Biochemistry, 1993 Aug 10, 32(31), 7893 - 903 Preparation and characterization of a bifunctionally spin-labeled mutant of murine epidermal growth factor for saturation-transfer electron paramagnetic resonance studies of the growth factor/receptor complex; Rousseau DL Jr et al.; In this report we describe the production of a {Lys3,Tyr22} murine epidermal growth factor (mEGF) mutant for spin-labeling with bis(sulfo-N-succinimidyl)-{15N,2H16}doxyl-2-spiro-4'-pimelate ({15N,2H16}BSSDP) in order to study the rotational dynamics of the EGF/EGF receptor complex by saturation-transfer electron paramagnetic resonance (ST-EPR) . Previous results {Faulkner-O'Brien et al . (1991) Biochemistry 30,8976-8985} indicated that the reaction of {15N,2H16}BSSDP with wild-type mEGF did not yield a product useful for ST-EPR studies of the EGF/EGF receptor complex because the major product, in which {15N,2H16}BSSDP was attached only at the amino terminus of mEGF, lacked rigid motional coupling of the spin probe to the protein, and the more tightly coupled bidentate product was unstable . Using oligonucleotide-mediated site-directed mutagenesis of a synthetic gene for mEGF, we replaced Tyr3 with Lys and His22 with Tyr in wild-type mEGF to produce a mutant mEGF suitable for {15N,2H16}BSSDP labeling . The {Lys3,Tyr22} mEGF was expressed in Escherichia coli HB101 transformed with a pIN-III-ompA3-{Lys3,Tyr22} mEGF plasmid and was purified from the bacterial periplasm using a simple two step purification method . The {15N,2H16}BSSDP reacted with {Lys3,Tyr22}mEGF in high yield, and EPR analysis of the major product revealed tight motional coupling between the spin probe and the protein . Biological activity, as assessed by stimulation of EGF receptor autophosphorylation and dimerization, was not affected by either the mutations or the addition of the spin label . The {15N,2H16}BSSDP-modified {Lys3,Tyr22} mEGF was shown to be equipotent with mEGF in EGF receptor competition binding assays using A431 cells; in EPR studies, mEGF also was shown to specifically block {15N,2H16}BSSDP-modified {Lys3,Tyr22}mEGF binding to the EGF receptor in A431 membrane vesicles . Using the {15N,2H16}BSSDP-modified {Lys3,Tyr22}mEGF, we now report the first measurement of the rotational dynamics of the EGF/EGF receptor complex in A431 membrane vesicles by ST-EPR. Biochemistry, 1993 Aug 10, 32(31), 7939 - 45 In vitro protein engineering using synthetic tRNA(Ala) with different anticodons; Ma C et al.; The use of synthetic tRNA for in vitro protein engineering was tested in a coupled transcription/translation system prepared from Escherichia coli . DNA sequences similar to the natural tRNA(Ala/UGC) gene from E . coli but with different anticodons were synthesized in vitro, cloned into a DNA plasmid, and then transcribed in vitro with T7 RNA polymerase . The UGC alanine anticodon was changed to CUA corresponding to the UAG stop codon, CCU corresponding to the rarely used AGG arginine codon, and two four-nucleotide anticodons used to suppress stop codons . Bacterial dihydrofolate reductase was the test protein . Its cloned coding sequence was mutagenized at the GUG codon for valine-75 to correspond to the anticodons of the tRNA constructs, and then the plasmids were used to direct the synthesis of dihydrofolate reductase in the coupled transcription/translation system containing the corresponding synthetic tRNA . The results indicate that all four synthetic tRNAs were functionally active in the synthesis of full-length, enzymatically active dihydrofolate reductase protein. Biochemistry, 1993 Aug 10, 32(31), 7866 - 71 Mutational effects on the cooperativity of Ca2+ binding in calmodulin; Waltersson Y et al.; The importance of the aspartate ligand in the +Y Ca2+ coordinating position of two EF-hands of calmodulin has been investigated . Synthetic calmodulin genes were used to produce engineered proteins with the wild-type bovine sequence as well as with aspartate 58 in Ca(2+)-binding site II and/or aspartate 95 in site III changed to asparagine . The macroscopic Ca(2+)-binding constants of the intact calmodulins and of tryptic fragments comprising the N- and C-terminal domains were determined from titrations with Ca2+ in the presence of 5,5'-Br2BAPTA . Substitution of aspartate by asparagine in Ca(2+)-binding site II led to a slight increase in the total free energy change on Ca2+ binding, and the cooperativity of Ca2+ binding to the N-terminal sites was substantially increased . The change from aspartate to asparagine in site III decreased the Ca2+ affinity and also appeared to decrease the positive cooperativity between the sites in the C-terminal domain . Thus, identical mutations in sites II and III were found to result in opposite effects . The data imply that involvement of liganding side chains in interactions other than direct calcium attraction and calcium coordination is of considerable importance for the Ca(2+)-binding process, particularly for the cooperativity. Biochemistry, 1993 Aug 10, 32(31), 7999 - 8003 A model for oxidative modification of glutamine synthetase, based on crystal structures of mutant H269N and the oxidized enzyme; Liaw SH et al.; Proteolytic degradation of glutamine synthetase (GS) in Escherichia coli is known to follow "marking" by oxidative modification . At an early stage of the degradative pathway, oxidation of His 269 and Arg 344 abolishes GS enzymatic activity . We propose a mechanism for the early stage of oxidative inactivation of GS on the basis of the crystal structure of H269N and tryptophan fluorescence spectra of H269N and H269NR344Q: (1) Oxidation of Arg 344, adjacent to the n2 metal ion site, decreases ATP binding . (2) Oxidation of His 269 to Asn destroys the n2 site, consistent with the function of His 269 as a ligand for the n2 metal . (3) Loss of Mn2+ at the n2 site destroys the integrity of the ATP binding site . (4) Destruction of the ATP site results in the observed low enzymatic activity of H269N and H269NR344Q . During later stages of oxidative modification, the n1 metal ion site is destroyed and the active site of the enzyme becomes flexible as suggested by X-ray data collected from an oxidized crystal of GS . Thus, studies of mutant and oxidized enzymes confirm that there are at least two stages of oxidative modification of GS . These studies suggest that the early modification occurs at the n2 metal ion site, eliminating enzyme activity, and the later modification occurs at the n1 metal ion site, relaxing the GS structure, perhaps enabling proteolytic degradation . These studies also illuminate the differing roles of the two bound metal ions: the tightly bound n1 ion enhances the stability of the catalytically active conformation, and the less tightly bound n2 ion participates in ATP binding. FEBS Lett, 1993 Aug 9, 328(1-2), 35 - 40 Expression of heterologous phosphofructokinase genes in yeast; Heinisch JJ; Genes encoding phosphofructokinases (PFK) from Escherichia coli and from the human muscle were expressed in PFK-deficient strains of Saccharomyces cerevisiae under the control of an inducible GAL1 promoter . They restored PFK activity under inducing conditions and complemented the galactose-negative growth phenotype of the recipient strains . The PFK enzymes expressed appear to be stable in yeast . The human muscle enzyme crossreacts with specific antibodies and shows the expected subunit size . As expected, its activity can be activated by fructose-2,6- bisphosphate, in contrast to the bacterial enzyme. FEBS Lett, 1993 Aug 9, 328(1-2), 130 - 8 Cloning and functional expression in Escherichia coli of a cyanobacterial gene for lycopene cyclase, the enzyme that catalyzes the biosynthesis of beta-carotene; Cunningham FX Jr et al.; Carotenoids with cyclic end groups are essential components of the photosynthetic membrane in all known oxygenic photosynthetic organisms . These yellow pigments serve the vital role of protecting against potentially lethal photo-oxidative damage . Many of the enzymes and genes of the carotenoid biosynthetic pathway in cyanobacteria, algae and plants remain to be isolated or identified . We have cloned a cyanobacterial gene encoding lycopene cyclase, an enzyme that converts the acyclic carotenoid lycopene to the bicyclic molecule beta-carotene . The gene was identified through the use of an experimental herbicide, 2-(4-methylphenoxy)triethylamine hydrochloride (MPTA), that prevents the cyclization of lycopene in plants and cyanobacteria . Chemically-induced mutants of the cyanobacterium Synechococcus sp . PCC7942 were selected for resistance to MPTA, and a mutation responsible for this resistance was mapped to a genomic DNA region of 200 bp by genetic complementation of the resistance in wild-type cells . A 1.5 kb genomic DNA fragment containing this MPTA-resistance mutation was expressed in a lycopene-accumulating strain of Escherichia coli . The conversion of lycopene to beta-carotene in these cells demonstrated that this fragment encodes the enzyme lycopene cyclase . The results indicate that a single gene product, designated lcy, catalyzes both of the cyclization reactions that are required to produce beta-carotene from lycopene, and prove that this enzyme is a target site of the herbicide MPTA . The cloned cyanobacterial lcy gene hybridized well with genomic DNA from eukaryotic algae, thus it will enable the identification and cloning of homologous genes for lycopene cyclase in algae and plants. FEBS Lett, 1993 Aug 9, 328(1-2), 111 - 4 Determination of a functional lysine residue of a plant cysteine synthase by site-directed mutagenesis, and the molecular evolutionary implications; Saito K et al.; Comparison of seven deduced amino acid sequences of cysteine synthase (O-acetyl-L-serine (thiol)-lyase, EC 4.2.99.8) from plants and bacteria disclosed the presence of 12 conserved Lys residues, which can be candidates for a functional binding site for pyridoxal phosphate cofactor . These 12 conserved Lys residues in a cDNA clone encoding spinach cysteine synthase A were replaced with Gly by oligonucleotide-directed in vitro mutagenesis . These Lys-->Gly mutated cDNAs were transferred into Escherichia coli NK3, a cysteine auxotroph lacking both cysteine synthase loci, cysK and cysM . One mutant replaced at Lys-49 could not complement the cysteine requirement of NK3, whereas other mutants and wild-type clone could . No enzymatic activity of cysteine synthase A was detected either in the cell-free extracts of E . coli NK3 transformed with the Lys-49 mutant . These results indicated that Lys-49 is a functional residue for the catalytic activity of cysteine synthase . This Lys residue is conserved in other evolutionarily related amino acid-metabolizing enzymes catalyzing reactions involving the beta-carbon of amino acids. Biochim Biophys Acta, 1993 Aug 7, 1164(3), 299 - 304 Identity and some properties of the L-threonine aldolase activity manifested by pure 2-amino-3-ketobutyrate ligase of Escherichia coli; Marcus JP et al.; 2-Amino-3-ketobutyrate ligase catalyzes the reversible, pyridoxal 5'-phosphate-dependent condensation of glycine with acetyl CoA forming the unstable intermediate, 2-amino-3-ketobutyrate . Several independent lines of evidence indicate that the pure protein obtained in the purification of this ligase from Escherichia coli also has L-threonine aldolase activity . The evidence includes: (a), a constant ratio of specific activities (aldolase/ligase) at all stages of purifying 2-amino-3-ketobutyrate ligase to homogeneity; (b), the same rate of loss of aldolase and ligase activities during controlled heat inactivation of the pure protein at 60 degrees C in the absence, as well as in the presence of acetyl CoA, a protective substrate; (c), ratios of the two enzymatic activities that are not significantly different during slow inactivation by iodoacetamide, with and without L-threonine added; (d), coincident rates of loss and essentially identical rates of recovery of aldolase activity and ligase activity during resolution of the holoenzyme with hydroxylamine followed by reconstitution with pyridoxal 5'-phosphate . No aldolase activity is observed with D-threonine as substrate and L-allothreonine is about 25% as effective as L-threonine . Whereas ligase activity has a sharp pH optimum at 7.5, the aldolase activity of this pure protein is maximal at pH 9.0 . Comparative apparent Km values for glycine (ligase) and L-threonine (aldolase) are 10 mM and 0.9 mM, respectively, whereas corresponding respective Vmax values were found to be 2.5 mumol of CoA released/min per mg vs . 