the F2-cDNA is recognized by a specific anti-NVFP serum . The temporal pattern of mRNA expression of the gene represented by the F2 clone follows that determined for the synthesis of NVFP . The data support the conclusion that NVFP is an hexamerin specific to the adult stage of Musca domestica. Cancer Res, 1997 Feb 1, 57(3), 461 - 5 In vivo gene therapy for alpha-fetoprotein-producing hepatocellular carcinoma by adenovirus-mediated transfer of cytosine deaminase gene; Kanai F et al.; The alpha-fetoprotein (AFP) gene is normally expressed in fetal liver and is transcriptionally silent in adult liver but overexpressed in human hepatocellular carcinoma (HCC) . Here, we demonstrate that replication defective recombinant adenoviral vectors, containing the human AFP promoter/enhancer, can be used to express the Escherichia coli cytosine deaminase (CD) gene (AdAFPCD) and the beta-galactosidase gene (AdAF-PlacZ) in AFP-producing HCC cell lines . Expression of the CD gene by adenovirus from the AFP promoter/enhancer (AdAFPCD) induced cells sensitive to 5-fluorocytosine (5FC) in the AFP-producing cells but not in the AFP-nonproducing cells . Transduction by an adenoviral vector harboring an ubiquitous strong promoter and CD gene showed enzymatic activity and 5FC killing in all cell lines . When AdAFPlacZ was injected into the s.c . established hepatoma in vivo, expression of the beta-galactosidase gene was confined to AFP-producing HCC xenografts . Moreover, HCC xenografts regressed by transduction with AdAFPCD and subsequently with 5FC treatment in vivo . These findings suggest that utilization of the AFP promoter/enhancer in an adenoviral vector can confer selective expression of a heterologous suicide gene in hepatocellular carcinoma cells in vitro and in vivo. Infect Immun, 1997 Feb, 65(2), 847 - 51 Adhesive factor/rabbit 2, a new fimbrial adhesin and a virulence factor from Escherichia coli O103, a serogroup enteropathogenic for rabbits; Fiederling F et al.; Enteropathogenic Escherichia coli-like E . coli strains belonging to serovar O103:K-:H2 and rhamnose-negative biotypes are highly pathogenic diarrhea-inducing strains for weaned European rabbits . We describe here the cloning and sequencing of the major subunit gene of a new fimbrial adhesin, adhesive factor/rabbit 2 (AF/R2), which confers on these strains the ability to attach to rabbit enterocytes and to HeLa cells in a diffuse manner and which is associated with in vivo virulence . The chromosomal operon that encodes functional AF/R2 has been cloned from strain B10 . The major subunit gene afr2G, as well as an adjacent open reading frame, afr2H, has been sequenced . The Afr2G protein shows homologies with FaeG and ClpG, which are the respective major subunits of fimbrial adhesin K88 (F4) and afimbrial adhesin CS31A . Plasmid carrying the operon transcomplements an AF/R2-negative TnphoA mutant for its ability to express AF/R2 . As a whole, AF/R2 is a new member of the E . coli K88 adhesin family which is associated with virulence and which may serve in the design of vaccines. Infect Immun, 1997 Feb, 65(2), 531 - 6 Escherichia coli K5 capsule expression enhances colonization of the large intestine in the gnotobiotic rat; Herias MV et al.; The role of capsule expression in the capacity of Escherichia coli to colonize in the large intestinal environment was studied in a gnotobiotic rat model . The rats were given perorally a mixture of two mutant strains differing in K5 expression . After 2 weeks, the rats were sacrificed, and subsequently intestinal contents, intestinal mucosae, and mesenteric lymph nodes were homogenized and bacterial numbers were quantified . Two E . coli mutant pairs were used, the first pair (972-998) lacking the O-specific side chain and the second pair (973-997) carrying the O75 lipopolysaccharide . The K5+ mutants established themselves at a higher level than the K5- mutants (10(9) versus 10(6) CFU/g {P < 0.001} for the first pair and 10(9) versus 10(8) CFU/g {P < 0.01} for the second pair, respectively) . The results were confirmed by serology showing a K5+ phenotype for practically all isolates . The bacterial population associated with the mucosa was similar to that in the luminal contents with respect to the proportions of the respective mutants, and translocation occurred in numbers proportional to the intestinal population densities of the respective mutants . All mutants were able to express type 1 as well as P fimbriae . After colonization, the expression of P fimbriae remained high whereas only a minority of the isolates expressed type 1 fimbriae . The results suggest that capsule expression and P fimbriae enhance intestinal colonization by E . coli and that these virulence factors, by increasing bacterial densities in the intestine, secondarily increase translocation. Infect Immun, 1997 Feb, 65(2), 507 - 13 A new putative fimbrial colonization factor, CS19, of human enterotoxigenic Escherichia coli; Grewal HM et al.; A gene probe derived from the colonization factor antigen I (CFA/I) operon cross-hybridized at very low stringency to plasmid DNA from coli surface antigen 17 (CS17)-producing enterotoxigenic Escherichia coli (ETEC) and from the ETEC strain F595C, which was negative for previously described CFAs, CSs, and putative colonization factors (PCFs) . A 16-kDa protein was identified in sodium dodecyl sulfate-polyacrylamide gel electrophoresis of heat extracts prepared after growth of strain F595C at 37 degrees C on CFA agar containing bile salts . Transmission electron microscopy revealed bile salt- and temperature-dependent expression of fimbriae with a diameter of 7 nm . After transformation with a recombinant plasmid harboring the cfaR gene, which encodes a positive regulator of several CFAs, PCFs, and CSs, the 16-kDa protein was hyperexpressed . Polyclonal antibodies raised against this protein bound to the fimbriae and inhibited the adhesion of F595C bacteria to tissue-cultured Caco-2 cells . Nucleotide sequence determination of the gene encoding the 16-kDa fimbrial subunit revealed a high degree of amino acid sequence homology to the CFA/I, CS1, CS2, CS4, CS14, and CS17 polypeptides . The term CS19 is proposed for the new fimbria. Infect Immun, 1997 Feb, 65(2), 412 - 21 Molecular characterization of a 6.6-kilodalton Borrelia burgdorferi outer membrane-associated lipoprotein (lp6.6) which appears to be downregulated during mammalian infection; Lahdenne P et al.; Isolated outer membranes of Borrelia burgdorferi 297 were utilized to obtain partial amino acid sequence information for a low-molecular-weight, outer membrane-associated polypeptide . Degenerate oligonucleotide primers based upon this information were used to amplify a 100-bp probe for detection of the corresponding full-length gene within a B . burgdorferi total genomic library . The relevant open reading frame (ORF) encoded a polypeptide comprised of a 17-amino-acid putative signal peptide terminated by LFVAC, a probable consensus sequence for lipoprotein modification, and a mature protein of 51 amino acids (predicted molecular mass of 5.8 kDa) . The DNA sequences of the corresponding ORFs in B . burgdorferi 297 and B31 were identical; the corresponding ORF in strain N40 differed by only one nucleotide . Assuming conventional processing and acylation, the molecular weight of the lipoprotein, designated lp6.6, is about 6,600 . The lp6.6 gene, which was localized to the 49-kb linear plasmid of B . burgdorferi, subsequently was cloned and expressed in Escherichia coli as a fusion protein with glutathione S-transferase . Immunoblot analysis with monoclonal antibody 240.7 revealed that lp6.6 was identical to a low-molecular-weight, highly conserved B . burgdorferi lipoprotein reported previously (L . I . Katona, G . Beck, and G . S . Habicht, Infect . Immun . 60:4995-5003, 1992) . Results of indirect immunofluorescence assays, growth inhibition assays, passive immunizations, and active immunizations indicated that this outer membrane-associated antigen is not surface exposed in B . burgdorferi . Particularly interesting was the finding that mice and rhesus monkeys chronically infected with B . burgdorferi failed to develop antibodies against this antigen . We propose that high-level expression of lp6.6 is associated with the arthropod phase of the spirochetal life cycle and that expression of the gene is downregulated during mammalian infection. Infect Immun, 1997 Feb, 65(2), 349 - 57 Identification of the gene family encoding the 160-kilodalton Trypanosoma cruzi complement regulatory protein; Norris KA et al.; Trypanosoma cruzi trypomastigotes are exquisitely resistant to the lytic effects of vertebrate complement, and this characteristic contributes to the survival of the parasites in the host bloodstream . Trypomastigotes avoid complement-mediated lysis by the production of a surface glycoprotein that inhibits the formation of the alternative and classical C3 convertase, thus preventing activation and amplification of the complement cascade at the parasite surface . We have developed a monoclonal antibody to the 160-kDa T . cruzi complement regulatory protein (CRP) and describe a one-step immunoaffinity purification procedure . The CRP was purified to homogeneity and subjected to amino-terminal peptide sequence analysis . Based on the protein sequence obtained, the CRP was identified as a member of a large family of trypomastigote-specific genes, and a complete cDNA was isolated and sequenced . The complete coding sequence was cloned in Escherichia coli, and antibodies raised against the full-length recombinant protein reacted specifically with a 160-kDa protein in trypomastigote membrane protein preparations as well as with native, purified CRP . Indirect immunofluorescence revealed that the protein is uniformly expressed at the cell surfaces of trypomastigotes. J Bacteriol, 1997 Feb, 179(3), 968 - 71 Purification and in vitro phosphorylation of HupT, a regulatory protein controlling hydrogenase gene expression in Rhodobacter capsulatus; Elsen S et al.; The HupT protein of Rhodobacter capsulatus, involved in negative regulation of hydrogenase gene expression, is predicted to be a histidine kinase on the basis of sequence comparisons . The protein was overproduced in Escherichia coli, purified to homogeneity, and demonstrated to autophosphorylate in vitro in the presence of {gamma-32P}ATP . An H217N hupt mutant was constructed, and the mutant protein was shown to have lost kinase activity . This result, and the fact that the phosphoryl group in phosphorylated HupT appeared to be bound to an N atom, support the suggestion from sequence comparisons that HupT is a histidine kinase, which can autophosphorylate on the His217 residue. J Bacteriol, 1997 Feb, 179(3), 959 - 63 Variation in RNA