Escherichia coli

Science, 1988 May 20, 240(4855), 1041 - 3
Escherichia coli secretion of an active chimeric antibody fragment; Better M et al.; A chimeric mouse-human Fab protein that binds specifically to the human carcinoma cell line C3347 has been expressed and secreted from Escherichia coli . This molecule, which contains functionally assembled kappa and Fd proteins, binds as effectively to sites on the surface of C3347 cells as Fab fragments prepared proteolytically from whole chimeric or mouse antibody . The production in Escherichia coli of foreign heterodimeric protein reagents, such as Fab, should prove useful in the management of human disease.

Science, 1988 May 20, 240(4855), 1038 - 41
Assembly of a functional immunoglobulin Fv fragment in Escherichia coli; Skerra A et al.; An expression system was developed that allows the production of a completely functional antigen-binding fragment of an antibody in Escherichia coli . The variable domains of the phosphorylcholine-binding antibody McPC603 were secreted together into the periplasmic space, where protein folding as well as heterodimer association occurred correctly . Thus, the assembly pathway for the Fv fragment in E . coli is similar to that of a whole antibody in the eukaryotic cell . The Fv fragment of McPC603 was purified to homogeneity with an antigen-affinity column in a single step . The correct processing of both signal sequences was confirmed by amino-terminal protein sequencing . The functionality of the recombinant Fv fragment was demonstrated by equilibrium dialysis . These experiments showed that the affinity constant of the Fv fragment is identical to that of the native antibody McPC603, that there is one binding site for phosphorylcholine in the Fv fragment, and that there is no inactive protein in the preparation . This expression system should facilitate future protein engineering experiments on antibodies.

J Mol Biol, 1988 May 20, 201(2), 455 - 7
Crystallization and preliminary X-ray investigation reveals that tumor necrosis factor is a compact trimer furnished with 3-fold symmetry; Hakoshima T et al.; Crystals of recombinant human tumor necrosis factor produced by Escherichia coli have been obtained under different conditions . Crystals suitable for X-ray studies are produced by a vapor diffusion technique using sodium phosphate as both precipitant and buffer at pH 6.5 . The crystals belong to the cubic space group, P2(1)3 with unit cell dimensions a = b = c = 95.7 A (1 A = 0.1 nm) . Preliminary photography reveals that the crystals are moderately stable to X-rays and diffract to at least 3 A resolution . The diffraction data for native crystals have been collected on a diffractometer at 3 A resolution . Another crystal form, which appeared in a solution containing sodium phosphate at pH 8.0, has the trigonal space group P3 with unit cell dimensions a = b = 63.8 A and c = 54.4 A, and produces measurable reflections to a resolution of 3 A . Hexagonal crystals also have been obtained by the use of polyethylene glycol as precipitant in the range pH 7.6 to 8.0; however, the crystals are fragile and unstable to X-rays . Conservation of 3-fold symmetry in the different crystal forms obtained could reflect the ability of tumor necrosis factor molecules to form trimers in solution and probably the nature of binding of the molecules to cellular receptors.

J Mol Biol, 1988 May 20, 201(2), 393 - 404
Three-dimensional structure of 50 S Escherichia coli ribosomal subunits depleted of proteins L7/L12; Carazo JM et al.; A structural study of Escherichia coli 50 S ribosomal subunits depleted selectively of proteins L7/L12 and visualized by low-dose electron microscopy has been carried out by multivariate statistical analysis, classification schemes and the new reconstruction technique from single-exposure, random-conical tilt series . This approach has allowed us to solve the three-dimensional structure of the depleted 50 S subunits at a resolution of 3 nm-1 . In addition, two distinct morphological populations of subunits (cores) have been identified in the electron micrographs analyzed and have been separately studied in three dimensions . Depleted subunits in the two morphological states present as main features common to these two structures but different from those of the non-depleted subunit (1) the absence of the stalk, (2) a rearrangement of the stalk-base that changes the overall structure of this region . This morphological change is quite noticeable and important, since this region is mapped as a part of the GTPase center . The two conformations differ mainly in the orientation of the area between the L1 region and the head (the probable localization of the peptidyl transferase center) and in the accessibility of the region located below the head . A possible relationship of these structural changes to the functional dynamics of the ribosome is suggested.