0.014 mumol of acetaldehyde formed (NADH oxidized)/min per mg. J Med Chem, 1993 Aug 6, 36(16), 2279 - 91 Benzoquinazoline inhibitors of thymidylate synthase: enzyme inhibitory activity and cytotoxicity of some 3-amino- and 3-methylbenzo{f}quinazolin-1(2H)-ones; Pendergast W et al.; The synthesis and thymidylate synthase (TS) inhibitory activity of a series of simple benzo{f}-quinazolin-1(2H)-ones are described . Fully aromatic 3-amino compounds with compact lipophilic substituents in the 9-position were found to have I50 values as low as 20 nM on the isolated enzyme, and represent the first examples of potent, folate-based TS inhibitors that completely lack any structural feature corresponding to the (p-aminobenzoyl)glutamate moiety of the cofactor . A number of the compounds also showed moderate growth inhibitory activity against a human colon adenocarcinoma cell line (SW480), with IC50 values as low as 2 microM. Science, 1993 Aug 6, 261(5122), 762 - 5 Circularly permuted tRNAs as specific photoaffinity probes of ribonuclease P RNA structure; Nolan JM et al.; Regions of Escherichia coli ribonuclease P (RNase P) RNA in proximity to a bound transfer RNA (tRNA) substrate were mapped by photoaffinity . A photoaffinity cross-linking reagent was introduced at specific sites in the interior of the native tRNA structure by modification of the 5' ends of circularly permuted tRNAs (cptRNAs) . The polymerase chain reaction was used for the production of cptRNA templates . After the amplification of a segment of a tandemly duplicated tRNA gene, the cptRNA gene was transcribed in vitro to produce cptRNA . Modified cptRNAs were cross-linked to RNase P RNA, and the conjugation sites in RNase P RNA were determined by primer extension . These sites occur in phylogenetically conserved structures and sequences and identify regions of the ribozyme that form part of the tRNA binding site . The use of circularly permuted molecules to position specific modifications is applicable to the study of many inter- and intramolecular interactions. Nature, 1993 Aug 5, 364(6437), 548 - 9 CAP interacts with RNA polymerase in solution in the absence of promoter DNA; Heyduk T et al.; Protein-protein interactions between transcription activator proteins and RNA polymerase or basal transcription factors have been suggested to be important for transcription activation . Interactions between catabolite gene activator protein (CAP) and RNA polymerase have been proposed based on face-of-helix-dependent transcription activation by CAP and based on face-of-helix-dependent cooperative binding of CAP and RNA polymerase to promoter DNA . Mutants of CAP specifically defective in transcription activation have been isolated (mutants defective in transcription activation, but not defective in DNA binding and DNA bending) . All such mutants contain amino-acid substitutions within a surface loop consisting of amino acids 152 to 166 of CAP . Here we use the thermodynamically rigorous technique of fluorescence polarization to show that CAP interacts with RNA polymerase in solution in the absence of promoter DNA (KD,app = 2.8 x 10(-7) M), whereas {Ala158}CAP, a mutant of CAP specifically defective in transcription activation, does not. J Biol Chem, 1993 Aug 5, 268(22), 16639 - 47 Effects of substitutions of closely related amino acids at the contact surface in an antigen-antibody complex on thermodynamic parameters; Ito W et al.; We constructed a library of 512 kinds of Fv fragment, derivatives of a monoclonal antibody, D1.3, specific for hen egg-white lysozyme, in which a total of nine of the original amino acids were replaced by closely related amino acids at positions in the complementarity-determining regions of the H chain . More than 80% of the clones in the library produced Fv fragments in Escherichia coli . Two wild-type and 13 mutant Fv fragments were prepared in large quantities and subjected to analysis by differential titration calorimetry . The association constants of the 15 Fv fragments with hen egg-white lysozyme were distributed between 0.12 x 10(7) and 1.59 x 10(8) M-1 . The changes in delta H0 and -T delta S0 caused by one-point mutation at each position did not have intrinsic values for each change . The same changes at one position had different effects on KA, delta H0, and -T delta S0 when differences had been introduced in other regions . The delta(delta G0) caused by a single-point mutation ranged from -0.56 to 1.56 kcal/mol . By contrast, the delta(delta H0) and delta(-T delta S0) caused by a single-point mutation ranged from -3.5 to 3.4 and from -3.8 to 3.4 kcal/mol, respectively . When antibodies gain the binding energy contributed by the effects of enthalpy, they lose the binding energy contributed by the effects of entropy and vice versa . In general, changes in entropy compensate for changes in enthalpy. J Biol Chem, 1993 Aug 5, 268(22), 16519 - 27 HIV nucleocapsid protein . Expression in Escherichia coli, purification, and characterization; You JC et al.; The single-stranded nucleocapsid protein that coats the RNA genome of human immunodeficiency virus within the virion core has been produced in Escherichia coli and purified to homogeneity . The mature 55-amino acid protein, normally generated from the gag polyprotein precursor by HIV protease-catalyzed processing of both its amino and carboxyl termini, was produced in E . coli with authentic termini directly, without the need for processing . The protein was purified 30-fold to apparent homogeneity, as determined by both amino acid analysis and SDS-polyacrylamide gel electrophoresis . Sequencing of each terminus of the purified protein indicated that no proteolytic degradation occurred . A molar extinction coefficient (epsilon 280 = 8350 cm-1 M-1) was determined . The purified nucleocapsid protein binds tightly to single-stranded RNA as judged by a nitrocellulose filter binding assay . A binding constant (Kw) of 1 x 10(8) M-1 was calculated . Using fluorescence quenching of nucleocapsid protein upon RNA binding as an assay, a binding site size of seven nucleotides was determined . These results contrast to a larger 15-nucleotide site measured by others for a larger form of nucleocapsid protein-containing sequences from its immature precursor . The possible relevance of these findings are discussed. J Biol Chem, 1993 Aug 5, 268(22), 16302 - 8 Energy transduction between membranes . TonB, a cytoplasmic membrane protein, can be chemically cross-linked in vivo to the outer membrane receptor FepA; Skare JT et al.; TonB, a cytoplasmic membrane protein, couples cytoplasmic membrane protonmotive force to active transport across the outer membrane of Escherichia coli . In vivo cross-linking studies were initiated to analyze TonB interactions with other cell envelope proteins . Four TonB-specific cross-linked complexes were detected with apparent molecular masses of 195, 77, 59, and 43.5 kDa . The 195-kDa complex was shown to contain both TonB and FepA, the outer membrane receptor for the siderophore enterochelin . The 195-kDa complex is absent in strains missing either TonB or FepA and can be detected by either TonB-specific or FepA-specific monoclonal antibodies . This is the first direct in vivo evidence that TonB can span the periplasmic space to interact physically with outer membrane receptors . Consistent with that observation, the outer membrane protease OmpT was shown to play a role in TonB turnover, both in the presence and absence of ExbB results in the rapid degradation of TonB . The absence of OmpT could be used to stabilize TonB in an exbB::Tn10 strain such that steady state levels of TonB protein are identical to a wild-type strain . Under those conditions, the absence of ExbB results in greatly reduced TonB activity, indicating that ExbB plays a direct role in energy transduction and probably secondarily protects TonB protein from proteolysis . The 59-kDa complex was absent in an exbB::Tn10 strain, suggesting either that ExbB is in the complex with TonB or that ExbB is required to form the 59-kDa complex . A tolQ nonsense mutation had no effect on the cross-linking profile observed, confirming that its participation in TonB-dependent phenomena is minor and most likely the result of evolutionary cross-talk. J Biol Chem, 1993 Aug 5, 268(22), 16259 - 64 Identification of the metal ligands and characterization of a putative zinc finger in methionyl-tRNA synthetase; Xu B et al.; A truncated form of the methionyl-tRNA synthetase (delta MTS), which has been cloned, overproduced, and characterized, was used in an attempt to better understand the role of the enzyme-bound zinc in the amino-acylation process . Apo-, Zn(2+)-, Co(2+)-, and 113Cd(2+)-substituted delta MTS proteins were prepared in vivo and purified to homogeneity . Apo-delta MTS was devoid of enzymatic activity in the aminoacylation of tRNA(fMet) and in the methionine-dependent ATP-pyrophosphate exchange reactions . Kinetic constants in both the aminoacylation and ATP-pyrophosphate exchange reactions for the Co(2+)- and 113Cd(2+)-substituted delta MTS proteins were found to be identical with those of the native Zn2+ protein . The low energy absorption spectrum of Co(2+)-substituted delta MTS resembles the d-d transition bands characteristic of tetrahedrally coordinated Co(2+)-substituted proteins . A strong S-->Co2+ charge transfer absorption at 350 nm was clearly evident having a molar absorptivity consistent with four thiolate ligands . The environment of the metal center was further probed by measuring the 113Cd chemical shift of 113Cd(2+)-substituted delta MTS . A single resonance at 759.6 ppm was observed . This chemical shift is consistent with Cd2+ coordinated to four thiolate ligands . The Escherichia coli methionyl-tRNA synthetase contains a potential metal binding sequence Cys-X2-Cys-X9-Cys-X2-Cys in a connecting polypeptide within the nucleotide fold . Titration of a 21-amino acid peptide corresponding to this putative metal binding site, Cys145-Cys161, was shown to bind Co2+ with a Kd of 120 +/- 11 microM . These results demonstrate that the isolated zinc finger binding domain is capable of specifically forming a stoichiometric complex with the divalent cation . Taken together, our studies identify the 4 cysteine residues in the zinc finger-like domain as the metal binding ligands in the E . coli methionyl-tRNA synthetase . The role of the enzyme-bound metal appears to be structural and not directly involved in catalysis. J Biol Chem, 1993 Aug 5, 268(22), 16241 - 7 Leucine/isoleucine/valine-binding protein contracts upon binding of ligand; Olah GA et al.; Small-angle x-ray scattering and computer modeling have been used to study the effects of ligand binding to the leucine/isoleucine/valine-binding protein, an initial component of the high-affinity active transport system for branched-chain aliphatic amino acids in Escherichia coli . Measurements were made with no ligand present and with either L-leucine or L-valine present . Upon binding of either leucine or valine, there is a decrease in the radius of gyration, from 23.2 +/- 0.2 to 22.2 +/- 0.2 A, and in the maximum particle dimension, from 82 +/- 3 to 73 +/- 3 A . The x-ray structure of the unbound form has been determined and gives a radius of gyration and a maximum dimension consistent with the values found for the solution structure in this study (Sack, J . S., Saper, M . A., and Quiocho, F . A . (1989) J . Mol . Biol . 206, 171-191) . The reduction in the radius of gyration and maximum dimension upon ligand binding can be accounted for by a substrate-induced cleft closure in a combined "hinge-twist" motion . Modeling of the substrate-bound state was done by comparison of this protein with another periplasmic binding protein (L-arabinose-binding protein), which possesses a similar two-lobe structure and for which the x-ray structure is known in its ligand-bound form. J Biol Chem, 1993 Aug 5, 268(22), 16216 - 22 A Kazal-type inhibitor with thrombin specificity from Rhodnius prolixus; Friedrich T et al.; A thrombin-specific inhibitor with an apparent molecular mass of 11 kDa has been purified from the insect Rhodnius prolixus . Amino-terminal protein sequence analysis allowed the molecular cloning of the corresponding cDNA . The open reading frame codes for a protein of about 103 amino acid residues and displays an internal sequence homology of residues 6-48 with residues 57-101 indicating a two-domain structure . Based on the amino acid sequence the two domains exhibit high homology to protease inhibitors belonging to the Kazal-type family . Model building suggests that the first domain binds to the active site with residue His10 pointing into the specificity pocket . From gel filtration and tight-binding inhibition experiments the inhibitor appears to form 1:1 complexes with thrombin . Periplasma-directed heterologous expression of the rhodniin cDNA in Escherichia coli yields the intact thrombin inhibitor . Natural and recombinant rhodniin both display inhibition constants of about 2 x 10(-13) M. J Biol Chem, 1993 Aug 5, 268(22), 16105 - 8 The DNA-binding subunit of human transcription factor IID can interact with the TATA box as a multimer; Jupp R et al.; Transcription initiation from eukaryotic protein-coding genes is a complex process that minimally requires RNA polymerase (pol) II (B) and at least seven general transcription factors . The 38-kDa subunit (TBP) of the human general transcription factor TFIID recognizes the TATA sequence element and initiates the assembly of the other general transcription factors and RNA pol II . It is believed, based on experiments with yeast recombinant protein, that TBP binds as a monomer to DNA . Using purified recombinant human TBP protein we find that TBP interacts with the TATA element as both a monomer and a dimer . The multimeric binding of TBP to DNA revealed by this study has important implications for the role of TBP in transcription initiation and suggests novel mechanisms whereby other transcription factors may interact with a RNA pol II preinitiation complex. J Biol Chem, 1993 Aug 5, 268(22), 16528 - 36 Human immunodeficiency virus reverse transcriptase . Expression in Escherichia coli, purification, and characterization of a functionally and structurally asymmetric dimeric polymerase; Thimmig RL et al.; Human immunodeficiency virus (HIV) reverse transcriptase isolated from viral particles contains two subunits, p51 and p66 . We have produced both subunits in separate Escherichia coli strains using expression vectors . Stop codons were placed immediately after the codon for the carboxyl-terminal residue of the mature processed p51 and p66 subunits found in viral particles . Insertion of a methionine in front of the HIV protease cleavage site in the recombinant protein enabled synthesis of both subunits with the natural amino-terminal proline, since E . coli methionine aminopeptidase cleaves a Met-Pro amino-terminal linkage . That this occurred to an extent greater than 95% was confirmed by sequencing the purified subunits . Examination of the activities of the individual p51 and p66 subunits on a variety of templates and under solution conditions optimized for each subunit revealed a significant catalytic activity for the natural p51 subunit . This result contrasts to results reported earlier for many recombinant forms without the natural amino and/or carboxyl termini . As expected from earlier work, the optimal homopolymeric template for the p66 subunit was poly(rA) . For the p51 subunit, poly(dC) was found to be the optimal template; its activity is 2- to 4-fold greater than p66 on poly(dC) . The p51 subunit is 13- to 50-fold less active on poly(rC) . These findings are discussed in the context of our earlier hypothesis (McHenry, C . S . (1989) in Molecular Biology of Chromosome Function (Adolph, K., ed) Chap . 