polymerase sigma subunit composition within different stocks of Escherichia coli W3110; Jishage M et al.; The composition of RNA polymerase sigma subunits was analyzed for stock strains of Escherichia coli K-12 W3110 in Japan . Heterogeneity was discovered with respect to two sigma subunits, sigma28 (sigmaF, the rpoF gene product) and sigma38 (sigmaS, the rpoS gene product) . Five different types of W3110 were identified: A-type lineages have both sigma subunits in intact forms; B-type lineages carry a truncated sigma38 subunit and an intact sigma28 subunit; C-type lineages carry an intact sigma28 subunit but lack a sigma38 subunit; D-type lineages have only a sigma38 subunit without a sigma28 subunit; and E-type stocks lack both sigma subunits . All the lineages examined, however, contain the intact forms of sigma70 (sigmaD, the rpoD gene product) and sigma54 (sigmaN, the rpoN gene product) . As expected from the lack of a sigma28 subunit, cells of D- and E-type lineages are nonmotile . The truncated form of the sigma38 subunit in B-type stocks carries two mutations near its N terminus and lacks C-terminal proximal region 4 due to an amber mutation . The failure of C- and E-type W3110 cells to express sigma38 and that of D- and E-type cells to express sigma28 were found to be due to defects in transcription even though the respective sigma subunit genes remain intact . These findings emphasize the importance of paying attention to possible variations in the genetic background between laboratory stocks originating from the same strain. J Bacteriol, 1997 Feb, 179(3), 956 - 8 Isolation and characterization of a priB mutant of Escherichia coli influencing plasmid copy number of delta rop ColE1-type plasmids; Berges H et al.; The lethality induced by the overproduction in Escherichia coli of a heterologous protein was used to select bacterial mutants . In one of these, the mutation responsible was mapped to priB . We describe the isolation of this mutant, the sequencing of the mutated gene, and its in vivo effect on plasmid replication. J Bacteriol, 1997 Feb, 179(3), 949 - 51 Dihydroneopterin triphosphate epimerase of Escherichia coli: purification, genetic cloning, and expression; Haussmann C et al.; The enzyme catalyzing the epimerization at position 2' of dihydroneopterin triphosphate was purified by a factor of about 10,000 from cell extract of Escherichia coli . The cognate gene was cloned, sequenced, expressed, and mapped to kb 2427 on the E . coli chromosome. J Bacteriol, 1997 Feb, 179(3), 909 - 18 The Escherichia coli stpA gene is transiently expressed during growth in rich medium and is induced in minimal medium and by stress conditions; Free A et al.; The transcriptional regulation of the stpA gene, encoding the Escherichia coli H-NS-like protein StpA, has been studied as a function of a variety of environmental conditions, and its response to trans-acting factors has been characterized . Chromosomally located stpA is expressed primarily from a promoter immediately upstream of the gene which is severely repressed by the homologous nucleoid-associated protein H-NS . However, we show here that even in a strain containing functional H-NS, stpA is transiently induced during growth of a batch culture in rich medium . It can also be induced strongly by osmotic shock and, to a lesser extent, by an increase in growth temperature . Moreover, when cells are grown in minimal medium, we observe a more sustained induction of stpA which is dependent on the leucine-responsive regulatory protein (Lrp) . This enhanced level of stpA transcription is virtually abolished in an H-NS-independent manner when the culture undergoes carbon starvation . A sensitivity of the stpA promoter to DNA topology may contribute to some of these responses . Results reported here show that cloned fragments of the stpA promoter region can confer H-NS and Lrp responsiveness upon a lacZ reporter gene and suggest that several hundred base pairs of DNA upstream of the transcriptional start may be required for regulation by these two proteins. J Bacteriol, 1997 Feb, 179(3), 880 - 8 Stability of linear DNA in recA mutant Escherichia coli cells reflects ongoing chromosomal DNA degradation; Kuzminov A et al.; To study the fate of linear DNA in Escherichia coli cells, we linearized plasmid DNA at a specific site in vivo and monitored its behavior in recA mutant cells deficient in recombinational repair . Earlier, we had found that in wild-type (WT) cells linearized DNA is degraded to completion by RecBCD nuclease . We had also found that in WT cells chi sites on linear DNA inhibit RecBCD degradation by turning off its nucleolytic activities . Now we report that chi sites do not work in the absence of the RecA protein, suggesting that RecA is required in vivo to turn off the degradative activities of the RecBCD enzyme . We also report that the degradation of linearized plasmid DNA, even devoid of chi sites, is never complete in recA cells . Investigation of this linear DNA stability indicates that a fraction of recA cells are recBC phenocopies due to ongoing chromosomal DNA degradation, which titrates RecBCD nuclease . A possible role for RecBCD-promoted DNA degradation in controlling chromosomal DNA replication in E . coli is discussed. J Bacteriol, 1997 Feb, 179(3), 825 - 30 Genetic analysis of the catalytic domain of the chemotaxis-associated histidine kinase CheA; Ellefson DD et al.; Escherichia coli cells express two forms of CheA, the histidine kinase associated with chemotaxis . The long form, CheA(L), plays a critical role in chemotactic signal transduction by phosphorylating two chemotaxis-associated response regulators, CheY and CheB . CheA(L) first autophosphorylates amino acid His-48 before its phosphoryl group is transferred to these response regulators . The short form, CheA(S), lacks the amino-terminal 97 amino acids of CheA(L) and therefore does not possess the site of phosphorylation . The centrally located transmitter domain of both forms of CheA contains four regions, called N, G1, F, and G2, highly conserved among histidine kinases of the family of two-component signal transduction systems . On the basis of sequence similarity to highly conserved regions of certain eukaryotic kinases, the G1 and G2 regions are purported to be involved in the binding and hydrolysis of ATP . We report here that alleles mutated in the G1, G2, or F region synthesize CheA variants that cannot autophosphorylate in vitro and which cannot support chemotaxis in vivo . We also show that in vitro, the nonphosphorylatable CheA(S) protein mediates transphosphorylation of a CheA(L) variant defective in both G1 and G2 . In contrast, CheA(L) variants defective for either G1 or G2 mediate transphosphorylation of each other poorly, if at all . These results are consistent with a mechanism by which the G1 and G2 regions of one protomer of a CheA dimer form a unit that mediates transphosphorylation of the other protomer within that dimer. J Bacteriol, 1997 Feb, 179(3), 784 - 93 Two polypeptide products of the Escherichia coli cell division gene ftsW and a possible role for FtsW in FtsZ function; Khattar MM et al.; Two new mutations in the cell division gene ftsW have been isolated and characterized . The ftsW263(Ts) mutation results in a block to division at the initiation stage, similar to that previously observed with the ftsW201(Ts) mutation . The ftsW1640(Ts) mutation, however, causes a block to division at a later stage . The ftsW201 and ftsW263 mutants were shown to be phenotypically sensitive to the genetic background and growth conditions and are possibly relA dependent . Immunofluorescence microscopy showed that the FtsZ protein can localize to presumptive division sites in strains carrying ftsW(Ts) mutations at the nonpermissive temperature, suggesting that FtsW is unlikely to be specifically required for the localization of FtsZ to the division site . Examination of the localization of FtsZ in an ftsW rodA double mutant (lemon-shaped cells) revealed several classes of cells ranging from a common class where an FtsZ ring structure is absent to a class where FtsZ forms a complete ring at the midpoint of a lemon-shaped cell, suggesting a role for FtsW in the establishment of a stable FtsZ-based septal structure . We further demonstrate that two FtsW peptides, FtsWL (large) and FtsWS (small), can be identified and that the expression of ftsWS is sufficient for complementation of ftsW(Ts) mutations. J Bacteriol, 1997 Feb, 179(3), 742 - 53 Importance of structural differences between complementary RNA molecules to control of replication of an IncB plasmid; Wilson IW et al.; Replication of the IncB miniplasmid pMU720 is dependent on the expression of repA, the gene encoding replication initiator protein RepA . Binding of a small antisense RNA (RNAI) to its complementary target (stem-loop I {SLI}) in the RepA mRNA prevents the participation of SLI in the formation of a pseudoknot that is an enhancer of translation of this mRNA . Thus, RNAI regulates the frequency of replication of pMU720 by controlling the efficiency of translation of the RepA mRNA . Mutational analysis of the two seven-base complementary sequences involved in formation of the pseudoknot showed that only the five central bases of each were critical for the formation of the pseudoknot . Physical analysis of SLI showed that despite the complete complementarity of its sequence to that of RNAI, the structures of the two molecules are different . The most prominent difference between the two structures is the presence of a 4-base internal loop immediately below the hairpin loop of SLI but not that of RNAI . Closure of this internal loop in SLI resulted in a 40-fold reduction in repA expression and loss of sensitivity of the residual expression to inhibition by RNAI . By contrast, repA expression was largely unaffected by the closure of a lower internal loop whose presence in SLI and RNAI is essential for effective interaction between these two molecules . These results suggest that the interaction of SLI with the distal pseudoknot bases is fundamentally different from the RNAI-SLI binding interaction and that the differences in structure between RNAI and SLI are necessary to allow SLI to be able to efficiently bind RNAI and to participate in pseudoknot formation. J Bacteriol, 1997 Feb, 179(3), 735 - 41 Role of conserved residues in hydrophilic loop 8-9 of the lactose permease; Pazdernik NJ et al.; A peptide motif, GXXX(D/E)(R/K)XG(R/K)(R/K), has been conserved in a large group of evolutionarily related membrane proteins that transport small molecules across the membrane . Within the superfamily, this motif is located in two cytoplasmic loops that connect transmembrane segments 2 and 3 and transmembrane segments 8 and 9 . In a previous study concerning the loop 2-3 motif of the lactose permease (A . E . Jessen-Marshall, N . J . Paul, and R . J . Brooker, J . Biol . Chem . 