J Mol Biol, 1988 May 20, 201(2), 261 - 71
Functional sites of the Ada regulatory protein of Escherichia coli . Analysis by amino acid substitutions; Takano K et al.; Specific cysteine residues at possible methyl acceptor sites of the Ada protein of Escherichia coli were converted to other amino acids by site-directed mutagenesis of the cloned ada gene of E . coli . Ada protein with the cysteine residue at 321 replaced by alanine was capable of accepting the methyl group from the methylphosphotriester but not from O6-methylguanine or O4-methylthymine of alkylated DNA, whereas the protein with alanine at position 69 accepted the methyl group from the methylated bases but not from the methylphosphotriester . These two mutants were used to elucidate the biological significance of repair of the two types of alkylation lesions . Introduction of the ada gene with the Ala69 mutation into an ada- cell rendered the cell more resistant to alkylating agents with respect to both killing and induction of mutations, but the gene with the Ala321 mutation exhibited no such activity . Replacement of the cysteine residue at position 69, but not at position 321, abolished the ability of Ada protein to promote transcription of both ada and alkA genes in vitro . These results are compatible with the idea that methylation of the cysteine residue at position 69 renders Ada protein active as a transcriptional regulator, whilst the cysteine residue at position 321 is responsible for repair of pre-mutagenic and lethal lesions in DNA . The actions of mutant Ada proteins on the ada and alkA promoters in vivo were investigated using an artificially composed gene expression system . When the ada gene with the Ala69 mutation was introduced into the cell, there was little induction of expression of either the ada or the alkA genes, even after treatment with an alkylating agent, in agreement with the data obtained from studies in vitro . With the Ala321 mutation, however, a considerable degree of ada gene expression occurred without adaptive treatment . The latter finding suggests that the cysteine residue at position 321, which is located near the C terminus of the Ada protein, is involved in regulating activity, as the transcriptional activator.

Biochemistry, 1988 May 17, 27(10), 3842 - 9
Interaction of tRNAs and of phosphorothioate-substituted nucleic acids with an organomercurial . Probing the chemical environment of thiolated residues by affinity electrophoresis; Igloi GL; The interactions of 4-thiouridine and 5-{(methylamino)methyl}-2-thiouridine in tRNA and of phosphorothioate esters in nucleic acids with an organomercurial have been investigated . For this purpose, an affinity electrophoretic system has been developed in which the mercury derivative has been covalently immobilized in a standard polyacrylamide gel . The retardation of thiolated macromolecules was found to be sensitive to the chemical environment of the sulfur atom, giving characteristic interaction constants dependent on the nature of the modification and its accessibility to binding . The interaction could, in the case of tRNA, be abolished by conventional specific chemical modification of the thiolated bases, as well as by irradiation with 32P-derived beta-emission . Not only has the fractionation of sulfur-modified from unmodified species been attained but a quantitative application of the technique has made it possible to study the binding of mercury and, by competition, that of magnesium in terms of the conformation of tRNA.

Biochemistry, 1988 May 17, 27(10), 3664 - 71
Expression and site-directed mutagenesis of human dihydrofolate reductase; Prendergast NJ et al.; A procaryotic high-level expression vector for human dihydrofolate reductase has been constructed and the protein characterized as a first step toward structure-function studies of this enzyme . A vector bearing the tac promoter, four synthetic oligodeoxynucleotides, and a restriction fragment from the dihydrofolate reductase cDNA were ligated in a manner which optimized the transcriptional and translational frequency of the enzyme mRNA . The reductase, comprising ca . 17% of the total soluble protein in the host bacteria, was purified to apparent homogeneity as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and characterized by amino acid composition, partial amino acid sequence, and steady-state kinetic analysis . This expression vector has been used as a template for double-stranded plasmid DNA site-specific mutagenesis . Functional studies on a Cys-6----Ser-6 mutant enzyme support the contention that Cys-6 is obligatory for organomercurial activation of human dihydrofolate reductase . The Ser-6 mutant enzyme was not activated to any extent following a 24-h incubation with p-(hydroxymercuri)benzoate and nicotinamide adenine dinucleotide phosphate (reduced) (NADPH), whereas the kcat for Cys-6 reductase increased 2-fold under identical conditions . The specific activities of the Cys-6 and Ser-6 enzymes were virtually identical as determined by methotrexate titration as were the Km values for both dihydrofolate and NADPH . The Ser-6 mutant showed a decreased temperature stability and was more sensitive to inactivation by alpha-chymotrypsin when compared to the wild-type enzyme . These results suggest that the Ser-6 mutant reductase is conformationally altered relative to the Cys-6 native enzyme.