5, Springer-Verlag, New York) that the HIV reverse transcriptase might be functionally asymmetric with distinct plus- and minus-strand polymerases. Biochemistry, 1993 Aug 3, 32(30), 7765 - 71 Kinetics of ATP hydrolysis during the DNA helicase II-promoted unwinding of duplex DNA; Yodh JG et al.; The ATP hydrolysis activity of DNA helicase II from Escherichia coli was examined in the presence of linear single-stranded DNA (ssDNA) and linear double-stranded DNA (dsDNA) . In the presence of ssDNA, the ATP hydrolysis reaction followed a linear time course until the ATP was depleted . In the presence of dsDNA, in contrast, there was a kinetic lag before a linear phase of ATP hydrolysis was achieved . The nonlinear kinetics of the dsDNA-dependent ATP hydrolysis reaction could be modeled by a kinetic scheme in which helicase II undergoes a time-dependent transition from an ATPase-inactive to an ATPase-active form . Order of addition experiments indicated that this transition was not due to a rate-limiting association event between helicase II and any other component of the reaction . Instead, agarose gel assays showed that progressive unwinding of the dsDNA occurs during the same time period as the lag phase of the ATP hydrolysis reaction . No significant ATP hydrolysis was observed when the linear dsDNA was replaced with closed circular dsDNA, suggesting that the ATP hydrolysis reaction requires a dsDNA substrate that can be unwound to the complementary single strands . These results are consistent with a model in which the lag phase of the dsDNA-dependent ATP hydrolysis reaction corresponds to progressive unwinding of the dsDNA, with the ATP hydrolysis reaction arising from helicase II molecules that are bound to the separated single strands. Biochemistry, 1993 Aug 3, 32(30), 7692 - 7 Genetic fusion of subunits I, II, and III of the cytochrome bo ubiquinol oxidase from Escherichia coli results in a fully assembled and active enzyme; Ma J et al.; The cytochrome bo ubiquinol oxidase from Escherichia coli is a five-subunit enzyme which is a member of the superfamily of heme-copper respiratory oxidases . Three of the subunits (I, II, and III) are homologous to the three mitochondrial encoded subunits of the eukaryotic aa3-type cytochrome c oxidase . Subunits, I, II, and III of the eukaryotic oxidase contain 12, 2, and 7 putative transmembrane spans, respectively . The hydropathy profiles of the subunits of most other members of this oxidase superfamily are consistent with these structures . However, subunit I from the E . coli oxidase contains 15 transmembrane spans, with one additional span at the N-terminus and two additional spans at the C-terminus in comparison to the eukaryotic oxidase . The additional transmembrane helix at the N-terminus predicts that the amino terminal residue should be on the periplasmic side of the membrane . By deleting the intergenic region between the cyoA and cyoB genes, an in-frame fusion between subunit II (cyoA) and subunit I (cyoB) was generated . This links the C-terminus of subunit II, known to be on the periplasmic side of the membrane, to the N-terminus of subunit I . The resulting oxidase is fully active, and supports the toplogical folding pattern previously suggested for subunit I with the N-terminus in the periplasm . Whereas subunit I of the E . coli oxidase has two additional membrane-spanning helices at the C-terminus, subunit III has two fewer helices than does the corresponding subunit III of the eukaryotic oxidase.(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1993 Aug 3, 32(30), 7753 - 8 Fluorescence anisotropy assays implicate protein-protein interactions in regulating trp repressor DNA binding; LeTilly V et al.; The study of interactions between proteins and nucleic acids is central to the understanding of the control of genetic expression . Fluorescence anisotropy has been used to measure, in solution, the equilibrium binding profiles of a bacterial repressor protein, the tryptophan repressor (TR), to a fluorescently labeled oligonucleotide containing one of its target operator sequences . Investigation of the effects of changing concentrations of corepressor, operator DNA, and protein implicate TR oligomers in the regulation of DNA binding . These studies also demonstrate that the relatively straightforward technique of fluorescence anisotropy can be applied to the study of the interactions between proteins and nucleic acids . The fluorescence technique exhibits sufficient sensitivity to replace radioactive methods of detection in most cases . In addition, since it is a solution-based methodology, it offers a true equilibrium measure of the protein-nucleic acid equilibria, and the effects of changes in solution conditions such as salt and ligand concentration, pH, and temperature can be readily evaluated . Data acquisition is relatively simple and rapid, and the data are of sufficient quality for detailed thermodynamic analyses of complex systems . Given these attributes, fluorescence anisotropy will find multiple applications in the area of genetic regulation. Biochemistry, 1993 Aug 3, 32(30), 7720 - 6 Orientation of functional and nonfunctional PTS permease signal sequences in lipid bilayers . A polarized attenuated total reflection infrared study; Tamm LK et al.; Synthetic peptides corresponding to the N-terminal 23 and 22 residues, respectively, of two integral plasma membrane proteins of Escherichia coli, namely the mannitol- and glucitol-specific permeases of the bacterial sugar phosphotransferase system, were incorporated into single planar phospholipid bilayers supported on germanium plates . Polarized attenuated total reflection infrared spectra were recorded, and order parameters were derived from the measured dichroic ratios . The order parameters of the two wild-type peptides which form amphiphilic alpha-helices in membranes were -0.4 to -0.5, indicating a preferential alignment of the alpha-helix long axis parallel to the membrane surface . Nonfunctional mutant peptides of the mannitol permease sequence in which serine-3 or aspartate-4 were substituted with prolines (S3P and D4P) or lysine (D4K), but which were still largely alpha-helical, exhibited peptide order parameters close to zero, indicating a high degree of disorder of these peptides in the lipid bilayers . The lipid was well ordered at low concentrations of peptides in the membranes but became disordered at high peptide concentrations . This effect of lipid disordering was more pronounced for the D4K mutant than for the wild-type mannitol peptide. Biochemistry, 1993 Aug 3, 32(30), 7650 - 7 Effects of mutations in the hinge region of serpins; Hopkins PC et al.; An expression system for alpha 1-antitrypsin in Escherichia coli was developed using a T7 RNA polymerase promoter . Addition of rifampicin to inhibit the E . coli RNA polymerase after induction of the T7 RNA polymerase gene resulted in about 30% of newly synthesized protein being alpha 1-antitrypsin . This expression system was then used to examine the effect of mutations in the hinge region of alpha 1-antitrypsin on its activity . The mutations were based on ones in antithrombin III that had previously been shown to have adverse effects on activity . Mutation of Ala347 to threonine in alpha 1-antitrypsin did not affect the kinetic behavior of the protein with trypsin or human leukocyte elastase . In contrast, mutation of Gly349 to proline converted the majority of the protein into a substrate for both proteinases . The small fraction of this mutant that was active, however, had kinetic parameters that were indistinguishable from wild-type alpha 1-antitrypsin . Cleavage within the reactive-site loop of wild-type alpha 1-antitrypsin causes a conformational change in the molecules (the S-to-R transition) and results in a marked increase in heat stability . This increase in heat stability was also seen upon cleavage within the reactive-site loops of both of the alpha 1-antitrypsin mutants . The results are discussed in terms of a kinetic mechanism for serpin-proteinase interactions, in which after the formation of an initial complex the serpin partitions between the formation of a stable complex and a cleavage reaction.(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1993 Aug 3, 32(30), 7623 - 9 Expression of CheA fragments which define domains encoding kinase, phosphotransfer, and CheY binding activities; Swanson RV et al.; The histidine protein kinase CheA is a central component of the Escherichia coli chemotaxis system . The autophosphorylation activity of CheA is controlled by membrane-bound chemoreceptors and by the CheW coupling protein . CheA phosphorylates the CheY and CheB proteins which respectively control the direction of flagellar rotation and the level of receptor adaptation, thereby regulating the cells' chemotactic response . Genes encoding three polypeptide fragments of CheA were constructed and expressed in order to better define the functional organization of the wild-type protein . These fragments allowed the identification of regions of the protein responsible for CheY binding, phosphotransfer, and kinase activity . The kinase domain was expressed as a 30-kDa polypeptide corresponding to the central portion of the wild-type protein which contains sequences homologous to other histidine kinases . It was able to phosphorylate a 15-kDa amino-terminal phosphotransfer domain which was separately expressed and purified . This latter domain is capable of phosphotransfer to CheY despite the fact that it lacks the ability to stably bind CheY . CheY was immobilized to a dextran matrix through a single cysteine residue which was introduced into the protein at a position far removed from the active site . A stable binding site for CheY was mapped to a segment between the site of autophosphorylation and the kinase domain by using surface plasmon resonance to detect binding to the immobilized CheY . The region of the kinase which tightly binds the unphosphorylated substrate may play an important role in regulating the specificity of the signal transducing system. Biochemistry, 1993 Aug 3, 32(30), 7703 - 11 Partially structured self-associating states of acidic fibroblast growth factor; Mach H et al.; A combination of near- and far-UV circular dichroism, Fourier-transform infrared spectroscopy, tryptophan fluorescence, size-exclusion chromatography, and a fluorescent extrinsic hydrophobic probe has been employed to characterize partially structured states of human recombinant acidic fibroblast growth factor (aFGF) . At low pH, the addition of specific polyanionic ligands or moderate amounts of salts induces states with high secondary but low tertiary structure content . At neutral pH, intermediate amounts of chaotropic agents impose similar partially structured conformational states which also display noncooperative unfolding transitions . Kinetic evidence indicates that similar forms of the protein exist in the first few hundred milliseconds in the refolding pathway of aFGF . The kinetics of their formation appear to be temperature-independent, implying lack of an energy barrier, which is characteristic for further slow folding into the native state . Unlike the native and fully unfolded states, these partially structured conformations exhibit very low solubility, resulting in irreversible aggregation . Potential physiological implications of the existence of such "molten globule" states with regard to the growth factor's transport and biological activity are considered. Biochemistry, 1993 Aug 3, 32(30), 7617 - 22 In vitro analysis of translational rate and accuracy with an unmodified tRNA; Harrington KM et al.; Escherichia coli tRNA(Phe) transcript lacking all the modified nucleosides was investigated in an in vitro translation system . To estimate the affinity of tRNA toward EF-Tu, Kd and K-1 were measured by the nuclease protection assay, and it was shown that the absence of modifications decreases ternary complex stability less than 2-fold . The activity of unmodified Phe-tRNA(Phe) on E . coli ribosomes was compared to modified Phe-tRNA(Phe) using the framework of the kinetic proofreading mechanism (Thompson & Dix, 1982) with both cognate and noncognate codons . Values of the individual rate constants in the elongation process showed that the modifications increased the accuracy of translation by (1) decreasing the rate of dipeptide synthesis and (2) increasing the rate of rejection with noncognate codons. Eur J Pharmacol, 1993 Aug 2, 248(2), 157 - 62 Inhibition of 5-hydroxytryptamine- and enterotoxin-induced fluid secretion by 5-HT receptor antagonists in the rat jejunum; Beubler E et al.; The effects of cholera toxin and heat stable Escherichia coli (E . coli) enterotoxin on intestinal fluid secretion are commonly considered to be mediated by cyclic nucleotides . It was demonstrated recently, by using the 5-hydroxytryptamine (5-HT)2 receptor antagonist ketanserin and the 5-HT3 receptor antagonist tropisetron, that 5-HT acts as an important mediator in cholera toxin- and heat stable E . coli enterotoxin-induced fluid secretion . In the present investigation ketanserin and tropisetron were compared with the newer 5-HT3 receptor antagonists ondansetron and granisetron versus 5-HT-, cholera toxin- and heat stable E . coli enterotoxin-induced fluid secretion in the rat jejunum in vivo . Both ondansetron and granisetron dose-dependently inhibited 5-HT- and enterotoxin-induced fluid secretion . Ketanserin blocked 5-HT-induced fluid secretion, but only diminished enterotoxin-induced effects even at higher doses . Tropisetron inhibited 5-HT- and cholera toxin-induced effects at high dose but only diminished heat stable E . coli enterotoxin-induced effects . We conclude that 5-HT3 receptors, located on enterochromaffin cells and nervous structures, are more important in mediating fluid secretion than 5-HT2 receptors, located on the