270:16251-16257, 1995), it was shown that the first-position glycine and the fifth-position aspartate are critical for transport activity since a variety of site-directed mutations greatly diminished the rate of transport . In the current study, a similar approach was used to investigate the functional significance of the conserved residues in the loop 8-9 motif . In the wild-type lactose permease, however, this motif has been evolutionarily modified so that the first-position glycine (an alpha-helix breaker) has been changed to proline (also a helix breaker); the fifth position has been changed to an asparagine; and one of the basic residues has been altered . In this investigation, we made a total of 28 single and 7 double mutants within the loop 8-9 motif to explore the functional importance of this loop . With regard to transport activity, amino acid substitutions within the loop 8-9 motif tend to be fairly well tolerated . Most substitutions produced permeases with normal or mildly defective transport activities . However, three substitutions at the first position (i.e., position 280) resulted in defective lactose transport . Kinetic analysis of position 280 mutants indicated that the defect decreased the Vmax for lactose uptake . Besides substitutions at position 280, a Gly-288-to-Thr mutant had the interesting property that the kinetic parameters for lactose uptake were normal yet the rates of lactose efflux and exchange were approximately 10-fold faster than wild-type rates . The results of this study suggest that loop 8-9 may facilitate conformational changes that translocate lactose. J Bacteriol, 1997 Feb, 179(3), 730 - 4 Oxidative inactivation of glutamine synthetase from the cyanobacterium Anabaena variabilis; Martin G et al.; In crude extracts of the cyanobacterium Anabaena variabilis, glutamine synthetase (GS) could be effectively inactivated by the addition of NADH . GS inactivation was completed within 30 min . Both the inactivated GS and the active enzyme were isolated . No difference between the two enzyme forms was seen in sodium dodecyl sulfate-gels, and only minor differences were detectable by UV spectra, which excludes modification by a nucleotide . Mass spectrometry revealed that the molecular masses of active and inactive GS are equal . While the Km values of the substrates were unchanged, the Vmax values of the inactive GS were lower, reflecting the inactivation factor in the crude extract . This result indicates that the active site was affected . From the crude extract, a fraction mediating GS inactivation could be enriched by ammonium sulfate precipitation and gel filtration . GS inactivation by this fraction required the presence of NAD(P)H, Fe3+, and oxygen . In the absence of the GS-inactivating fraction, GS could be inactivated by Fe2+ and H2O2 . The GS-inactivating fraction produced Fe2+ and H2O2, using NADPH, Fe3+, and oxygen . Accordingly, the inactivating fraction was inhibited by catalase and EDTA . This GS-inactivating system of Anabaena is similar to that described for oxidative GS inactivation in Escherichia coli . We conclude that GS inactivation by NAD(P)H is caused by irreversible oxidative damage and is not due to a regulatory mechanism of nitrogen assimilation. J Bacteriol, 1997 Feb, 179(3), 721 - 9 Nitrate- and nitrite-sensing protein NarX of Escherichia coli K-12: mutational analysis of the amino-terminal tail and first transmembrane segment; Williams SB et al.; Nitrate and nitrite control of anaerobic respiratory gene expression is mediated by dual two-component regulatory systems . The sensors NarX and NarQ each communicate nitrate and nitrite availability to the response regulators NarL and NarP . In the presence of nitrate, the NarX protein acts as a positive regulator ("kinase") of both NarL and NarP activity . In the presence of nitrite, the NarX protein acts primarily as a negative regulator ("phosphatase") of NarL activity but remains a positive regulator of NarP activity . In other topologically similar sensory proteins, such as the methyl-accepting chemotaxis proteins, the transmembrane regions are important for signal transduction . We therefore used localized mutagenesis of the amino-terminal coding region to isolate mutations in narX that confer an altered signaling phenotype . Five of the mutations studied alter residues in the amino-terminal cytoplasmic tail, and five alter residues in the first transmembrane segment . Based on patterns of target operon expression in various regulatory mutant strain backgrounds, most of the mutant NarX proteins appear to have alterations in negative control function . One mutant, with a change of residue Leu-11 to Pro in the cytoplasmic tail, exhibits strikingly altered patterns of NarL- and NarP-dependent gene expression . We conclude that the amino terminus of the NarX protein is important for the differential response to nitrate and nitrite. J Bacteriol, 1997 Feb, 179(3), 620 - 6 Detection and comparison of specific hemin binding by Porphyromonas gingivalis and Prevotella intermedia; Tompkins GR et al.; A radioligand assay was designed to detect and compare specific hemin binding by the periodontal anaerobic black-pigmenting bacteria (BPB) Porphyromonas gingivalis and Prevotella intermedia . The assay included physiological concentrations of the hemin-binding protein rabbit serum albumin (RSA) to prevent self-aggregation and nonspecific interaction of hemin with cellular components . Under these conditions, heme-starved P . intermedia cells (two strains) expressed a single binding site species (4,100 to 4,600 sites/cell) with a dissociation constant (Kd) of 1.0 x 10(-9) M . Heme-starved P . gingivalis cells (two strains) expressed two binding site species; the higher-affinity site (1,000 to 1,500 sites/cell) displayed a Kd of between 3.6 x 10(-11) and 9.6 x 10(-11) M, whereas the estimated Kd of the lower-affinity site (1.9 x 10(5) to 6.3 x 10(5) sites/cell) ranged between 2.6 x 10(-7) and 6.5 x 10(-8) M . Specific binding was greatly diminished in heme-replete cells of either BPB species and was not displayed by iron-replete Escherichia coli cells, which bound as much hemin in the absence of RSA as did P . intermedia . Hemin binding by BPB was reduced following treatment with protein-modifying agents (heat, pronase, and N-bromosuccinimide) and was blocked by protoporphyrin IX and hemoglobin but not by Congo red . Hemopexin also inhibited bacterial hemin binding . These findings indicate that both P . gingivalis and P . intermedia express heme-repressible proteinaceous hemin-binding sites with affinities intermediate between those of serum albumin and hemopexin . P . gingivalis exhibited a 10-fold-greater specific binding affinity and greater heme storage capacity than did P . intermedia, suggesting that the former would be ecologically advantaged with respect to heme acquisition. J Clin Microbiol, 1997 Feb, 35(2), 527 - 30 Distribution of colonization factor antigens among enterotoxigenic Escherichia coli strains isolated from patients with diarrhea in Nepal, Indonesia, Peru, and Thailand; Nirdnoy W et al.; Samples (1,318) of enterotoxigenic Escherichia coli (ETEC) isolated in 1994-1995 from children with diarrhea from Nepal, Indonesia, Peru, and Thailand were examined for colonization factor antigen (CFA) and coli surface (CS) antigens . Fifty-five percent of 361 heat-labile and heat-stable (LT-ST), 14% of 620 LT-only, and 48% of 337 ST-only ETEC had CFA/CS antigens . LT-ST ETEC strains were predominantly in the CFA II group, and ST only strains were in the CFA IV group . Additional studies are needed to identify ETEC strains that do not have CFA/CS antigens. Mol Cell Biol, 1997 Feb, 17(2), 977 - 88 Cleavage of membrane-associated pref-1 generates a soluble inhibitor of adipocyte differentiation; Smas CM et al.; pref-1 is an epidermal growth factor-like repeat protein present on the surface of preadipocytes that functions in the maintenance of the preadipose state . pref-1 expression is completely abolished during 3T3-L1 adipocyte differentiation . Bypassing this downregulation by constitutive expression of full-length transmembrane pref-1 in preadipocytes drastically inhibits differentiation . For the first time, we show processing of cell-associated pref-1 to generate both a soluble pref-1 protein of approximately 50 kDa that corresponds to the ectodomain and also smaller products of 24 to 25 kDa and 31 kDa . Furthermore, while all four of the alternately spliced forms of pref-1 produce cell-associated protein, only the two largest of the four alternately spliced isoforms undergo cleavage in the juxtamembrane region to release the soluble 50-kDa ectodomain . We demonstrate that addition of Escherichia coli-expressed pref-1 ectodomain to 3T3-L1 preadipocytes blocks differentiation, thus overriding the adipogenic actions of dexamethasone and methylisobutylxanthine . The inhibitory effects of the pref-1 ectodomain are blocked by preincubation of the protein with pref-1 antibody . That the ectodomain alone is sufficient for inhibition demonstrates that transmembrane pref-1 can be processed to generate an inhibitory soluble form, thereby greatly extending its range of action . Furthermore, we present evidence that alternate splicing is the mechanism that governs the production of transmembrane versus soluble pref-1, thereby determining the mode of action, juxtacrine or paracrine, of the pref-1 protein. Mol Cell Biol, 1997 Feb, 17(2), 571 - 83 The human Myt1 kinase preferentially phosphorylates Cdc2 on threonine 14 and localizes to the endoplasmic reticulum and Golgi complex; Liu F et al.; Entry into mitosis requires the activity of the Cdc2 kinase . Cdc2 associates with the B-type cyclins, and the Cdc2-cyclin B heterodimer is in turn regulated by phosphorylation . Phosphorylation of threonine 161 is required for the Cdc2-cyclin B complex to be catalytically active, whereas phosphorylation of threonine 14 and tyrosine 15 is inhibitory . Human kinases that catalyze the phosphorylation of threonine 161 and tyrosine 15 have been identified . Here we report the isolation of a novel human cDNA encoding a dual-specificity protein kinase (designated Myt1Hu) that preferentially phosphorylates Cdc2 on threonine 14 in a cyclin-dependent manner . Myt1Hu is 46% identical to Myt1Xe, a kinase recently characterized from Xenopus laevis . Myt1Hu localizes to the endoplasmic reticulum and Golgi complex in HeLa cells . A stretch of hydrophobic and uncharged amino acids located outside the catalytic domain of Myt1Hu is the likely membrane-targeting domain, as its deletion results in the localization of Myt1Hu