epithelial cells. Photochem Photobiol, 1993 Aug, 58(2), 219 - 25 In vivo repair of cytosine hydrates in DNA of cultured human lymphoblasts; Weiss RB et al.; Ultraviolet irradiation of DNA in vitro results in the production of a wide variety of pyrimidine base alterations, including cytosine hydrates . Enzymes that initiate the repair of monomeric pyrimidine damage have been identified in both bacterial and mammalian systems; however, the in vivo formation and repair of cytosine photohydrates has not been demonstrated in cellular DNA . Using Escherichia coli endonuclease III as a damage-specific probe, we have shown that ring-saturated pyrimidines are formed in cultured human cells by irradiation with broad-spectrum UV light . In addition, these types of base damage are removed from the DNA of human lymphoblasts within 5 h following the irradiation . Analysis of the action spectrum for the formation of cytosine hydrates in DNA reveals that these photoproducts are formed most efficiently by irradiation in the range of 255-265 nm light, coinciding with the wavelengths that are maximally absorbed by the DNA bases. Mol Microbiol, 1993 Aug, 9(3), 557 - 68 Escherichia coli endoribonuclease RNase E: autoregulation of expression and site-specific cleavage of mRNA; Mudd EA et al.; Mutations in the Escherichia coli rne (ams) gene have a general effect on the rate of mRNA decay in vivo . Using antibodies we have shown that the product of the rne gene is a polypeptide of relative mobility 180 kDa . However, proteolytic fragments as small as 70 kDa, which can arise during purification, also exhibit RNase E activity . In vitro studies demonstrate that the rne gene product, RNase E, is an endoribonuclease that cleaves mRNA at specific sites . RNase E cleaves rne mRNA and autoregulates the expression of the rne gene . In addition we demonstrate RNase E-dependent endonucleolytic cleavage of ompA mRNA, at a site known to be rate-determining for degradation and reported to be cleaved by RNase K . Our data are consistent with RNase K being a proteolytic fragment of RNase E. Mol Microbiol, 1993 Aug, 9(3), 443 - 57 The genes of the glutamine synthetase adenylylation cascade are not regulated by nitrogen in Escherichia coli; van Heeswijk WC et al.; Regulation of glutamine-synthetase (GS) activity in enteric bacteria involves a complex cascade of events . In response to nitrogen limitation, a transferase catalyses the uridylylation of the PII protein, which in turn stimulates deadenylylation of GS . Deadenylylated GS is the more active form of the enzyme . Here we characterize in detail the genes from Escherichia coli encoding uridylyl-transferase (glnD), the PII protein (glnB), and adenylyl-transferase (glnE) . glnD is transcribed from its own promoter, glnE is contranscribed with another gene, orfXE, whereas glnB is partly contranscribed with a gene encoding a homologue of the transcription activator NtrC . All three gln regulatory genes were constitutively expressed at a low level, i.e . their expression was independent of the nitrogen status and the RNA polymerase sigma factor sigma 54 . We conclude that the functioning of the GS adenylylation cascade is regulated by modulation of the activities of uridylyl-transferase and adenylyl-transferase, rather than by changes in the expression of their genes. Mol Microbiol, 1993 Aug, 9(3), 425 - 34 Nitrate regulation of anaerobic respiratory gene expression in Escherichia coli; Stewart V; Synthesis of most anaerobic respiratory pathways is subject to dual regulation by anaerobiosis and nitrate . Anaerobic induction is mediated by the FNR protein . Dual interacting two-component regulatory systems mediate nitrate induction and repression . The response regulator protein NARL binds DNA to control nitrate induction and repression of genes encoding nitrate respiration enzymes and alternate anaerobic respiratory enzymes, respectively . The homologous protein NARP controls nitrite induction of at least two operons . Nitrate and nitrite signalling are both mediated by the homologous sensor proteins NARX and NARQ . Recent mutational analyses have defined a heptamer sequence necessary for specific DNA binding by the NARL protein . These heptamers are located at different positions in the control regions of different operons . The NARL protein-binding sites in the narG (nitrate reductase) and narK (nitrate-nitrite antiporter) operon control regions are located approximately 200bp upstream of the transcription initiation site . The integration host factor (IHF) greatly stimulates nitrate induction of these operons, indicating that a specific DNA loop brings NARL protein, bound at the upstream region, into the proximity of the promoter for transcription activation . Other NARL protein-dependent opersons do not require IHF for nitrate induction, and the arrangement of NARL heptamer sequences in these control regions is quite different . This complexity of signal transduction pathways, coupled with the diversity of control region architecture, combine to provide many interesting areas for future investigation . An additional challenge is to determine how or if the FNR and NARL proteins interact to mediate dual positive control of transcription initiation. Mol Microbiol, 1993 Aug, 9(3), 419 - 24 Assembly of the Escherichia coli F1F0 ATPase, a large multimeric membrane-bound enzyme; Brusilow WS; The F1F0 proton translocating ATPase of Escherichia coli is a large membrane-bound enzyme complex consisting of more than 20 polypeptides that are encoded by the unc operon . Besides being a system for analysing the enzymology of ATP synthesis and energy coupling, the ATPase is a model system for determining how large oligomeric membrane-bound proteins are synthesized and assembled . The assembly of the ATPase involves differential gene expression and assembly of the subunits within the membrane and with each other . This review discusses the influence of F1 subunits on the assembly and proton permeability of the F0 proton channel, and the possible advantages to assembly of the particular arrangement of genes in the unc operon. J Gen Microbiol, 1993 Aug, 139 ( Pt 8), 1869 - 75 Molybdenum uptake in Escherichia coli K12; Lopez Corcuera G et al.; Molybdenum uptake was examined in Escherichia coli K12 using the radionuclide 99Mo . The molybdenum uptake system was characterized in an unusual chlD strain, which appeared to be normal in uptake of the MoO4(2-) ion but altered in subsequent molybdenum processing . As a consequence, molybdenum could be chased from cells in the chlD strain, while it was irreversibly assimilated in the wild-type strain . Molybdenum uptake showed a biphasic kinetic curve, with a very rapid binding followed by a slow uptake phase . The uptake appeared to involve an active transport system . Molybdenum, probably in the form of molybdate, accumulated by a factor of about 30 in the cells . An energy source was necessary and uptake was inhibited by arsenate, but not by CCCP (carbonyl cyanide m-chlorophenylhydrazone) . The uptake system saturated with a Km of 2.5-2.7 x 10(-8) M . Uptake seemed to depend on a periplasmic binding protein, since cold shock treatment and arsenate abolished uptake . A molybdate binding protein activity was detected in the periplasmic fluid with a KD of 9 nM . Sulphate inhibited uptake and the uptake activity was pH dependent, with an apparent pK of 6.7 . These results imply that molybdate transport belongs to the family of energy-dependent periplasmic binding protein systems . An explanation for the peculiar behaviour of the chlD strain used in this work is proposed. J Gen Microbiol, 1993 Aug, 139 ( Pt 8), 1829 - 40 Identification of the formate dehydrogenases and genetic determinants of formate-dependent nitrite reduction by Escherichia coli K12; Darwin A et al.; The formate dehydrogenases of Escherichia coli involved in electron transfer from formate to nitrite (Nrf activity: nitrite reduction by formate) have been identified . No previously undescribed selenoprotein was detected in bacteria grown under conditions optimal for the expression of Nrf activity . The Nrf activities of single mutants defective in either FdhN or FdhH were between 50 and 60% that of the parental strain . A double mutant defective in both FdhN and FdhH retained less than 10% of the activity of the FdhN+ FdhH+ strain . No Nrf activity was detected in a triple mutant defective in FdhN, FdhH and FdhO or in the selC strain . It is concluded that all three of the known formate dehydrogenases of E . coli can contribute to the transfer of electrons from formate to the Nrf pathway . Mutants defective in Nrf activity and cytochrome c552 synthesis were isolated by insertion mutagenesis or identified amongst strains received from the E . coli Genetic Stock Center . The mutations were located in at least three regions of the chromosome, including the 92 to 94 minute region which includes fdhF, the gene encoding FdhH required for formate hydrogenlyase activity . Fine structure mapping by P1 transduction established that the nrf mutations in the fdhF region were due to defects in three separable loci, all of which were independent of but close to fdhF . Clones were isolated from a cosmid library that complemented a deletion extending from fdhF into a region essential for Nrf activity . From these clones, plasmids were isolated that complemented only some of the Nrf- mutations in the 92 to 94 minute region, confirming the presence of different operons essential for Nrf activity and cytochrome c552 synthesis in this region . Suggested reasons for this genetic complexity include the need for proteins involved in electron transfer from the various formate dehydrogenases to cytochrome c552, for the attachment of the haem group to the apocytochrome and for cytochrome c552 export into the periplasm. J Gen Microbiol, 1993 Aug, 139 ( Pt 8), 1807 - 15 Purification and properties of cyanide hydratase from Fusarium lateritium and analysis of the corresponding chy1 gene; Cluness MJ et al.; The filamentous fungus Fusarium lateritium is cyanide tolerant, due, at least in part, to the induction by cyanide of the enzyme formamide hydrolyase (EC 4.2.1.66) . This enzyme, more commonly known as cyanide hydratase, catalyses the hydration of cyanide to formamide . The enzyme was purified from F . lateritium and showed a subunit molecular mass of 43 kDa (as judged by SDS-PAGE), while the native protein appeared to form aggregates of up to 1217 kDa (as judged by gel-filtration and non-denaturing PAGE) . mRNA samples from cultures grown with and without cyanide were in vitro translated and immunoprecipitated . This demonstrated that, in this species, the gene encoding the enzyme designated chy1, is cyanide inducible . Differential screening was used to isolate a cyanide hydratase cDNA clone which was subsequently used to obtain the corresponding genomic clone . A fragment of the cDNA clone encoding all but the first seven amino acids of the protein was expressed in E . coli using the expression vector pGEX-2T . Features of F . lateritium cyanide hydratase together with an analysis of the nucleotide sequence encoding this enzyme are presented. J Gen Microbiol, 1993 Aug, 139 ( Pt 8), 1715 - 21 Cloning, nucleotide sequence and expression in Escherichia coli of a gene (ompM) encoding a 25 kDa major outer-membrane protein (MOMP) of legionella pneumophila; High AS et al.; A genomic library derived from a virulent isolate of Legionella pneumophila was constructed in Escherichia coli JM 83 using the cloning vector pUC19 . The clones were screened by filter immunoassay using L . pneumophila rabbit polyclonal antisera and in the absence of in situ bacterial lysis one such clone, LP 116, expressed L . pneumophila-specific antigens on the surface of E . coli . Restriction endonuclease digest analysis and agarose gel electrophoresis revealed a fragment measuring approximately 750 bp . Southern hybridization confirmed that the fragment was L . pneumophila DNA . Sequencing data showed that the fragment was 810 bp in length with an open reading frame (ORF) of 678 bp . The outer-membrane profiles of the E . coli parent, the L . pneumophila DNA-contributing strain and clone LP 116 were compared by SDS-PAGE . A protein of 25 kDa was found in outer-membrane preparations of both the clone LP 116 and L . pneumophila but not in E . coli JM 83 . This was in agreement with the molecular mass of the deduced peptide of the mature protein . Immunoblots using L . pneumophila-specific polyclonal antiserum confirmed that this 25 kDa outer-membrane protein (OMP) was a L . pneumophila polypeptide . Both direct immunofluorescence assay and immunoblots using the commercially produced monoclonal antibody specific for the common antigen of the major outer-membrane protein (MOMP) confirmed that the 25 kDa protein produced by LP 116 was involved with the MOMP complex . The gene encoding this protein has been designated ompM.(ABSTRACT TRUNCATED AT 250 WORDS) J Gen Microbiol, 1993 Aug, 139 ( Pt 8), 1707 - 14 The Escherichia coli serA-linked capsule locus and its flanking sequences are polymorphic, genetic evidence for the existence of more than two groups of capsule gene clusters; Drake CR et al.; Two families of Escherichia coli capsules, termed groups I and II, have been defined previously on the basis of a number of biochemical and genetic criteria . Recently, a third group of capsules, termed I/II has been suggested on the basis of chemical structure and mode of expression . In this paper, we show that group I capsule-producing strains lack the serA-linked group II capsule genes . In addition, group I/II capsule-producing strains lack the group II capsule genes despite the former genes also mapping near to serA . Therefore, the genetic data presented in this paper support the existence of three groups of capsule gene clusters, two of which are linked to serA . Sequences flanking the K4 capsule genes were found in the chromosome of all E . coli strains examined and were sometimes present in multiple copies at different loci, indicating that this chromosomal region is highly polymorphic. Kansenshogaku Zasshi, 1993 Aug, 67(8), 753 - 7 {Serotypes of enteroadherent Escherichia coli exhibiting diffuse pattern of adherence isolated from diarrheal patients and healthy controls in Brazil, Myanmar and Japan}; Tsukamoto T et al.; We tried to detect enteroadherent Escherichia coli exhibiting a diffuse pattern of adherence to HeLa cells from stock strains derived from patients with or without diarrhea in Brazil, Myanmar and Japan (Osaka) . Enteroadherent E . coli was found from 16 (23 strains) out of 126 (384 strains) in diarrheal infant cases (12.7%), 26 (29 strains) out of 126 (348 strains) in healthy control cases (20.6%) in Brazil, from 15 (18 strains) out of 221 (542 strains) in diarrheal infant cases (6.8%), 4 (4 strains) out of 87 (212 strains) in healthy control cases (4.6%) in Myanmar . In Japan (Osaka), enteroadherent E . coli was detected from 7 (7 strains) out of 123 (198 strains) in diarrheal cases (5.7%) . These results show that enteroadherent E . coli (diffuse) may not be associated with diarrhea . Forty-four (62.9%) out of 70 strains belonged to 19 O serogroups or 21 O:H serotypes . All strains were H antigen serotypable, and 26 strains were found to be nonmotile . The predominant O:H serotypes were O11:H15, O11:H-, O20:H34, O21:H5, O89:H-, O99:H33 and O154:H45 . Only one strain belonged to enteropathogenic E . coli serogroup O127. FEMS Microbiol Lett, 1993 Aug 1, 111(2-3), 189 - 95 Transient repression of the synthesis of OmpF and aspartate transcarbamoylase in Escherichia coli K12 as a response to pollutant stress; Faber F et al.; The synthesis of total cellular proteins in Escherichia coli K12 was studied in batch culture following exposure of cells to low concentrations of monochlorophenol, pentachlorophenol and cadmium chloride . Changes in protein patterns were identified after pulse-chase labelling of proteins with {35S}methionine and subsequent two-dimensional gel electrophoresis (2D-PAGE) . We demonstrated that besides the induction of some stress proteins, also a transient decrease in the rate of synthesis of other proteins occurred . Two of these proteins were identified as OmpF and aspartate transcarbamoylase (ATCase) . Their transient repression appeared to be a general response to stress elicited by different pollutants and may therefore be used as a general and sensitive early warning system for pollutant stress. Electrophoresis, 1993 Aug, 14(8), 753 - 8 Analysis of plasmid-DNA and cell protein of recombinant Escherichia coli using capillary gel electrophoresis; Hebenbrock K et al.; Plasmid DNA prepared from cultivation samples of recombinant Escherichia coli was analyzed by capillary gel electrophoresis . We used this method to control the genetic stability during a fed-batch culture . The plasmid DNA and a standard DNA mixture with molecular weights in the size range of 3000-22,000 base pairs (bp) were analyzed in capillaries that were filled with solutions of non-cross-linked polyacrylamide . With this method the plasmid DNA from recombinant E . coli cultivation samples was analyzed within 30 min . Separation parameters such as gel concentration, capillary length and current were optimized for that purpose . The method was modified to allow the analysis of proteins . Crude cell lysate samples could be screened for the protein pattern within 15 min in sodium dodecyl sulfate-polyacrylamide filled capillaries . The method was also used to verify product purity after the down-stream process. Electrophoresis, 1993 Aug, 14(8), 713 - 9 Analysis of trp repressor-DNA interactions using gel electrophoresis; Lewis DE et al.; Quantitative analysis of the DNA-binding equilibria of E . coli trp repressor by gel electrophoresis led to reevaluation of our understanding of this complex system . In this review, the data leading to controversy about the trp system are discussed, and our current understanding is presented. Electrophoresis, 1993 Aug, 14(8), 704 - 12 Effects of anomalous migration and DNA to protein ratios on resolution of equilibrium constants from gel mobility-shift assays; Senear DF et al.; Numerical resolution of binding constants from data generated by the gel mobility-shift assay is dependent on the experimental resolution of the different ligation states of the DNA . Previously we showed that the populations of the intermediate ligation states at partial saturation with the protein ligand are extremely sensitive to cooperativity (Senear, D . F . and Brenowitz, M . J . Biol . Chem . 1991, 266, 13661-13671) . This makes accurate gel mobility-shift data extremely useful to the demonstration of cooperativity . However, the accuracy with which the intermediate ligation state populations are resolved has been questioned . Thus, two additional and related questions are now considered . First, what information is available if the intermediate ligation state populations are not used in the analysis of binding constants . Second, is accurate information obtained from those states under conditions of high DNA concentration . These questions are addressed by using the interactions of the lambda cI repressor protein with the three site operator, OR, and the interaction of the E . coli GalR protein with the single site operator, OE . Both simulated and experimental data are analyzed . The results point to two conclusions . First, precise resolution of all macroscopic constants for binding of proteins to DNA is critically dependent on the intermediate ligation state populations; resolution is limited to at most two DNA sites if these states are not used in the analysis . Second, when the DNA and protein concentrations used in the titrations are comparable, the resolution of binding constants is extremely sensitive to experimental uncertainty in the macromolecule concentrations. Electrophoresis, 1993 Aug, 14(8), 693 - 8 Electrophoretic analysis of protein-induced DNA bending and twist changes; Niederweis M et al.; DNA fragments with a varied phasing of two intrinsic bends at their ends and a tet operator in-between were constructed to determine Tet repressor-induced twist changes in the operator DNA . These distance variants show a sinusoidal dependence of their electrophoretic mobilities on the phasing of their bends in polyacrylamide gels . Complex formation with Tet repressor leads to a displacement of the first minimum, indicating an unwinding of the tet operator DNA . Model calculations were performed to assess the contribution of Tet repressor-induced DNA bending to this result . They revealed that the amplitude of the electrophoretic mobilities of the distance variants may be used as a parameter to separate the effects of Tet repressor-induced twist change and bending . The systematic analysis presented here may help to improve the quantitative interpretation of gel shifts and promote the use of this highly sensitive method to gain structural information about protein-DNA complexes. Electrophoresis, 1993 Aug, 14(8), 669 - 79 Theoretical studies on the mobility-shift behavior of binary protein-DNA complexes; Cann JR; The theory of mass transport coupled to reversible interactions under chemical kinetic control forms the basis for computer simulation of the electrophoretic mobility-shift behavior of binary protein-DNA complexes . Several systems have been modeled in terms of either (i) specific binding of a protein molecule to a single site on the DNA molecule; (ii) cooperative binding to two or three sites; (iii) noncooperative binding to two sites, both of which bind protein with equal affinity; (iv) statistical binding to multiple sites having identical intrinsic binding constants; or (v) protein-induced DNA loop formation . Both models (iii) and (v) embody the concept of reversible isomerization of protein-DNA complexes . The resulting simulations have provided fundamental information concerning (i) the factors governing the electrophoretic persistence and separation of protein-DNA complexes; (ii) the shape of experimental mobility-shift patterns; (iii) the generation of the protein-DNA ladder upon titration, for example, of the 203-base pair operator with lac repressor; and (iv) the theoretical bases for quantitative interpretation of the patterns in terms of thermodynamic and kinetic parameters . The practical implications of these findings are discussed. Comput Appl Biosci, 1993 Aug, 9(4), 435 - 40 CURVATURE: software for the analysis of curved DNA; Shpigelman ES et al.; Software is presented to plot the sequence-dependent spatial trajectory of the DNA double helix and/or distribution of curvature along the DNA molecule . The nearest-neighbor wedge model is implemented to calculate overall DNA path using local helix parameters: helix twist angle, wedge (deflection) angle and direction (of deflection) angle . The procedures described proved to be very convenient as tools for investigation of a relationship between overall DNA curvature and its gel electrophoretic mobility . All parameters of the model had been estimated from experimental data . Using these wedge parameters the program takes, as input, any DNA sequence and calculates the likely degree of curvature at each point along the molecule . This information is displayed both graphically and in the form of simplified representations of curved double helices . The Software, CURVATURE, can thus be used to investigate possible roles of curvature in modulation of gene expression and for location of curved portions of DNA, which may play an important role in sequence-specific protein--DNA interactions. Protein Sci, 1993 Aug, 2(8), 1320 - 30 Functional consequences of mutations at the allosteric interface in hetero- and homo-hemoglobin tetramers; Baudin V et al.; A seminal difference exists between the two types of chains that constitute the tetrameric hemoglobin in vertebrates . While alpha chains associate weakly into dimers, beta chains self-associate into tightly assembled tetramers . While heterotetramers bind ligands cooperatively with moderate affinity, homotetramers bind ligands with high affinity and without cooperativity . These characteristics lead to the conclusion that the beta 4 tetramer is frozen in a quaternary R-state resembling that of liganded HbA . X-ray diffraction studies of the liganded beta 4 tetramers and molecular modeling calculations revealed several differences relative to the native heterotetramer at the "allosteric" interface (alpha 1 beta 2 in HbA) and possibly at the origin of a large instability of the hypothetical deoxy T-state of the beta 4 tetramer . We have studied natural and artificial Hb mutants at different sites in the beta chains responsible for the T-state conformation in deoxy HbA with the view of restoring a low ligand affinity with heme-heme interaction in homotetramers . Functional studies have been performed for oxygen equilibrium binding and kinetics after flash photolysis of CO for both hetero- and homotetramers . Our conclusion is that the "allosteric" interface is so precisely tailored for maintaining the assembly between alpha beta dimers that any change in the side chains of beta 40 (C6), beta 99 (G1), and beta 101 (G3) involved in the interface results in increased R-state behavior . In the homotetramer, the mutations at these sites lead to the destabilization of the beta 4 hemoglobin and the formation of lower affinity noncooperative monomers. Protein Sci, 1993 Aug, 2(8), 1210 - 9 Synthetic chimeras of mouse growth factor-associated glandular kallikreins . I . Kinetic properties; Blaber M et al.; A series of six chimeric proteins, composed of fragments corresponding to either one or the other of the growth factor-associated mouse glandular kallikreins-epidermal growth factor binding protein (EGF-BP) and the gamma-subunit of nerve growth factor (gamma-NGF)--were expressed in Escherichia coli and isolated, and their kinetic properties were characterized . The assembly of these synthetic proteases involved the substitution of regions of the proteins containing four specific surface loops that have been postulated to influence both kinetic specificity and the formation of growth factor complexes . The substrates utilized in the kinetic characterization of these chimeric kallikreins were tripeptide nitroanilides representing carboxyl termini of both the EGF and beta-NGF mature hormones, putative processing sites for these kallikreins in the precursors . Characterization of these hybrid enzymes demonstrates that Km and kcat kinetic constants may be independently affected by the regions utilized in construction of these chimeric kallikreins . Specifically, loop 1, located in the amino terminal region (Bode, W., et al., J . Mol . Biol . 