primarily to the nucleus. J Virol, 1997 Feb, 71(2), 1538 - 46 The barley stripe mosaic virus 58-kilodalton beta(b) protein is a multifunctional RNA binding protein; Donald RG et al.; The barley stripe mosaic virus (BSMV) beta(b) gene product is the major viral nonstructural protein synthesized during early stages of the infection cycle and is required for systemic movement of the virus . To examine the biochemical properties of beta(b), a histidine tag was engineered at the amino terminus and the protein was purified from BSMV-infected barley tissue by metal affinity chromatography . The beta(b) protein bound ATPs in vitro, with a preference for ATP over dATP, and also exhibited ATPase activity . In addition, beta(b) bound RNA without detectable sequence specificity . However, binding was selective, as the beta(b) protein had a strong affinity for both single-stranded (ss) and double-stranded (ds) RNAs but not for tRNA or DNA substrates . Mutational analyses of beta(b) purified from Escherichia coli indicated that the protein has multiple RNA binding sites . These sites appear to contribute differently, because mutants that were altered in their binding affinities for ss and ds RNA substrates were recovered. J Chromatogr A, 1997 Jan 31, 760(2), 165 - 71 Role of polyethyleneimine in the purification of recombinant human tumour necrosis factor beta; Loh KC et al.; The chromatographic behaviour of recombinant human tumour necrosis factor beta (rhTNF-beta) (pI approximately 9.0) during cation-exchange chromatography at pH 7.5 is investigated . Without prior treatment of the Escherichia coli cell extract with polyethyleneimine (PEI), very little rhTNF-beta was bound to the column . However, upon addition of 5% PEI (100 microliters ml-1) to the cell lysate, rhTNF-beta was shown to bind to cation-exchange columns normally . TNF-beta was readily precipitated from the clarified cell extract by 20% ammonium sulphate, but ony ca . 25% of this precipitate could be re-solubilized for further purification . However, when 5% PEI was included in the solubilization buffer, the balance of the rhTNF-beta could be recovered . It is proposed that charge interaction between rhTNF-beta and nucleic acids in the cell extract is responsible for both of these anomalous phenomena, and that PEI (a cationic polyelectrolyte) was able to disrupt this interaction by displacing rhTNF-beta from the charge complex. Mutat Res, 1997 Jan 31, 383(1), 31 - 7 Different repair of O6-methylguanine occurring in DNA modified by MMS in vivo or in vitro; Sledziewska-Gojska E et al.; Lack of the adaptive response effect on the level of GC-->AT transitions induced by methyl methanesulfonate (MMS) in E . coli {Sledziewska-Gojska, E . (1993) The level of GC-->AT transitions induced by MMS is not affected by adaptive response of Escherichia coli K12 . Mutation Res., 294, 1-8.} can be explained by MMS inactivation of the ada encoded O6-methylguanine-DNA methyltransferase {Takahashi, K.Y., Kawazoe, K., Sakumi, Nakabeppu Y . and M . Sekiguchi (1988) Activation of Ada protein as a transcriptional regulator by direct alkylation with methylating agents, J . Biol . Chem., 263, 13490-13492; Sledziewska-Gojska, E . (1995) Inactivation of O6-methylguanine-DNA methyltransferase in vivo by SN2 alkylating agents, Mutation Res., 336, 61-67} . To evaluate this explanation and clarify the origin of MMS-induced GC-->AT transitions, we compared the repair of DNA treated by MMS in vivo or in vitro . Replication forms of lacZ mutants of E . coli phage M13mp18 were used to analyse the effect of the adaptive response on the frequency of GC-->AT transitions occurring in control and mismatch repair deficient strains . It was shown that DNA lesions, leading to GC-->AT transitions, induced by MMS in vivo are not repaired in adapted E . coli cells . In contrast, induction of the adaptive response causes efficient repair of these DNA lesions induced by MMS in vitro . This repair is consistent with the assumption that GC-->AT transitions induced by MMS are originated by O6-methylguanine and that MMS treatment of the cells during in vivo mutagenesis interfere with the adaptation mediated repair of the lesion . In agreement with this we have shown that treatment of the adapted cultures with 5 mM MMS completely blocks repair of in vitro modified DNA . Increased level of GC-->AT transitions induced by MMS occurs in mutS- strains . These mutations are avoided in adapted mutS- cells, when induced by MMS in vitro . This confirms that mismatch repair system of E . coli recognises mismatches formed in DNA by O6-methylguanine. Mutat Res, 1997 Jan 31, 383(1), 21 - 30 Rejoining of DNA double-strand breaks after the introduction of chromosome 11 into a radiosensitive bladder carcinoma cell line; Hofseth LJ et al.; Insertion of a normal chromosome 11 into tumour cell lines can protect against a sensitivity to irradiation and oxidative stress . A possible mechanism underlying this effect is that there is a correction of a defect in the rejoining of double-strand breaks (dsb) by the chromosome insertion . In order to explore this hypothesis, three cell lines were evaluated for their ability to rejoin dsb: (1) a bladder carcinoma cell line ('parent') previously shown to be sensitive to irradiation and radical generating species; (2) a derivative of this cell line into which a normal chromosome 11 had been inserted by microcell fusion ('hybrid') showing corrected radiosensitivity; and (3) a 'revertant' cell line that had spontaneously lost the insert and reverted to the radiosensitive phenotype . Nuclear extracts from the 3 lines were isolated and evaluated for their capacity to rejoin plasmid (pUC18) DNA broken at defined restriction sites (SalI, EcoRI, KpnI, SmaI) in the lacZ gene . The extent of rejoining was determined by gel electrophoresis and the fidelity of rejoining determined by expression of the lacZ gene in E . coli DH5 alpha bacteria . Results suggest there is no difference between the 'parent', 'hybrid' and 'revertant' nuclear extracts in the fidelity and the total extent of rejoining, regardless of the type of break . However, there is an alteration in the distribution of rejoined products . Nuclear extracts from 'hybrid' cells tend to rejoin linear DNA into circular monomers with a greater efficiency than extracts from both 'parent' and 'revertant' cells . This alteration in distribution is observed when 3'- or 5'-protruding ends are rejoined but not in the rejoining of blunt ends . The results suggest that loci on chromosome 11 are involved in the rejoining of dsb, affecting the relative amount of the different rejoined products . Whether this alteration plays a role in the 'parent' cell's radiosensitivity is yet to be determined. J Mol Biol, 1997 Jan 31, 265(4), 385 - 93 Role of the spacer boxA of Escherichia coli ribosomal RNA operons in efficient 23 S rRNA synthesis in vivo; Pfeiffer T et al.; A boxA sequence, known to be important for transcriptional antitermination, is found in both the leader region and in the spacer between the 16 S and 23 S genes of Escherichia coli ribosomal RNA operons . We have shown that a functional leader boxA is important for efficient completion of 16 S rRNA transcription . In this study, point mutations were introduced into the 16S-23S spacer boxA of a plasmid-encoded E . coli rrnB operon in order to study the contribution of this conserved sequence element to ribosomal RNA synthesis in vivo . The rrnB mutant constructs contained an additional point mutation in each of the 16 S and 23 S genes, which were used to distinguish rRNA derived from plasmid and chromosomal rrn operons by primer extension analysis . Mutations in the spacer boxA reduced the proportion of plasmid-derived 23 S rRNA without affecting synthesis of plasmid-derived 16 S rRNA or spacer boxA RNA, indicating that premature termination of transcription occurred during 23 S rRNA synthesis . Reductions in plasmid-derived 23 S rRNA were very similar for total cellular RNA, 50 S subunits and 70 S ribosomes, suggesting that plasmid-derived rRNAs from mutant operons were functional in ribosome biogenesis . In the presence of a wild-type leader boxA, single nucleotide exchanges in the spacer boxA reduced the proportion of plasmid-derived 23 S rRNA from 70% to about 55% under conditions of exponential growth in rich medium . This proportion further decreased to 20 to 25% with an additional point mutation in the leader boxA . We conclude that modification of RNA polymerase into a termination-resistant form has to be renewed at the spacer boxA in order to ensure the faithful completion of full-length 23 S rRNA. Gene, 1997 Jan 31, 185(1), 111 - 7 A simple screening for mutant DNA binding proteins: application to murine transcription factor PEBP2alpha subunit, a founding member of the Runt domain protein family; Akamatsu Y et al.; Mouse transcription factor PEBP2 (polyomavirus enhancer-binding protein (2) is composed of two distinct subunits alpha and beta . The alpha subunit has an ability to bind the specific DNA sequences, which is enhanced by formation of a heterodimer with the beta subunit . The DNA binding and heterodimerization activities of the alpha subunit are both localized within a 128-amino-acid (aa) region termed as the Runt domain for its homology to the Drosophila segmentation gene runt . To characterize the molecular determinants for these activities, the Runt domain was randomly mutagenized and produced in E . coli as a secreted form . Using E . coli culture supernatant, the DNA binding and heterodimerization of mutant Runt domains were analyzed by gel retardation assay . Nine randomly picked single-aa substitution mutants showed various functional alterations in DNA binding and heterodimerization either separately or simultaneously . This observation suggests that the structure of Runt domain is highly ordered and is quite sensitive to modulations in its primary structure . The method presented here provides a simple and quick method to characterize a large number of mutant DNA binding proteins. Gene, 1997 Jan 31, 185(1), 105 - 9 Cloning and expression of AatII restriction-modification system in Escherichia coli; Nwankwo DO et al.; The genes encoding the AatII restriction endonuclease and methylase from Acetobacter aceti have been cloned and expressed in Escherichia coli . The nucleotide sequences of aatIIM and aatIIR genes were determined . The aatIIM and aatIIR genes are 996 bp and 1038 bp, respectively, encoding the 331-aa methylase with a predicted molecular mass of 36.9 kDa, and the 345-aa AatII restriction endonuclease with a predicted molecular mass of 38.9 kDa . The two genes overlap by 4 base pairs and are transcribed in the same orientation . The aatIIRM genes are located next to a putative gene for plasmid