164, 237-282, 1983), in gamma-NGF enhanced the kcat for substrates containing threonine in the P2 position, as is the case during the processing of the carboxy terminus of the beta-NGF precursor . Also, the central regions of the kallikreins containing loop 2 and the kallikrein loop dictated the generally inverted Km and kcat kinetic constants observed between EGF-BP and gamma-NGF . Finally, in gamma-NGF the autolysis loop, found in the carboxyl terminal region, functions to lower the Km kinetic constant for a variety of substrates . The results allow previously characterized kinetic differences between EGF-BP and gamma-NGF to be interpreted in terms of specific regions of the proteins and identify a subset of amino acid positions responsible for these functional characteristics. J Pharmacol Toxicol Methods, 1993 Aug, 29(4), 185 - 93 The use of cyclic voltammetry for the evaluation of oxidative damage in biological samples; Kohen R; A method using cyclic voltammetry to evaluate oxidative damage in biological systems is presented . Three biological systems were tested: Escherichia coli cells, the rat jejunal mucosa, and the enzyme, lactate dehydrogenase . Exposure of E . coli cells to oxidative stress resulted in a rapid decrease in their survival and a decrease in their ability to accumulate 14C-leucine . This was accompanied by a significant increase in the oxidation potential of the cells . Similar results were obtained when the rat jejunal mucosa was exposed in a perfusion system to oxidative stress induced by the hydroxyl radical produced by either hydrogen peroxide and ferrous ions or the combination of ascorbic acid and copper ions . Loss of cellular potassium was taken as an indication of damage to the rat jejunum . Exposure of lactate dehydrogenase to oxidative stress induced by hydroxyl and peroxyl radicals also resulted in a significant loss of enzyme activity along with a pronounced change in the cyclic voltammogram of the enzyme . It was concluded that measurement of the oxidation potentials of these biological systems can give an indication of the occurrence of oxidative damage. Biokhimiia, 1993 Aug, 58(8), 1240 - 6 {Thioredoxin-reductase: structure, properties, and function}; Labudova O et al.; Thioredoxin reductase (TR-RED) pertains to the family of pyridine nucleotide disulfide oxidoreductases distinguished by their remarkable structural homology . The enzyme is a constituent component of the thioredoxin complex which is present in all types of organisms and is universal in respect of the numerous physiological functions it performs . The ability of TR-RED to protect the skin from UV-generated free oxygen species has been found . Owing to its ability to control melanin biosynthesis, the enzyme "doses" the suntan. J Vet Med Sci, 1993 Aug, 55(4), 607 - 11 Changes in reticuloendothelial function in dogs with endotoxin-induced shock; Okano S et al.; A shock model was experimentally produced by intravenous injection of a lethal dose (3 mg/kg) of endotoxin under general anesthesia induced by pentobarbital sodium using 7 beagles . The effect of this endotoxic shock on the reticuloendothelial function was investigated . The blood endotoxin concentration peaked immediately after administration and decreased subsequently . However, the value still remained on an increased level (1,051 pg/ml) even at 360 min after endotoxin treatment . The lipid emulsion test as an index of reticuloendothelial phagocytotic activity and the arterial ketone body ratio as an index of the energy charge in the liver decreased after endotoxin treatment and failed to recover during the experiment . Fibronectin, one of opsonic proteins, tended to decrease after injection of the endotoxin and was significantly (p < 0.01) low at 180 and 360 min compared with the value before injection of the endotoxin . These results suggested the depression of the reticuloendothelial function during endotoxin-induced shock. Hum Gene Ther, 1993 Aug, 4(4), 403 - 9 Assessment of recombinant adenoviral vectors for hepatic gene therapy; Li Q et al.; Recombinant adenoviral vectors have recently been used to transfer genes into a number of different cell types in vitro and in vivo . A recombinant adenoviral vector bearing the Escherichia coli beta-galactosidase (beta-gal) gene was used to quantitate the frequency of hepatocyte transduction in the mouse after direct viral infusion into the portal vein . When 10(10) adenoviral particles were infused, over 95% of the hepatocytes were transduced in vivo as determined by x-gal staining . The transduction protocol is relatively safe in that there is no detectable helper virus production in transduced animals and that very few extrahepatic cells are transduced by this method . There is also no evidence of significant liver pathology unless substantially greater quantities of virus are used . However, the transduced hepatocytes do not appear to persist in vivo because the percentage of hepatocytes expressing beta-gal declined over time . Four months after the procedure, 0.5-10% of the hepatocytes contain detectable beta-gal activity in vivo . The change in beta-gal-positive cells correlates with decreasing amounts of adenoviral DNA . Thus, current recombinant adenoviral vectors may have clinical applications in gene therapy for acute hepatic disorders. Am J Physiol, 1993 Aug, 265(2 Pt 1), G370 - 8 Na(+)-K(+)-2Cl- cotransport and Cl- secretion evoked by heat-stable enterotoxin is microfilament dependent in T84 cells; Matthews JB et al.; We previously reported that adenosine 3',5'-cyclic monophosphate-mediated stimulation of Cl- secretion in the human intestinal epithelial cell line T84 is accompanied by significant remodeling of F-actin and that both the secretory and cytoskeletal responses may be inhibited by phalloidin derivatives, agents that polymerize actin and prevent dynamic reorganization of microfilaments . In contrast, the carbachol-elicited Cl- secretory response (Ca2+ mediated) was not attenuated by phalloidin (J . Clin . Invest . 87: 1903-1909, 1991) . In the present study, we examine the effect of phalloidin on the Cl- secretory response elicited by the heat-stable enterotoxin of Escherichia coli (STa), which induces elevations in intracellular guanosine 3',5'-cyclic monophosphate . We find that apical administration of 1 microM STa results in a regionally restricted redistribution of F-actin confined to the basal pole of the cells . In monolayers pretreated with phalloidin, the Cl- secretory response to STa was inhibited by > 60% . Sequential treatment of phalloidin-loaded monolayers with STa followed by carbachol resulted in a synergistic secretory response that was not different from control (unloaded) monolayers . Examination of efflux/uptake through specific membrane transport pathways involved in STa-stimulated Cl- secretion indicated normal activation of apical Cl- and basolateral K+ channels under phalloidin-loaded conditions . The ability of STa-treated monolayers to pump Na+ in an absorptive direction was also unaffected by phalloidin . der phalloidin-loaded conditions, STa-stimulated Na(+)-K(+)-2Cl- cotransporter activity was reduced by approximately 60%, sufficient to account for the observed inhibition of net Cl- secretory response.(ABSTRACT TRUNCATED AT 250 WORDS) Arch Biochem Biophys, 1993 Aug 1, 304(2), 367 - 70 A new approach to measurement of redox-cycling activity in Escherichia coli; McManus DC et al.; Redox-cycling agents catalyze the flow of reducing equivalents to oxygen; this process generates superoxide ion and other reduced oxygen species . Measurements of redox-cycling activity have been performed previously by studying cyanide-resistant oxygen consumption (respiration) of Escherichia coli cells . E . coli strain GK100, lacking both terminal oxidases, has almost no measurable respiration . We show that the use of this strain eliminates the requirement for cyanide in measurements of redox-cycling activity . The addition of either menadione sodium bisulfite or plumbagin, well-known redox-cycling agents, to GK100 cells resulted in high levels of oxygen consumption . The rate of menadione bisulfite-induced oxygen consumption in this respiration-deficient strain, in the absence of cyanide, was comparable to the cyanide-resistant respiration of isogenic respiration-proficient E . coli strains . In GK100 cells, cyanide increased menadione bisulfite-induced oxygen consumption but had no effect on plumbagin-induced oxygen consumption. Arch Biochem Biophys, 1993 Aug 1, 304(2), 338 - 44 Cryogenic solvents inhibit the binding of the Escherichia coli heat-stable enterotoxin to intestinal brush border membranes; Katwa LC et al.; The cryogenic solvents ethylene glycol, glycerol, dimethyl sulfoxide (Me2SO) and dimethylformamide (Me2FM), with increasing potency, produced concentration-dependent inhibition of the binding of the Escherichia coli heat-stable enterotoxin (STa) to pig intestinal brush border membranes . Inhibition increased with time, and both Me2SO and Me2FM appeared to decrease both the affinity of the STa receptor and the effective receptor number . Both solvents stimulated the release of previously bound 125I-STa (Me2FM > Me2SO), 3 M Me2FM inducing 93% release by 120 min . These effects were reversible, and preincubation of membranes with up to 3 M Me2SO or Me2FM at 37 degrees C for 30 min, followed by washing, did not alter subsequent 125I-STa binding . Also, 125I-STa released from membranes by 3 M Me2FM was shown to rebind to the membranes after 10-fold dilution of Me2FM . Since pretreating membranes with the thiol reagent p-chloromercuribenzenesulfonate had no effect on the release of bound 125I-STa by Me2SO or Me2FM, and since neither of these can reduce disulfide bonds, the formation of mixed disulfides between STa and receptor is unlikely . Me2SO inhibition of 125I-STa binding was greater with membranes than with a partially purified receptor preparation, which may result from the substitution of detergent for the phospholipid normally associated with the receptor(s). Proc Natl Acad Sci U S A, 1993 Aug 1, 90(15), 7029 - 33 Guanosine pentaphosphate phosphohydrolase of Escherichia coli is a long-chain exopolyphosphatase; Keasling JD et al.; An exopolyphosphatase {exopoly(P)ase; EC 3.6.1.11} activity has recently been purified to homogeneity from a mutant strain of Escherichia coli which lacks the principal exopoly(P)ase . The second exopoly(P)ase has now been identified as guanosine pentaphosphate phosphohydrolase (GPP; EC 3.6.1.40) by three lines of evidence: (i) the sequences of five tryptic digestion fragments of the purified protein are found in the translated gppA gene, (ii) the size of the protein (100 kDa) agrees with published values for GPP, and (iii) the ratio of exopoly(P)ase activity to GPP activity remains constant throughout a 300-fold purification in the last steps of the procedure . The enzyme liberates orthophosphate by processive hydrolysis of the phosphoanyhydride bonds of polyphosphate {poly(P)} chains (1000 residues) or by hydrolysis of the 5'-gamma-phosphate of guanosine 5'-triphosphate 3'-diphosphate (pppGpp) to guanosine 5'-diphosphate 3'-diphosphate (ppGpp or "magic spot") . The Km for long-chain poly(P) as a substrate (approximately 0.5 nM) is far lower than that for pppGpp (0.13 mM); the kcat for the poly(P)ase activity is 1.1 s-1 and that for pppGpp hydrolase is 0.023 s-1 . These and other findings direct attention to possible functions of poly(P) in the response of E . coli to stresses and deprivations. J Gen Virol, 1993 Aug, 74 ( Pt 8), 1649 - 52 Inducible expression of a foreign gene inserted into the human cytomegalovirus genome; Takekoshi M et al.; We previously described the insertion of a foreign gene into a non-essential region of human cytomegalovirus (HCMV) by homologous recombination . Here we report insertion of the Escherichia coli lacZ gene downstream of the mouse metallothionein promoter into the HindIII-O region of HCMV by replacement-type recombination . Expression of the lacZ gene in the recombinant was independent of viral growth, but dependent on induction by heavy metals . Of several metals tested for beta-galactosidase induction and also for their toxicity to HEL cells, Zn was found to be the most suitable for use as an inducer . In HEL cells infected with the recombinant in the presence of 50 microsM-Zn, beta-galactosidase activity was maximal 3 days after infection, and reached levels 27 times higher than the value obtained in the absence of Zn. EMBO J, 1993 Aug, 12(8), 3123 - 32 The cdc2-related protein p40MO15 is the catalytic subunit of a protein kinase that can activate p33cdk2 and p34cdc2; Poon RY et al.; Activation of the cyclin-dependent protein kinases p34cdc2 and p33cdk2 requires binding with a cyclin partner and phosphorylation on the first threonine residue in the sequence THEVVTLWYRAPE . We present evidence that this threonine residue, number 160 in p33cdk2, can be specifically phosphorylated by a cdc2-related protein kinase from Xenopus oocytes called p40MO15 . Binding to cyclin A and phosphorylation of this threonine are both required to activate fully the histone H1 kinase activity of p33cdk2 . In cell extracts, a portion of p40MO15 is found in a high molecular weight complex that is considerably more active than a lower molecular weight form . Wild-type MO15 protein expressed in bacteria does not possess kinase activity, but acquires p33cdk2-T160 kinase activity after incubation with cell extract and ATP . We conclude that p40MO15 corresponds to CAK (cdc2/cdk2 activating kinase) and speculate that, like p33cdk2 and p34cdc2, p40MO15 requires activation by phosphorylation and association with a companion subunit. Virology, 1993 Aug, 195(2), 638 - 48 Selection of Marek's disease virus recombinants expressing the Escherichia coli gpt gene; Marshall DR et al.; We developed a positive selection method for recovering Marek's disease virus (MDV) recombinants . The Escherichia coli xanthine-guanine phosphoribosyltransferase gene (gpt), under the control of the major immediate-early promoter from cytomegalovirus, was inserted into the inverted repeats flanking the unique long (UL) region of a non-pathogenic serotype 2 MDV strain 281MI/1 . In a second demonstration of the usefulness of the positive selection system, the gpt gene was inserted into the inverted repeats flanking the unique short (US) region of the turkey herpesvirus (HVT) strain FC126 . The targeted insertion site in 281MI/1 was in a previously established nonessential site for virus replication . The targeted insertion site for FC126, at the junction of the UL and US regions, is a nonessential site for in vitro replication of herpes simplex virus . Recombinant viruses were easily selected by incubating the transfected cells in mycophenolic acid (MPA)-containing medium . Purification of recombinants resulted from a series of trypsinization and sonication steps combined with the culturing of virus in MPA-containing medium to inhibit wild-type virus replication . This simple technique for recovering MDV and HVT recombinants should increase the efficiency of identifying nonessential sites and gene function analysis by insertional mutagenesis. J Bacteriol, 1993 Aug, 175(15), 4905 - 6 Delayed expression of in vivo restriction activity following conjugal transfer of Escherichia coli hsdK (restriction-modification) genes; Prakash-Cheng A et al.; Following conjugal transfer of the hsdK genes (hsdRK, hsdMK, and hsdSK) of Escherichia coli K-12, restriction activity was first detected only after approximately 15 generations, whereas modification activity was observed immediately . This sequential expression explains the establishment of hsdK genes in a nonmodified host and suggests regulation of restriction activity after conjugal transfer. J Bacteriol, 1993 Aug, 175(15), 4843 - 50 Plasmid pKM101 encodes two nonhomologous antirestriction proteins (ArdA and ArdB) whose expression is controlled by homologous regulatory sequences; Belogurov AA et al.; The IncN plasmid pKM101 (a derivative of R46) encodes the antirestriction protein ArdB (alleviation of restriction of DNA) in addition to another antirestriction protein, ArdA, described previously . The relevant gene, ardB, was located in the leading region of pKM101, about 7 kb from oriT . The nucleotide sequence of ardB was determined, and an appropriate polypeptide was identified in maxicells of Escherichia coli . Like ArdA, ArdB efficiently inhibits restriction by members of the three known families of type I systems of E . coli and only slightly affects the type II enzyme, EcoRI . However, in contrast to ArdA, ArdB is ineffective against the modification activity of the type I (EcoK) system . Comparison of deduced amino acid sequences of ArdA and ArdB revealed only one small region of similarity (nine residues), suggesting that this region may be somehow involved in the interaction with the type I restriction systems . We also found that the expression of both ardA and ardB genes is controlled jointly by two pKM101-encoded proteins, ArdK and ArdR, with molecular weights of about 15,000 and 20,000, respectively . The finding that the sequences immediately upstream of ardA and ardB share about 94% identity over 218 bp suggests that their expression may be controlled by ArdK and ArdR at the transcriptional level . Deletion studies and promoter probe analysis of these sequences revealed the regions responsible for the action of ArdK and ArdR as regulatory proteins . We propose that both types of antirestriction proteins may play a pivotal role in overcoming the host restriction barrier by self-transmissible broad-host-range plasmids . It seems likely that the ardKR-dependent regulatory system serves in this case as a genetic switch that controls the expression of plasmid-encoded antirestriction functions during mating. J Bacteriol, 1993 Aug, 175(15), 4764 - 71 DNA topology affects transcriptional regulation of the pertussis toxin gene of Bordetella pertussis in Escherichia coli and in vitro; Scarlato V et al.; The bvg locus of Bordetella pertussis encodes an environmentally inducible operon essential for the expression of virulence genes . We show that in Escherichia coli, the PTOX promoter cloned in cis of the bvg locus is activated and environmentally regulated . Cotransformation of E . coli with the bvg locus cloned in a low-copy-number plasmid and with the PTOX promoter cloned in a high-copy-number plasmid can give rise to two different results . If the PTOX promoter is cloned in the pGem-3 vector, transcription is absent . If the PTOX promoter is cloned in the plasmid pKK232, containing the PTOX promoter between two ribosomal gene terminators of transcription, transcription occurs, although regulation of transcription is abolished . Under these conditions, the intracellular amount of RNA transcripts is increased by adding to the culture medium novobiocin, an inhibitor of bacterial gyrases . In vitro, the transcription of the PTOX promoter is activated on E . coli RNA polymerase supplemented with cell extracts from wild-type B . pertussis . Addition of DNA gyrase to the mixture dramatically reduces the amount of RNA synthesized . Our data show that the products of the bvg locus, BvgA and BvgS, are directly involved in the regulation of the PTOX promoter in E . coli and that DNA topology may play a role in the induction of transcription. J Bacteriol, 1993 Aug, 175(15), 4744 - 55 Identification and molecular characterization of csrA, a pleiotropic gene from Escherichia coli that affects glycogen biosynthesis, gluconeogenesis, cell size, and surface properties; Romeo T et al.; Current evidence suggests that a few global regulatory factors mediate many of the extensive changes in gene expression that occur as Escherichia coli enters the stationary phase . One of the metabolic pathways that is transcriptionally activated in the stationary phase is the pathway for biosynthesis of glycogen . To identify factors that regulate glycogen biosynthesis in trans, a collection of transposon mutants was generated and screened for mutations which independently increase or decrease glycogen levels and the expression of a plasmid-encoded glgC'-lacZ fusion . The glycogen excess mutation TR1-5 was found to be pleiotropic . It led to increased expression of the genes glgC (ADPglucose pyrophosphorylase) and glgB (glycogen branching enzyme), which are representative of two glycogen synthesis operons, and the gluconeogenic gene pckA (phosphoenolpyruvate carboxykinase), and it exhibited effects on cell size and surface (adherence) properties . The mutated gene was designated csrA for carbon storage regulator . Its effect on glycogen biosynthesis was mediated independently of cyclic AMP (cAMP), the cAMP receptor protein, and guanosine 3'-bisphosphate 5'-bisphosphate (ppGpp), which are positive regulators of glgC expression . A plasmid clone of the native csrA gene strongly inhibited glycogen accumulation and affected the ability of cells to utilize certain carbon sources for growth . Nucleotide sequence analysis, complementation experiments, and in vitro expression studies indicated that csrA encodes a 61-amino-acid polypeptide that inhibits glycogen biosynthesis . Computer-assisted data base searches failed to identify genes or proteins that are homologous with csrA or its gene product. J Bacteriol, 1993 Aug, 175(15), 4670 - 80 A second chromosomal gene necessary for intimate attachment of enteropathogenic Escherichia coli to epithelial cells; Donnenberg MS et al.; Enteropathogenic Escherichia coli (EPEC) is capable of attaching intimately to epithelial cells and effacing their microvilli . A chromosomal locus, eaeA (originally eae), is required for the intimate attachment aspect of this effect . We report the mapping of a region of the EPEC chromosome that is located immediately downstream of the eaeA gene and that is also necessary for intimate attachment . An isogenic in-frame deletion mutation in one of the open reading frames identified in this region was engineered . Because the resulting mutant, like an eaeA deletion mutant, is deficient in the ability to attach intimately to epithelial cells, the mutated gene is designated eaeB . Full activity is restored to the eaeB mutant when the cloned gene is reintroduced on a plasmid . The eaeB mutant remains capable of producing intimin, the product of the eaeA gene . No differences in the fractionation properties or electrophoretic mobility of intimin are apparent in the eaeB mutant . The product of the eaeB locus was identified by in vitro transcription-translation . The nucleotide sequence of the eaeB gene predicts a protein that contains a sequence motif common to several aminotransferase enzymes . These results indicate that the attaching and effacing effect is a complex phenotype dependent on a gene cluster present on the EPEC chromosome. J Virol, 1993 Aug, 67(8), 4566 - 79 Efficient generation of infectious recombinant baculoviruses by site-specific transposon-mediated insertion of foreign genes into a baculovirus genome propagated in Escherichia coli; Luckow VA et al.; The construction and purification of recombinant baculovirus vectors for the expression of foreign genes in insect cells by standard transfection and plaque assay methods can take as long as 4 to 6 weeks . This period can be reduced to several days by using a novel baculovirus shuttle vector (bacmid) that can replicate in Escherichia coli as a plasmid and can infect susceptible lepidopteran insect cells . The bacmid is a recombinant virus that contains a mini-F replicon, a kanamycin resistance marker, and attTn7, the target site for the bacterial transposon Tn7 . Expression cassettes comprising a baculovirus promoter driving expression of a foreign gene that is flanked by the left and right ends of Tn7 can transpose to the target bacmid in E . coli when Tn7 transposition functions are provided in trans by a helper plasmid . The foreign gene is expressed when the resulting composite bacmid is introduced into insect cells. J Virol, 1993 Aug, 67(8), 4464 - 73 Identification of the phosphorylation sequence in the cytoplasmic tail of the varicella-zoster virus Fc receptor glycoprotein gpI; Yao Z et al.; Varicella-zoster virus (VZV) glycoprotein gpI, the homolog of herpes simplex virus gE, functions as a receptor for the Fc portion of immunoglobulin G . Like other cell surface receptors, this viral receptor is highly phosphorylated in cell culture . To identify the precise location of the cellular kinase-mediated phosphorylation, we generated a tailless deletion mutant and several point mutants which had altered serine and threonine residues within the cytoplasmic domain of gpI . The mutated and wild-type genes of gpI were transfected and expressed within a vaccinia virus-T7 polymerase transfection system in order to determine what effect these mutations had on the phosphorylation state of the protein in vivo and in vitro . Truncation of the cytoplasmic domain of gpI diminished the phosphorylation of gpI in vivo . Examination of the point mutants established that the major phosphorylation sequence of gpI was located between amino acids 593 and 598, a site which included four phosphorylatable serine and threonine residues . Phosphorylation analyses of the mutant and wild-type glycoproteins confirmed that gpI was a substrate for casein kinase II, with threonines 596 and 598 being critical residues . Although the mutant glycoproteins were phosphorylated by casein kinase I, protease V8 partial digestion profiles suggested that casein kinase II exerted the major effect . Thus, these mutagenesis studies demonstrated that the gpI cytoplasmic sequence Ser-Glu-Ser-Thr-Asp-Thr was phosphorylated in mammalian cells in the absence of any other herpesvirus products . Since the region defined by transfection was consistent with results obtained with in vitro phosphorylation by casein kinase II, we propose that VZV gpI is a physiologic substrate for casein kinase II . Immunofluorescence and pulse-chase experiments demonstrated that the mutant glycoproteins were processed and transported to the outer cell membrane. J Urol, 1993 Aug, 150(2 Pt 2), 759 - 62 The diagnosis of acute pyelonephritis in the piglet using single photon emission computerized tomography dimercaptosuccinic acid scintigraphy: a pathological correlation; Giblin JG et al.; Single photon emission computerized tomography (SPECT) scintigraphy has proved to be an extremely sensitive renal imaging modality in children with genitourinary pathology, including pyelonephritis, particularly when compared to 2-dimensional planar imaging . This study was undertaken to corroborate SPECT dimercaptosuccinic acid (DMSA) scintigraphic findings with specific histopathology in acute pyelonephritis . Unilateral vesicoureteral reflux was produced in 19 Yorkshire piglets 3 to 4 weeks old . The bladders of 12 animals were inoculated with Escherichia coli 2 weeks later, after baseline SPECT DMSA scans had been obtained . The animals were then re-imaged at 3 (4), 7 (4) or 14 (4) days after infection and sacrificed for histological evaluation . Seven purposefully uninfected piglets with unilateral reflux served as controls and were followed for up to 6 weeks before imaging and sacrifice . SPECT proved to be 97% sensitive and 93% specific in providing the diagnosis of acute pyelonephritis . The SPECT findings were manifest by a spectrum of abnormal findings (mottling, striations, inner cortical scalloping and focal cortical defects), which correlated precisely with the extent and severity of cortical involvement in the acute pyelonephritic process . We propose a new classification scheme for SPECT DMSA renal scintigraphic imaging, and believe that this modality is exquisitely sensitive in providing the diagnosis as well as in evaluating the extent of renal parenchymal involvement when acute pyelonephritis is induced in the animal model. Nippon Geka Gakkai Zasshi, 1993 Aug, 94(8), 809 - 15 {Experimental study on the effects of endotoxemia as a retardation-factor influencing on the decrease of serum bilirubin after the relief of obstructive jaundice}; Idei T; I examined the effects of endotoxemia influencing on obstructive jaundice and the decrease of serum bilirubin after the relief of it . Experimental obstructive jaundice and its alleviation by external biliary drainage was made in Donryu-rats . Serum bilirubin was higher (p < 0.01) in the group treated by continuous infusion of low-dose endotoxin (LPS E . coli 0111:B4, 6 micrograms/hr/100gBW) during 72 hours biliary obstruction (10.48 +/- 1.54mg/dl) than in the control group (6.76 +/- 0.71mg/dl) . After the relief of biliary obstruction, 4 kinds of experimental conditions were set up as follows: CONTROL; infusion of sterile saline, ET; continuous infusion of low-dose endotoxin, ET + UDCA; continuous infusion of endotoxin and ursodeoxycholic acid (UDCA, 10mg/hr/100gBW) through another intravenous tube simultaneously, ET + MP; continuous infusion of endotoxin after one-shot injection of methylprednisolone (MP, 3mg/100gBW) . Serum bilirubin 7.5 hours after the relief of biliary obstruction was as follows: CONTROL: 0.48 +/- 0.18mg/dl, ET: 3.41 +/- 1.13mg/dl, ET + UDCA: 2.80 +/- 1.28mg/dl, ET + MP: 1.18 +/- 0.50mg/dl . ET-group showed retardation of the decrease of serum bilirubin (p < 0.01) . MP showed improvement of the impaired decreasing rate of serum bilirubin by endotoxemia (p < 0.01) . Bile-output from the external biliary drainage after the relief of biliary obstruction was decreased significantly in the ET-group . ET + UDCA-group showed remarkable increase of the bile-output, but no increase of excretion of bilirubin in the bile compared with ET-group . While ET + MP-group showed improvement of the bile-output and increase of excretion of bilirubin in the bile compared with ET-group.