mobilization . A stable overproducing strain was constructed, in which the aatIIM gene was expressed from a pSC101-derived plasmid . The aatIIR gene was inserted into a modified T7 expression vector that carries transcription terminators upstream from the T7 promoter . The recombinant AatII restriction endonuclease was purified to near homogeneity by chromatography through DEAE Sepharose, Heparin Sepharose, and phosphocellulose columns. J Biol Chem, 1997 Jan 31, 272(5), 2834 - 40 Overexpression, purification, and characterization of the catalase-peroxidase KatG from Mycobacterium tuberculosis; Johnsson K et al.; Wild-type catalase-peroxidase KatG from Mycobacterium tuberculosis as well as a specific mutant (R463L) frequently found in isoniazid-resistant strains have been overexpressed in Escherichia coli, allowing purification of sufficient quantities of enzyme for physical and kinetic characterization . Optical absorption and EPR spectroscopies indicate that KatG is similar to a growing class of bacterial catalase-peroxidases . Optical and EPR spectra of KatG in the presence of either a strong field or weak field ligand suggest that, like horseradish peroxidase and metmyoglobin, KatG is likely to have a histidine as a proximal ligand . The wild-type enzyme functions as a highly active catalase as well as a broad specificity peroxidase . Wild-type KatG and the R463L mutant of KatG exhibit identical spectroscopic and kinetic properties . Furthermore, both enzymes are equally capable of metabolizing the important antituberculosis drug isoniazid. J Biol Chem, 1997 Jan 31, 272(5), 2744 - 52 Siroheme biosynthesis in higher plants . Analysis of an S-adenosyl-L-methionine-dependent uroporphyrinogen III methyltransferase from Arabidopsis thaliana; Leustek T et al.; Siroheme, the prosthetic group for both nitrite and sulfite reductases, is a methylated, iron-containing modified tetrapyrrole . Here we report the first molecular characterization of the branch point enzyme in higher plants, which directs intermediates toward siroheme synthesis . A cDNA was cloned from Arabidopsis thaliana (UPM1) that functionally complements an Escherichia coli cysG mutant, a strain that is unable to catalyze the conversion of uroporphyrinogen III (Uro'gen-III) to siroheme . UPM1 is 1484 base pairs and encodes a 369-amino acid, 39.9-kDa protein . The UPM1 product contains two regions that are identical to consensus sequences found in bacterial Uro'gen-III and precorrin methyltransferases . Recombinant UPM1 protein was found to catalyze S-adenosyl-L-methionine-dependent transmethylation by UPM1 in a multistep process involving the formation of a covalently linked complex with S-adenosyl-L-methionine . The UPM1 product has a sequence at the amino terminus that resembles a transit peptide for localization to mitochondria or plastids . The protein produced by in vitro expression is able to enter isolated intact chloroplasts but not mitochondria . Genomic blot analysis showed that UPM1 is encoded in the A . thaliana genome . The genomic DNA corresponding to UPM1 was cloned and sequenced and found to contain at least five introns. J Biol Chem, 1997 Jan 31, 272(5), 2714 - 21 Real time conformational changes in the retinal phosphodiesterase gamma subunit monitored by resonance energy transfer; Berger AL et al.; The gamma subunit of the retinal cGMP phosphodiesterase (gammaPDE) acts as an inhibitor of phosphodiesterase (PDE) catalytic activity and mediates enzyme regulation by the alpha subunit of the GTP-binding protein transducin (alphaT) . In order to characterize conformational changes in the 87-amino acid gammaPDE subunit that may accompany the activation of the holoenzyme, gammaPDE was labeled with the fluorescent probes 5-iodoacetamidofluorescein and eosin-5-isothiocyanate for use in resonance energy transfer measurements . 5-Iodoacetamidofluorescein specifically labeled a cysteine residue at position 68 and served as a resonance energy transfer donor . The site of modification of eosin-5-isothiocyanate, which served as the resonance energy transfer acceptor, was determined to be within the first seven residues of the amino terminus of gammaPDE . Energy transfer between the labeled sites on free, unbound gammaPDE indicated that they were separated by a distance of 63 A, consistent with a random conformation . Upon binding the catalytic alphabeta subunits of the PDE, the distance between the two probes on gammaPDE increased to 77 A . Binding of the labeled gammaPDE by alphaT.guanosine 5'-3-O-(thio)triphosphate did not affect the distance between the probes under conditions where the PDE was activated . These data are consistent with the view that the binding of activated alphaT to gammaPDE, which is essential for the stimulation of PDE activity, does not impart significant alterations in the tertiary structure of the gammaPDE molecule . They also support a model for PDE activation that places active alphaT in a complex with the holoenzyme. J Biol Chem, 1997 Jan 31, 272(5), 2682 - 7 Biosynthesis of the escherichia coli K4 capsule polysaccharide . A parallel system for studies of glycosyltransferases in chondroitin formation; Lidholt K et al.; Escherichia coli K4 bacteria synthesize a capsule polysaccharide (GalNAc-GlcA(fructose))n with the carbohydrate backbone identical to chondroitin . GlcA- and GalNAc-transferase activities from the bacterial membrane were assayed with acceptors derived from the capsule polysaccharide and radiolabeled UDP-{14C}GlcA and UDP-{3H}GalNAc, respectively . It was shown that defructosylated oligosaccharides (chondroitin) could serve as substrates for both the GlcA- and the GalNAc-transferases . The radiolabeled products were completely degraded with chondroitinase AC; the {14C}GlcA unit could be removed by beta-D-glucuronidase, and the {3H}GalNAc could be removed by beta-N-acetylhexosaminidase . A fructosylated oligosaccharide acceptor tested for GlcA-transferase activity was found to be inactive . These results indicate that the chain elongation reaction of the K4 polysaccharide proceeds in the same way as the polymerization of the chondroitin chain, by the addition of the monosaccharide units one by one to the nonreducing end of the polymer . This makes the biosynthesis of the K4 polysaccharide an interesting parallel system for studies of chondroitin sulfate biosynthesis . In the biosynthesis of capsule polysaccharides from E . coli, a similar mechanism has earlier been demonstrated for polysialic acid (NeuNAc)n (Rohr, T . E., and Troy, F . A . (1980) J . Biol . Chem . 255, 2332-2342) and for the K5 polysaccharide (GlcAbeta1-4GlcNAcalpha1-4)n (Lidholt, K., Fjelstad, M., Jann, K., and Lindahl, U . (1994) Carbohydr . Res . 255, 87-101) . In contrast, chain elongation of hyaluronan (GlcAbeta1-3GlcNAcbeta1-4)n is claimed to occur at the reducing end (Prehm, P . (1983) Biochem . J . 211, 181-189). Nature, 1997 Jan 30, 385(6615), 442 - 6 Crosstalk between G proteins and protein kinase C mediated by the calcium channel alpha1 subunit; Zamponi GW et al.; The modulation of voltage-dependent Ca2+ channels at presynaptic nerve terminals is an important factor in the control of neurotransmitter release and synaptic efficacy . Some terminals contain multiple Ca2(+)-channel subtypes (N and P/Q), which are differentially regulated by G-protein activation and by protein kinase C (PKC)-dependent phosphorylation . Regulation of channel activity by crosstalk between second messenger pathways has been reported although the molecular mechanisms underlying crosstalk have not been described . Here we show that crosstalk occurs at the level of the presynaptic Ca2(+)-channel complex . The alpha1 subunit domain I-II linker, which connects the first and second transmembrane domains, contributes to the PKC-dependent upregulation of channel activity, while G-protein-dependent inhibition occurs through binding of Gbetagamma to two sites in the I-II linker . Crosstalk results from the PKC-dependent phosphorylation of one of the Gbetagamma binding sites which antagonizes Gbetagamma-induced inhibition . The results provide a mechanism for the highly regulated and dynamic control of neurotransmitter release that depends on the integration of multiple presynaptic signals. Biochemistry, 1997 Jan 28, 36(4), 932 - 40 Equilibrium and kinetic analyses of unfolding and refolding for the conserved proline mutants of tryptophan synthase alpha subunit; Ogasahara K et al.; To elucidate the role of conserved proline residues of the tryptophan synthase alpha subunit from Escherichia coli in stability and folding, equilibrium and kinetic studies of the unfolding-refolding induced by guanidine hydrochloride for six mutant alpha subunits (Pro-->Ala) were carried out by peptidyl circular dichroism and aromatic fluorescence measurements at pH 7 and 25 degrees C . These results were analyzed assuming the presence of one intermediate (I) state in the denaturation process . (I) For all mutant and wild-type proteins, the Gibbs energy change (delta Gni(H2O)) in water between the native (N) and I states coincided with the difference (delta G++u(H2O)-delta G++r(H2O)) between the activation Gibbs energy changes in water for the unfolding (delta G++u(H2O) and refolding (delta G++r(H2O) reactions . This means that the early folding intermediate of the alpha subunit corresponds to the equilibrium intermediate . Delta Gni(H2O) values of all mutant proteins decreased compared with that of the wild-type protein . Gibbs energy change (delta Gid(H2O) in water between I and the denatured (D) states was not substantially affected by the substitutions . Delta G++u(H2O) and delta G++r(H2O) decreased and increased, respectively, for all mutant proteins . (2) Six conserved prolines played roles in stability and folding of the alpha subunit in a different manner: prolines 28 and 96 by stabilizing the N state and prolines 28, 96, 132, and 207 by destabilizing the I state . The contributions of prolines 57 and 62 to the stability were marginal . (3) Cis proline 28 was not the origin of the slow phase in the refolding kinetics assumed to arise from the cis-trans isomerization reaction of proline. Biochemistry, 1997 Jan 28, 36(4), 894 - 902 Uncompetitive substrate inhibition and noncompetitive inhibition by 5-n-undecyl-6-hydroxy-4,7-dioxobenzothiazole (UHDBT) and 2-n-nonyl-4-hydroxyquinoline-N-oxide (NQNO) is observed for the cytochrome bo3 complex: implications for a Q(H2)-loop proton translocation mechanism; Musser SM et al.; The cytochrome bo3 ubiquinol oxidase complex from Escherichia coli contains two binding sites for ubiquinone(ol) (UQ(H2)) . One of these binding sites, the ubiquinol oxidation site, is clearly in dynamic equilibrium with the UQ(H2) pool in the membrane . The second site has a high affinity for ubiquinone (UQ), stabilizes a semiquinone species, and is located physically close to the low-spin heme b component of the enzyme . The UQ molecule in this site has been proposed to remain strongly bound to the enzyme during enzyme turnover and to act as a cofactor facilitating the transfer of electrons from the substrate ubiquinol to heme b {Sato-Watanabe et al . (1994) J . Biol . Chem . 