(ABSTRACT TRUNCATED AT 250 WORDS) Genetics, 1993 Aug, 134(4), 1031 - 8 The mutational specificity of two Escherichia coli dnaE antimutator alleles as determined from lacI mutation spectra; Schaaper RM; In a companion study we have described the isolation of a series of mutants of Escherichia coli that replicate their DNA with increased fidelity . These mutants carry a mutation in the dnaE gene, encoding the alpha (polymerase) subunit of DNA polymerase III holoenzyme, which is responsible for the faithful replication of the bacterial chromosome . The mutants were detected as suppressors of the high mutability of a mutL strain (defective in postreplicative mismatch correction), in which mutations may be considered to arise predominantly from errors of DNA replication . To investigate the specificity of these antimutator effects, we have analyzed spectra of forward mutations in the N-terminal part of the lacI gene (i-d mutations) for two of the mutL dnaE derivatives (dnaE911 and dnaE915), as well as the control mutL strain . DNA sequencing of over 600 mutants revealed that in the mutL background both antimutator alleles reduce specifically transition mutations (A.T-->G.C and G.C-->A.T) . However, the two alleles behave differently in this respect . dnaE911 reduces A.T-->G.C more strongly than it does G.C-->A.T, whereas the reverse is true for dnaE915 . Second, dnaE911 does not appear to affect either transversion or frameshift mutations, whereas dnaE915 displays a distinct mutator effect for both . This mutator effect of dnaE915 for frameshift mutations was confirmed by the frequency of reversion of the trpE9777 frameshift mutation . The discovery that dnaE antimutator alleles possess distinct specificities supports the notion that DNA polymerases discriminate against errors along multiple pathways and that these pathways can be influenced independently. Circ Shock, 1993 Aug, 40(4), 268 - 75 Repeated plasma therapy induces fatal shock in experimental septicemia; Busund R et al.; Plasma therapy in severe septicemia, either as part of plasma exchange or alone, was evaluated in a model of lethal septic shock induced with live Escherichia coli in pigs . The following groups were studied: group I, septic animals treated with repeated plasma exchanges, n = 8; group II, nontreated septic controls, n = 8; group III, septic animals treated with repeated plasma infusions, n = 14; and group IV, nonseptic animals treated with repeated plasma infusions, n = 7 . In the septic animals treated with plasma (groups I and III), a rapid fatal response was observed between 2 and 5 min after the start of plasma therapy, while the septic controls (group II) showed a progressively longer lasting septic shock . The nonseptic animals (group IV) were unaffected by the plasma infusions . Plasma levels of endotoxin above 2 ng/ml were associated with rapid death during plasma therapy . Ionized Ca2+ fell abruptly in this situation . This study indicates that commonly used plasma therapies (exchanges or transfusions) in septic animals may have acute deleterious effects . These effects may be explained by depletion of ionized calcium. Circ Shock, 1993 Aug, 40(4), 235 - 42 Effect of tirilazad mesylate (U74006F) on eicosanoid and tumor necrosis factor generation in healthy and endotoxemic neonatal calves; Semrad SD et al.; The effects of a 21-aminosteroid, tirilazad mesylate (U74006F; The Upjohn Co., Kalamazoo, MI), developed for treatment of central nervous system trauma in human beings, on eicosanoid and tumor necrosis factor (TNF) generation were evaluated in both healthy and endotoxin-challenged neonatal calves . Endotoxemia was induced in 24 neonatal calves by intravenous infusion of Escherichia coli lipopolysaccharide (3.25 micrograms/kg) over 3 hr . Group I calves received the endotoxin infusion alone; group II calves received an infusion of 0.9% saline and were treated with tirilazad mesylate (1.5 mg/kg) 1 hr after the infusion was started, group III and IV calves were treated with tirilazad mesylate 1 hr after or before endotoxin infusion was started . Tirilazad mesylate effectively suppressed production of plasma prostacyclin (6-keto-PGF1 alpha) and TNF in endotoxin-challenged neonatal calves . In addition, production of thromboxane B2 was mitigated by treatment with tirilazad mesylate both 1 hr before and 1 hr after initiation of endotoxin infusion . It appears that tirilazad mesylate effectively suppresses eicosanoid and TNF generation induced by endotoxin, and thus may be beneficial in the treatment of endotoxemia and septicemia in neonatal calves. Protein Expr Purif, 1993 Aug, 4(4), 337 - 44 Syrian hamster 3-hydroxy-3-methylglutaryl-coenzyme A reductase expressed in Escherichia coli: production of homogeneous protein; Frimpong K et al.; When overexpressed in Escherichia coli, the catalytic domain of Syrian hamster 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA reductase, EC 1.1.1.34) is catalytically active, but exhibits major heterogeneity . This heterogeneity reflects deletion of about 60 aminoacyl residues from the C-terminus, presumably a result of proteolytic cleavage or premature termination of translation . With the intent of separating the intact and truncated proteins via immunoaffinity chromatography, we constructed the expression phagemid pKFT7-21 . This construct encodes the catalytic domain of Syrian hamster HMG-CoA reductase with the C-terminal extension Glu-Glu-Phe, an epitope recognized by a specific antibody . Following overexpression, the modified catalytic domain RcatEEF had high catalytic activity and exhibited no heterogeneity . It therefore was possible to purify RcatEEF to over 95% homogeneity without resorting to immunoaffinity chromatography . The yield of homogeneous protein averaged 20-25 mg per liter of cells with a final specific activity of up to 40 mumol NADPH oxidized per minute per milligram . The EEF modification thus should prove useful for the purification of the catalytic domains of other eukaryotic HMG-CoA reductases which exhibit heterogeneity. Protein Expr Purif, 1993 Aug, 4(4), 320 - 7 Use of nonreducing SDS-PAGE for monitoring renaturation of recombinant protein synthesis initiation factor, eIF-4 alpha; Stern BD et al.; The purification of biologically active human protein synthesis initiation factor 4 alpha, eIF-4 alpha, overexpressed in Escherichia coli, is complicated by its localization in insoluble inclusion bodies, as well as its possession of four cysteines . Two of these cysteines have been reported to be reduced in the native molecule and two form a disulfide bond . A method is described, using nonreducing sodium dodecyl sulfate polyacrylamide gel electrophoresis, for monitoring renaturation of the polypeptide during staged dialyses in decreasing urea concentrations . The production of biologically active eIF-4 alpha only occurs when the polypeptide is totally reduced during solubilization of the inclusion bodies in 8 M urea . This requires a minimum dithiothreitol concentration of 50 mM . Conversely, reformation of the disulfide bonds only occurs when the staged dialyses are performed at lower concentrations of sulfhydryl reagents . Once renatured, as described, eIF-4 alpha can be purified by affinity chromatography on m7GTP-Sepharose . Approximately 20 micrograms of biologically active eIF-4 alpha per milliliter of bacterial culture can be obtained . The affinity-purified eIF-4 alpha has activity equivalent to that reported for purified native human eIF-4 alpha, as measured by its activity in a rabbit reticulocyte translation system . The method described is applicable to the purification of other cysteine-containing polypeptides that accumulate to high levels in inclusion bodies. Protein Expr Purif, 1993 Aug, 4(4), 282 - 9 Expression, purification, and characterization of recombinant ornatin E, a potent glycoprotein IIb-IIIa antagonist; Mazur P et al.; A synthetic gene encoding ornatin E (OrnE), a 50-amino acid glycoprotein IIb-IIIa (GP IIb-IIIa) antagonist and platelet aggregation inhibitor isolated from the leech Placobdella ornata, was designed, constructed, and expressed in Escherichia coli . The OrnE gene was fused to the heat stable enterotoxin stII signal sequence and expressed under the transcriptional control of the E . coli alkaline phosphatase promoter . This construction directed secretion of recombinant ornatin E (rOrnE) into the extracellular medium at levels of 7-19 mg/liter . The protein was purified to apparent homogeneity in 18-38% yields by ammonium sulfate precipitation, Q-Sepharose and S-Sepharose ion exchange chromatography, and reverse-phase HPLC . Purified rOrnE was found to be indistinguishable from leech-derived OrnE as judged by amino acid composition, N-terminal sequencing, mass spectroscopic analysis, and HPLC coelution . In addition, rOrnE exhibits similar activity in fibrinogen/GP IIb-IIIa ELISA and platelet aggregation assays . Purified rOrnE possesses three disulfide bonds, the reduction and carboxymethylation of which results in a ca . 60-fold reduction in biological activity . A misfolded variant of rOrnE was characterized and shown to have a ca . 6-fold reduction in activity . These data demonstrate that the native disulfide bonds are required for the optimal GP IIb-IIIa antagonist activity of the ornatins. Protein Expr Purif, 1993 Aug, 4(4), 275 - 81 High yield of active STb enterotoxin from a fusion protein (MBP-STb) expressed in Escherichia coli; Handl CE et al.; Maltose binding protein (MBP) fused to STb, a heatstable enterotoxin of Escherichia coli, was secreted into the periplasm . A factor Xa cleavage site is present between MBP and STb allowing MBP to be cleaved from STb . The gene fusion is under the control of the strong and inducible Ptac promoter . Three hours after induction with IPTG, cells were harvested . Following osmotic shock treatment of the cells, the MBP-STb fusion protein was released and affinity-purified using an amylose resin . The fusion protein purified in this way was biologically active in ligated intestinal segments of rats . Digestion of MBP-STb with factor Xa released native STb which was purified to homogeneity by reverse-phase chromatography using a PepRPC column . The toxin was eluted at approximately 38% acetonitrile . The 5000-Da toxin was shown to be pure by SDS-PAGE and immunoblotting . The recovered enterotoxin was active in the rat loop assay . Amino acid sequence analysis showed that the first eight residues were identical to those of native STb, confirming the identity of STb . The ultraviolet absorption spectra of purified STb revealed low absorption at 254 and 280 nm compared to 210-230 nm . Isoelectric focusing under nondenaturing conditions indicated a pI of 9.6 . Typically, 8 liters of bacterial culture resulted in 2.2 mg of pure STb . This genetic construction provides a readily obtainable source of biologically active STb toxin. Protein Expr Purif, 1993 Aug, 4(4), 265 - 74 Gene fusions with human carbonic anhydrase II for efficient expression and rapid single-step recovery of recombinant proteins: expression of the Escherichia coli F1-ATPase epsilon subunit; Van Heeke G et al.; A new expression vector was constructed which allows the overproduction in Escherichia coli of tripartite proteins consisting of human carbonic anhydrase isozyme II (hCAII), a peptide linker containing an enterokinase cleavage site, and a target protein of interest . Carbonic anhydrase is soluble and stable in E . coli and serves as a highly specific purification tag in the recovery of the fusion protein by a single affinity chromatography step . The enterokinase cleavage site was engineered into the construct to allow accurate and efficient release of the target protein . To demonstrate the practical value of this vector, the E . coli F1-ATPase epsilon subunit was expressed as a fusion with hCAII . After a single purification step, biologically active recombinant E . coli F1-ATPase epsilon subunit was recovered following proteolytic removal of the hCAII moiety. Biotechniques, 1993 Aug, 15(2), 264 - 6 In vitro transcription of transfer RNAs with 3'-end modifications; Liu M et al.; It is difficult to modify the 3'-end of RNA transcribed in vitro from recombinant plasmid or phagemid DNA because of the need to retain a restriction endonuclease recognition site at the downstream end of the template in order to linearize the DNA before transcription . To avoid limitations on the 3'-sequence of RNA transcripts, we have modified a template-containing phagemid by inserting a FokI site outside the RNA gene sequence, positioned to cleave the template DNA to yield the desired 3' terminus . We demonstrate that this phagemid is an efficient template for tRNA transcription and use it to prepare mutants of Escherichia coli tRNA(Val) with modifications at the 3'-CCA end . Phagemids containing a FokI site should be suitable for in vitro transcription of large quantities of RNA of any length and with an unlimited variety of 3'-terminal sequences. J Clin Microbiol, 1993 Aug, 31(8), 2163 - 6 Evaluation of conventional media for detection of colonization factor antigens of enterotoxigenic Escherichia coli; Ghosh AR et al.; Strains of enterotoxigenic Escherichia coli producing either colonization factor antigen (CFA) I or II were tested for expression of CFA when grown on 16 different agar media by using agglutination and coagglutination with monoclonal antibodies, mannose-resistant hemagglutination, and a salt aggregation assay . CFA was detected from the CFA-positive strains when CFA agar was used, and it was also detected when other commercially available media were used, notably nutrient agar . CFA was not detected when other commercial media such as MacConkey agar were used . The use of nutrient agar with monoclonal antibody-based coagglutination reagents offers a potentially simple and rapid method for detecting E . coli whic