269, 28908-28912} . In this paper, the steady-state turnover of the enzyme is examined in the presence and absence of inhibitors (UHDBT and NQNO) that appear to be recognized as ubisemiquinone analogs . It is found that the kinetics are accounted for best by a noncompetitive inhibitor binding model . Furthermore, at high concentrations, the substrates ubiquinol-1 and ubiquinol-2 inhibit turnover in an uncompetitive fashion . Together, these observations strongly suggest that there must be at least two UQ(H2) binding sites that are in rapid equilibrium with the UQ(H2) pool under turnover conditions . Although these data do not rule out the possibility that a strongly bound UQ molecule functions to facilitate electron transfer to heme b, they are more consistent with the behavior expected if the two UQ(H2) binding sites were to function in a Q(H2)-loop mechanism (similar to that of the cytochrome bc1 complex) as originally proposed by Musser and co-workers {(1993) FEBS Lett . 327, 131-136} . In this model, ubiquinol is oxidized at one site and ubiquinone is reduced at the second site . While the structural similarities of the heme-copper ubiquinol and cytochrome c oxidase complexes suggest the possibility that these two families of enzymes translocate protons by similar mechanisms, the current observations indicate that the Q(H2)-loop proton translocation mechanism for the heme-copper ubiquinol oxidase complexes should be further investigated and experimentally tested. Biochemistry, 1997 Jan 28, 36(4), 812 - 22 Spectroscopic properties of Escherichia coli UDP-N-acetylenolpyruvylglucosamine reductase; Axley MJ et al.; Purified uridine diphosphate N-acetylenolpyruvylglucosamine reductase (E.C . 1.1.1.158) was analyzed by circular dichroism (CD) and UV-visible spectroscopy to establish the spectral properties of its tightly bound flavin adenine dinucleotide (FAD) cofactor . The polypeptide backbone displayed a single circular dichroic minimum at 208 nm and a single maximum at 193 nm . The CD spectrum of bound flavin exhibited a single major negative Cotton peak at 364 nm and two minor negative Cotton peaks at 464 and 495 nm . The protein was reversibly unfolded in 9.8 M urea and refolded in buffer in the presence of excess FAD . The refolded enzyme incorporated FAD and catalyzed full activity . The bound FAD displayed an absorption maximum at 464 nm with an extinction coefficient of epsilon 464 = 11700 M-1 cm-1 . Anaerobic reduction with dithionite was complete at 1 equiv . Anaerobic reduction with nicotinamide adenine dinucleotide phosphate, reduced form (NADPH), also was essentially complete at 1 equiv and produced a long-wavelength absorbance band characteristic of an FAD-pyridine nucleotide charge transfer complex . Photochemical bleaching in the presence of ethylenediaminetetraacetic acid (EDTA) followed exponential kinetics . None of the anaerobic reductive titrations produced a spectral intermediate characteristic of a flavin semiquinone, and all reduced enzyme species could be fully reoxidized by oxygen, with full recovery of catalytic activity . Photochemically reduced enzyme was reoxidized by titration with either NADP+ or uridine diphospho N-acetylglucosamine enolpyruvate (UNAGEP) . Reoxidation by NADP+ reached a chemical equilibrium, whereas reoxidation by UNAGEP was stoichiometric . Binding of NADP+ or UNAGEP to the oxidized form of the enzyme produced a dead-end complex that could be titrated by following a 10-nm red shift in the absorption spectrum of the bound FAD . The Kd of NADP+ for oxidized enzyme was 0.7 +/- 0.3 microM and the Kd of UNAGEP was 2.7 +/- 0.3 microM . Solvent deuterium isotope effects on binding were observed for both NADP+ and UNAGEP, depending on the pH . At pH 8.5, the HKd/DKd was 2.2 for NADP+ and 3.9 for UNAGEP . No spectral changes were observed in the presence of a 40-fold excess of uridine diphospho N-acetylmuramic acid (UNAM) either aerobically or anaerobically . These studies have identified spectral signals for five steps in the kinetic mechanism, have indicated that product formation is essentially irreversible, and have indicated that hydrogen bonding or protonation contributes significantly to ground-state complex formation with the physiological substrate. Biochemistry, 1997 Jan 28, 36(4), 699 - 710 Phosphotransfer site of the chemotaxis-specific protein kinase CheA as revealed by NMR; Zhou H et al.; Bacterial chemotaxis involves autophosphorylation of a histidine kinase and transfer of the phosphoryl group to response regulators to control flagellar rotation and receptor adaptation . The phosphotransfer domain, CheA1-134, of the chemotaxis-specific histidine autokinase CheA from Escherichia coli contains the site of phosphorylation, His48, and two other histidine residues, His26 and His67 . Two-dimensional 1H-15N NMR techniques were applied to characterize the protonation states of these histidine residues and to evaluate the structural changes in the domain that occur upon phosphorylation of His48 . The pKa of His48 was determined to be 7.8 (in 50 mM NaPO4 buffer at 30 degrees C) . At high pH, its imidazole ring exists primarily as the normally unfavored N delta 1H tautomer, suggesting hydrogen bond formation to the ring nitrogen atom(s) to stabilize this state . The pKa values and predominant tautomeric states of the imidazole rings of His26 (pKa approximately 7.1, N epsilon 2H tautomer) and His67 (pKa approximately 6.5, N delta 1H tautomer) were also determined . His48 of CheA1-134 and CheA1-233 was phosphorylated by full-length CheA . The phosphorylation site was confirmed to be the N epsilon 2 position in the imidazole ring . Phosphorylation of His48 only results in small changes in the amide 1H and 15N chemical shifts of a few residues from helices B and C, suggesting that only very small changes in structure are associated with phosphorylation of the phosphotransfer domain of CheA . These residues occupy a small surface area of the helix bundle and form the active site of the protein . At the active site, in addition to His48, residues Gly52, His67, and Glu70 are conserved in the CheA homologous phosphotransfer domains from 10 different organisms . Sequence comparison of these CheA homologs suggest that the phosphotransfer domains likely fold in a similar helix-bundle structure and the structural features at the active site are well-conserved. Mol Gen Genet, 1997 Jan 27, 253(4), 515 - 9 Functional relationship between Escherichia coli RNase E and the CafA protein; Wachi M et al.; We analyzed the functional relationship between the Escherichia coli RNase E and the CafA protein, which show extensive sequence similarity . The temperature-sensitive growth of the RNase E mutant strain ams1 was partially suppressed by multicopy plasmids bearing the cafA gene . Introduction of a cafA::cat mutation enhanced the temperature sensitivity of the ams1 mutant . These results suggest that there is a functional homology between these two proteins. FEBS Lett, 1997 Jan 27, 402(1), 62 - 6 Cloning of the fabF gene in an expression vector and in vitro characterization of recombinant fabF and fabB encoded enzymes from Escherichia coli; Edwards P et al.; Analysis of the beta-ketoacyl-ACP synthase (KAS) encoded by the fabF gene of Escherichia coli has been hampered by a reported instability of the cloned gene . Here we describe biochemical characterization of purified, active protein from the recombinant fabF gene . This enzyme has the properties ascribed to KAS II and not those of a putative KAS IV reported to be encoded by fabJ, a genomic clone with DNA sequence identical to that of fabF . We also characterize active protein from a recombinant fabB gene and suggest that this method may have a general utility for analysis of KAS enzymes. J Mol Biol, 1997 Jan 24, 265(3), 302 - 9 Leading versus lagging strand mutagenesis induced by 7,8-dihydro-8-oxo-2'-deoxyguanosine in Escherichia coli; Wagner J et al.; We have previously shown that a single N-2-acetylaminofluorene (AAF) adduct bound to the C-8 position of a guanine residue located within plasmids containing the unidirectional ColE1 origin of replication induces a 20-fold higher mutation frequency when the adduct is located in the lagging strand as compared to the leading strand . In this study, single 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxodG) lesions have been introduced in the leading and lagging strand orientation within the same sequence context as for the AAF adducts . The induced frequency of guanine to thymine transversions has been measured, using a specific PCR-based quantitative assay, in strains deficient in the repair of the oxidative lesion . The potential involvement of the UvrABC excision repair system in the removal of 8-oxodG has also been investigated and ruled out . Concerning the mutation frequency asymmetry, in contrast to AAF adducts, 8-oxodG adducts induce the same mutation frequency, irrespective of their location in the leading or lagging strands . This striking difference between 8-oxodG and dGuo-C8-AAF adducts is discussed in terms of their differential capacity to block DNA replication. Cell, 1997 Jan 24, 88(2), 277 - 85 Endostatin: an endogenous inhibitor of angiogenesis and tumor growth; O'Reilly MS et al.; We previously identified the angiogenesis inhibitor angiostatin . Using a similar strategy, we have identified endostatin, an angiogenesis inhibitor produced by hemangioendothelioma . Endostatin is a 20 kDa C-terminal fragment of collagen XVIII . Endostatin specifically inhibits endothelial proliferation and potently inhibits angiogenesis and tumor growth . By a novel method of sustained release, E . coli-derived endostatin was administered as a nonrefolded suspension . Primary tumors were regressed to dormant microscopic lesions . Immunohistochemistry revealed blocked angiogenesis accompanied by high proliferation balanced by apoptosis in tumor cells . There was no toxicity . Together with angiostatin data, these findings validate a strategy for identifying endogenous angiogenesis inhibitors, suggest a theme of fragments of proteins as angiogenesis inhibitors, and demonstrate dormancy therapy. Cell, 1997 Jan 24, 88(2), 235 - 42 The solution structure of the S1 RNA binding domain: a member of an ancient nucleic acid-binding fold; Bycroft M et al.; The S1 domain, originally identified in ribosomal protein S1, is found in a large number of RNA-associated proteins . The structure of the S1 RNA-binding domain from the E . coli polynucleotide phosphorylase has been determined using NMR methods and consists of a five-stranded antiparallel beta barrel . Conserved residues on one face of the barrel and adjacent loops form the putative RNA-binding site . The structure of the S1 domain is very similar to that of cold shock protein, suggesting that they are both derived from an ancient nucleic acid-binding protein . Enhanced sequence searches reveal hitherto unidentified S1 domains in RNase E, RNase II, NusA, EMB-5, and other proteins. J Biol Chem, 1997 Jan 24, 272(4), 2559 - 69 Abasic translesion synthesis by DNA polymerase beta violates the "A-rule" . Novel types of nucleotide incorporation by human DNA polymerase beta at an abasic lesion in different sequence contexts; Efrati E et al.; The "A-rule" reflects the preferred incorporation of dAMP opposite abasic lesions in Escherichia coli in vivo . DNA polymerases (pol) from procaryotic and eucaryotic organisms incorporate nucleotides opposite abasic lesions in accordance with the A-rule . However, recent in vivo data demonstrate that A is not preferentially incorporated opposite abasic lesions in eucaryotes . Purified human DNA polymerases beta and alpha are used to measure the specificity of nucleotide incorporation at a site-directed tetrahydrofuran abasic lesion, in 8-sequence contexts, varying upstream and downstream bases adjacent to the lesion . Extension past the lesion is measured in 4 sequence contexts, varying the downstream template base . Pol alpha strongly favors incorporation of dAMP directly opposite the lesion . In marked contrast, pol beta violates the A-rule for incorporation directly opposite the lesion . In addition to incorporation taking place directly opposite the lesion, we also analyze misalignment incorporation directed by a template base downstream from the lesion . Lesion bypass by pol beta occurs predominantly by "skipping over" the lesion, by insertion of a nucleotide complementary to an adjacent downstream template site . Misalignment incorporation for pol beta occurs by a novel "dNTP-stabilized" mechanism resulting in both deletion and base substitution errors . In contrast, pol alpha shows no propensity for this type of synthesis . The misaligned DNA structures generated during dNTP-stabilized lesion bypass do not conform to misaligned structures reported previously. J Biol Chem, 1997 Jan 24, 272(4), 2429 - 36 Dissection of glutathionylspermidine synthetase/amidase from Escherichia coli into autonomously folding and functional synthetase and amidase domains; Kwon DS et al.; The bifunctional glutathionylspermidine synthetase/amidase from Escherichia coli catalyzes both the ATP-dependent formation of an amide bond between N1 of spermidine (N-(3-amino)propyl-1, 4-diaminobutane) and the glycine carboxylate of glutathione (gamma-Glu-Cys-Gly) and the opposing hydrolysis of this amide bond (Bollinger, J . M., Jr., Kwon, D . S., Huisman, G . W., Kolter, R., and Walsh, C . T . (1995) J . Biol . Chem . 270, 14031-14041) . In our previous work describing its initial characterization, we proposed that the 619-amino acid (70 kDa) protein might possess separate amidase (N-terminal) and synthetase (C-terminal) domains . In the present study, we have confirmed this hypothesis by expression of independently folding and functional amidase and synthetase modules . A fragment containing the C-terminal 431 amino acids (50 kDa) has synthetase activity only, with steady-state kinetic parameters similar to the full-length protein . A fragment containing the N-terminal 225 amino acids (25 kDa) has amidase activity only and is significantly activated relative to the full-length protein for hydrolysis of glutathionylspermidine analogs . This observation suggests that the amidase activity in the full-length protein is negatively autoregulated . The amidase active site catalyzes hydrolysis of amide and ester derivatives of glutathione (e.g . glutathione ethyl ester and glutathione amide) but lacks activity toward acetylspermidine (N1 and N8) and acetylspermine (N1), indicating that glutathione provides the primary recognition determinants for glutathionylspermidine amide bond cleavage . No metal ion is required for the amidase activity . A tetrahedral phosphonate analogue of glutathionylspermidine, designed as a mimic of the proposed tetrahedral intermediate for either reaction, inhibits the synthetase activity (Ki approximately 10 microM) but does not inhibit the amidase activity. J Biol Chem, 1997 Jan 24, 272(4), 2259 - 67 Influence of Mg2+ and temperature on formation of the transcription bubble; Zaychikov E et al.; The transcription bubble formed in the binding complex of T7A1 promoter upon Escherichia coli RNA polymerase was analyzed by chemical probes, namely by single-strand specific reagents, to map the unpaired bases in the bubble, and by FeEDTA, to analyze the accessibility of the DNA backbone . The latter probe could also be used as a local hydroxyl radical probe placed close to the Mg2+-binding site in the active center . The data show that the transcription bubble consists of two parts, an Mg2+-dependent part and an Mg2+-independent part, both having individual transition temperatures . The data further suggest that formation of a transcription active open complex is preceded by a transition state complex having enhanced affinity for those Mg2+ ions presumably participating in the formation of the catalytic site . Our data also suggests that the three catalytically active Mg2+ ions in RNA polymerase are functionally not equivalent . One/two of the three Mg2+ ions are responsible for the polymerization, the other two/one for enlargement of the transcription bubble. Biochem Biophys Res Commun, 1997 Jan 23, 230(3), 582 - 6 Escherichia coli rnpB promoter mutants altered in stringent response; Jung YH et al.; The promoter of the rnpB gene (encoding the RNA component of Escherichia coli RNase P) shares a consensus discriminator sequence, located between the -10 hexamer sequence and the transcription start site, with other promoters whose activities are repressed upon stringent condition . Under stringent conditions induced by seryl-tRNA starvation the transcription of the rnpB gene was repressed in wild type E . coli but not in a relaxed strain carrying a relA- mutation . Site-directed mutagenesis was carried out to examine sequences of the rnpB promoter necessary for stringent control . The results indicate that the discriminator region is responsible for the transcription repression of the rnpB gene during the stringent response and that both the content and position of GC pairs in the region determine the strength of negative stringent signals. Nature, 1997 Jan 23, 385(6614), 365 - 8 Crystal structure of the NG domain from the signal-recognition particle receptor FtsY; Montoya G et al.; Newly synthesized proteins destined either for secretion or incorporation into membranes are targeted to the membrane translocation machinery by a ubiquitous system consisting of a signal-recognition particle (SRP) and its receptor . Both the SRP receptor and the protein within the SRP that binds the signal sequence contain GTPases . These two proteins, together with the RNA component of the SRP, form a complex and thereby regulate each other's GTPase activity . Here we report the structure of the GTPase-containing portion of FtsY, the functional homologue of the SRP receptor of Escherichia coli, at 2.2 A resolution without bound nucleotide . This so-called NG domain displays similarities to the Ras-related GTPases, as well as features unique to the SRP-type GTPases, such as a separate amino-terminal domain, an insertion within the p21ras (Ras) effector domain, and a wide-open GTP-binding region . The structure explains the low affinity of FtsY for GTP, and suggests rearrangements that may occur on nucleotide binding . It also identifies regions potentially involved in the transmission of signals between domains and in interactions with regulatory proteins. Nature, 1997 Jan 23, 385(6614), 361 - 4 Structure of the conserved GTPase domain of the signal recognition particle; Freymann DM et al.; The signal-recognition particle (SRP) and its receptor (SR) function in the co-translational targeting of nascent protein-ribosome complexes to the membrane translocation apparatus . The SRP protein subunit (termed Ffh in bacteria) that recognizes the signal sequence of nascent polypeptides is a GTPase, as is the SR-alpha subunit (termed FtsY) . Ffh and FtsY interact directly, each stimulating the GTP hydrolysis activity of the other . The sequence of Ffh suggests three domains: an amino-terminal N domain of unknown function, a central GTPase G domain, and a methionine-rich M domain that binds both SRP RNA and signal peptides . Sequence conservation suggests that structurally similar N and G domains are present in FtsY . Here we report the structure of the nucleotide-free form of the NG fragment of Ffh . Consistent with a role for apo Ffh in protein targeting, the side chains of the empty active-site pocket form a tight network of interactions which may stabilize the nucleotide-free protein . The structural relationship between the two domains suggests that the N domain senses or controls the nucleotide occupancy of the GTPase domain . A structural subdomain unique to these evolutionarily conserved GTPases constitutes them as a distinct subfamily in the GTPase superfamily. Nature, 1997 Jan 23, 385(6614), 353 - 7 Bcl-x(L) forms an ion channel in synthetic lipid membranes; Minn AJ et al.; Bcl-2-related proteins are critical regulators of cell survival that are localized to the outer mitochondrial, outer nuclear and endoplasmic reticulum membranes . Despite their physiological importance, the biochemical function of Bcl-2-related proteins has remained elusive . The three-dimensional structure of Bcl-xL, an inhibitor of apoptosis, was recently shown to be similar to the structures of the pore-forming domains of bacterial toxins . A key feature of these pore-forming domains is the ability to form ion channels in biological membranes . Here we demonstrate that Bcl-xL shares this functional feature . Like the bacterial toxins, Bcl-xL can insert into either synthetic lipid vesicles or planar lipid bilayers and form an ion-conducting channel . This channel is pH-sensitive and becomes cation-selective at physiological pH . The ion-conducting channel(s) formed by Bcl-xL display multiple conductance states that have identical ion selectivity . Together, these data suggest that Bcl-xL may maintain cell survival by regulating the permeability of the intracellular membranes to which it is distributed. Biochim Biophys Acta, 1997 Jan 21, 1344(2), 139 - 52 Characterization of human apolipoprotein A-I expressed in Escherichia coli; Bergeron J et al.; Human apolipoprotein A-I (apoA-I), with an additional N-terminal extension (Met-Arg-Gly-Ser-(His)6-Met) (His-apoA-I), has been produced in Escherichia coli with a final yield after purification of 10 mg protein/1 of culture medium . We have characterized the conformation and structural properties of His-apoA-I in lipid-free form, and in reconstituted lipoproteins containing two apoA-I per particle (Lp2A-I) by both immunochemical and physicochemical techniques . The lipid-free forms of the two proteins present very similar secondary structure and stability, and have also very similar kinetics of association with dimyristoyl phosphatidylcholine . His-apoA-I and native apoA-I can be complexed with 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC) to form similar, stable, either discoidal or spherical (sonicated) Lp2A-I particles . Lipid-bound native apoA-I and His-apoA-I showed very similar alpha-helical content (69% and 66%, respectively in discoidal Lp2A-I and 54% and 51%, respectively in spherical Lp2A-I) . The conformation of His-apoA-I in lipid-free form and in discoidal or spherical Lp2A-I has also been shown to be similar to native apoA-I by immunochemical measurements using 13 monoclonal antibodies recognizing distinct apoA-I epitopes . In the free protein and in reconstituted Lp2A-I, the N-terminal has no effect on the affinity of any of the monoclonal antibodies and minimal effect on immunoreactivity values . Small differences in the exposure of some apoA-I epitopes are evident on discoidal particles, while no difference is apparent in the expression of any epitope of apoA-I on spherical Lp2A-I . The presence of the N-terminal extension also has no effect on the reaction of LCAT with the discoidal Lp2A-I or on the ability of complexes to promote cholesterol efflux from fibroblasts in culture . In conclusion, we show that His-apoA-I expressed in E . coli exhibits similar physicochemical properties to native apoA-I and is also identical to the native protein in its ability to interact with phospholipids and to promote cholesterol esterification and cellular cholesterol efflux. Proc Natl Acad Sci U S A, 1997 Jan 21, 94(2), 463 - 8 Activities of human recombination protein Rad51; Gupta RC et al.; Homologous pairing and strand exchange, which are catalyzed by Escherichia coli RecA protein, are central to homologous recombination . Homologs of this protein are found in eukaryotes; however, little has been reported on the recombinase activities of the mammalian homologs, including the human protein, denoted HsRad51 . For the studies described here, we purified HsRad51 form E . coli . Although the activities of HsRad51 and RecA were qualitatively similar in the presence of ATP, there were also striking differences . The stoichiometry of binding to DNA and the rate of renaturation of complementary strands were similar for the two proteins, but rates of ATP hydrolysis, homologous pairing, and subsequent strand exchange promoted by HsRad51 were less than 1/10 those of RecA . In addition, HsRad51 bound gamma-thio-ATP and formed stable presynaptic complexes that promoted renaturation as rapidly as RecA, but the recombinant human protein catalyzed neither strand exchange nor homologous pairing of a single strand with duplex DNA in the presence of the ATP analog . By contrast, RecA promoted both of the latter reactions in control experiments . These observations suggest that among RecA-like proteins, HsRad51 may be a variant in which homologous pairing and strand exchange are more closely linked to the hydrolysis of ATP. Proc Natl Acad Sci U S A, 1997 Jan 21, 94(2), 391 - 6 Discrimination of a single base change in a ribozyme using the gene for dihydrofolate reductase as a selective marker in Escherichia coli; Fujita S et al.; For use of ribozymes in vivo, it is desirable to select functional ribozymes in the cellular environment (in the presence of inhibitory factors and limited concentrations of mandatory Mg2+ ions, etc.) . As a first step toward this goal, we developed a new screening system for detection in vivo of an active ribozyme from pools of active and inactive ribozymes using the gene for dihydrofolate reductase (DHFR) as a selective marker . In our DHFR expression vector, the sequence encoding either the active or the inactive ribozyme was connected to the DHFR gene . The plasmid was designed such that, when the ribozyme was active, the rate of production of DHFR was high enough to endow resistance to trimethoprim (TMP) . We demonstrated that the active ribozyme did indeed cleave the primary transcript in vivo, whereas the inactive ribozyme had no cleavage activity . Cells that harbored the active-ribozyme-coding plasmid grew faster in the presence of a fixed concentration of TMP than the corresponding cells that harbored the inactive-ribozyme-coding plasmid . Consequently, when cells were transformed by a mixture that consisted of active- and inactive-ribozyme-coding plasmids at a ratio of 1:1, (i) mainly those cells that harbored active ribozymes survived in the presence of TMP and (ii) both active- and inactive-ribozyme-harboring cells grew at an identical rate in the absence of TMP, a demonstration of a positive selection system in vivo . If the background "noise" can be removed completely in the future, the selection system might usefully complement existing selection systems in vitro. Biochemistry, 1997 Jan 21, 36(3), 615 - 25 Use of 1H-15N heteronuclear multiple-quantum coherence NMR spectroscopy to study the active site of aspartate aminotransferase; Mollova ET et al.; Aspartate aminotransferase from Escherichia coli, an 88 kDa enzyme, was uniformly and selectively enriched with 15N and was studied by heteronuclear multiple-quantum coherence NMR spectroscopy in H2O . Good resolution was obtained for the downfield region (above 9.5 ppm chemical shift in the 1H dimension) for NH protons in the amide, indole, imidazole, and guanidinium group regions and several resonances were tentatively assigned . Two downfield resonances, at 12.6 and 11.36 ppm, appear to belong to oxygen- or sulfur-bound protons . The most downfield amide resonance at 11.78 ppm was assigned to the active site cysteine 192 whose peptide proton is 2.9 A away from the negatively charged carboxyl group of aspartate 199 . Large downfield shifts (up to 1.15 ppm) of the indole NH resonance of the active site tryptophan 140 were observed upon binding of dicarboxylic inhibitors to the pyridoxal 5'-phosphate (PLP) form and of inorganic dianions to the pyridoxamine 5'-phosphate (PMP) form of the enzyme . We discuss these striking differences in the light of the available crystallographic data . Active sites of proteins, as well as specific inhibitory molecules, often contain negatively charged groups . These may be able to form hydrogen-bonds to NH groups and to shift the NH resonances downfield into a less crowded and therefore more readily observable region for many large proteins . Our approach, which makes use of both HMQC spectroscopy and NOE observations, should be widely applicable. Biochemistry, 1997 Jan 21, 36(3), 604 - 14 Escherichia coli dimethylallyl diphosphate:tRNA dimethylallyltransferase: a binding mechanism for recombinant enzyme; Moore JA et al.; Escherichia coli dimethylallyl diphosphate:tRNA dimethylallyltransferase (DMAPP-tRNA transferase) catalyzes the first step in the biosynthesis of the hypermodified A37 residue in tRNAs that read codons beginning with uridine . The enzyme, encoded by the miaA gene, was overproduced and purified to apparent homogeneity in three steps by ion-exchange (DE52 and Mono-Q) and size exclusion chromatography . Affinity-tagged DMAPP-tRNA transferase containing a C-terminal tripeptide alpha-tubulin epitope also was overproduced and purified to apparent homogeneity in two steps by ion-exchange and immunoaffinity chromatography . Addition of the C-terminal tripeptide alpha-tubulin epitope to DMAPP-tRNA transferase did not affect the activity of the enzyme . Undermodified tRNA(Phe) used as substrate in the DMAPP-tRNA transferase-catalyzed reaction was isolated and purified from an overexpressing clone in a miaA deficient strain of E . coli . Active recombinant E . coli DMAPP-tRNA transferase is monomeric . The enzyme transferred the dimethylallyl moiety of DMAPP to A37, located adjacent to the anticodon in undermodified tRNA(Phe) . The enzyme required Mg2+ for activity and exhibited a broad pH optimum . Michaelis constants for tRNA(Phe) and DMAPP are 96 +/- 11 nM and 3.2 +/- 0.5 microM, respectively, and Vmax = 0.83 +/- 0.02 micromol min-1 mg-1 . DMAPP-tRNA transferase bound tRNA(Phe) with a dissociation constant of 5.2 +/- 1.2 nM . In contrast, DMAPP did not bind to the enzyme in the absence of tRNA . However, DMAPP was bound with a dissociation constant of 3.4 +/- 0.6 microM in the presence of a minihelix analogue of the anticodon stem-loop of tRNA(Phe) where the base corresponding to A37 was replaced by inosine . These results suggest an ordered sequential mechanism for substrate binding. Biochemistry, 1997 Jan 21, 36(3), 461 - 8 Escherichia coli RNA polymerase activity observed using atomic force microscopy; Kasas S et al.; Fluid tapping-mode atomic force microscopy (AFM) was used to observe Escherichia coli RNA polymerase (RNAP) transcribing two different linear double-stranded (ds) DNA templates . The transcription process was detected by observing the translocation of the DNA template by RNAP on addition of ribonucleoside 5'-triphosphates (NTPs) in sequential AFM images . Stalled ternary complexes of RNAP, dsDNA and nascent RNA were adsorbed onto a mica surface and imaged under continuously flowing buffer . On introduction of all four NTPs, we observed some DNA molecules being pulled through the RNAP, some dissociating from the RNAP and others which did not move relative to the RNAP . The transcription rates were observed to be approximately 0.5-2 bases/s at our NTP concentrations, approximately 5 microM . The RNA transcripts were not unambiguously imaged in fluid . However, in experiments using a small single-stranded (ss) circular DNA template, known as a rolling circle, transcripts up to 1 or 2 microns long could be observed with tapping mode AFM once the samples were dried and imaged in air . This confirmed our observations of the transcriptional activity of RNAP adsorbed onto mica . This work illustrates that the development of tapping-mode in fluid has made it possible to use AFM to follow biological processes at the molecular level and get new insights about the variability of activity of individual molecules bound to a surface. Circulation, 1997 Jan 21, 95(2), 455 - 62 Recombinant staphylokin