Biochemistry, 1988 May 17, 27(10), 3834 - 42
Determination of the rate-limiting segment of aminoglycoside nucleotidyltransferase 2''-I by pH and viscosity-dependent kinetics; Gates CA et al.; Aminoglycoside nucleotidyltransferase 2''-I follows a Theorell-Chance kinetic mechanism in which turnover is controlled by the rate-limiting release of the final product (Q), a nucleotidylated aminoglycoside {Gates, C . A., & Northrop, D . B . (1988) Biochemistry (second of three papers in this issue)} . The effects of viscosity on the kinetic constants of netilmicin, gentamicin C1, and sisomicin aminoglycoside substrates are as follows: no change in the substrate inhibition constants of all three antibiotics, a small but significant and highly unusual increase in Vmax/Km for netilmicin but large, normal decreases for gentamicin C1 and sisomicin, and marked decreases in the maximal velocities for all three . The lack of effect on substrate inhibition provides essential control experiments, signifying that glycerol does not interfere with binding of aminoglycosides to EQ and that the steady-state distribution of EQ does not increase as the release of Q is slowed by a viscosogen . The decrease in the Vmax/Km of better substrates indicates dominance by a diffusion-controlled component in the catalytic segment, attributed to the release of pyrophosphate . The presence of an increase in the Vmax/Km of the poor substrate, however, is inexplicable in terms of either single or multiple diffusion-controlled steps . Instead, it is here attributed to an equilibrium between conformers of the enzyme-nucleotide complex in which glycerol favors the conformation necessary for binding of aminoglycosides . The decrease in Vmax is consistent with the diffusion-controlled release of the final product determining enzymatic turnover.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1988 May 17, 27(10), 3850 - 7
Effect of apurinic/apyrimidinic endonucleases and polyamines on DNA treated with bleomycin and neocarzinostatin: specific formation and cleavage of closely opposed lesions in complementary strands; Povirk LF et al.; Bleomycin and neocarzinostatin induce modified apurinic/apyrimidinic (AP) sites by oxidation of the sugar moiety in DNA . In order to quantitatively assess the susceptibility of these lesions to repair endonucleases, drug-treated 3H-labeled colE1 DNA was mixed with 14C-labeled heat-depurinated DNA, and endonuclease-susceptible sites in the mixture were titrated with various AP endonucleases or with polyamines . Single- and double-strand breaks were quantitated by determining the fractions of supercoiled, nicked circular, and linear molecules . Exonuclease III and endonucleases III and IV of Escherichia coli, as well as putrescine, produced a nearly 2-fold increase in single-strand breaks in bleomycin-treated DNA, indicating cleavage of drug-induced AP sites . The bleomycin-induced AP sites were comparable to heat-induced sites in their sensitivity to E . coli endonucleases III and IV but were cleaved by exonuclease III only at high concentrations . Bleomycin-induced AP sites were much more sensitive to cleavage by putrescine than heat-induced sites . Treatment with putrescine or very high concentrations of endonuclease III also increased the number of double-strand breaks in bleomycin-treated DNA, suggesting a minor class of lesion consisting of an AP site accompanied by a closely opposed break in the complementary strand . These complex lesions were resistant to cleavage by endonuclease IV . However, when colE1 DNA was treated with neocarzinostatin, subsequent treatment with putrescine, endonuclease IV, or very high concentrations of endonuclease III produced a dramatic increase in double-strand breaks but no detectable increase in single-strand breaks . These results suggest that virtually all neocarzinostatin-induced AP sites are accompanied by a closely opposed strand break.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochem Biophys Res Commun, 1988 May 16, 152(3), 1144 - 50
Phosphorylation of secreted forms of human beta 2-interferon/hepatocyte stimulating factor/interleukin-6; May LT et al.; "Beta 2-Interferon/hepatocyte stimulating factor/interleukin-6" (IFN-beta 2) has emerged as a major mediator of the plasma protein response to tissue injury (the acute phase response) in addition to its numerous effects on cells of the immune system . Human fibroblasts and monocytes induced with tumor necrosis factor, interleukin-1, bacterial lipopolysaccharide (endotoxin) or virus infection secrete multiple forms of differentially glycosylated IFN-beta 2 polypeptides: at least a doublet of molecular mass approximately 25 kD and a triplet of mass approximately 30 kD . We report that immunoprecipitation analyses of medium from {32P}orthophosphate- labeled cultures of induced fibroblasts carried out using a rabbit polyclonal antibody to recombinant E . coli-derived human IFN-beta 2 reveal that the secreted gp23-25 and gp28-30 forms of IFN-beta 2 are phosphorylated . IFN-beta 2 gp23-25 secreted by induced monocytes is phosphorylated whereas the monocytic gp28-30 is poorly labeled with {32P}orthophosphate suggesting tissue-specific differences in IFN-beta 2 phosphorylation . Phosphoamino acid analyses indicate that all of the detected phosphate is in phosphoserine residues . Furthermore, IFN-beta 2 can be completely dephosphorylated by alkaline phosphatase (E.C . No . 3.1.3.1); thus all of the phosphate label is in readily accessible sites . These observations suggest the possibility that differential phosphorylation of IFN-beta 2 forms may be a mechanism to modulate its functions in a tissue-specific manner.

J Immunol, 1988 May 15, 140(10), 3528 - 33
Characterization of a murine monoclonal antibody that detects a C-terminal fragment of the raf oncogene product; Giardina SL et al.; A murine mAb, STEGI 1, was generated against a 30-kDa raf protein purified from an Escherichia coli expression vector . Immunoblot analysis confirmed that this antibody recognized the original immunizing protein as well as a 44- to 48-kDa protein from several raf-transformed cell lines . Immunoprecipitation experiments isolated a 48-kDa protein from a cell line transfected with a c-raf construct as well as from normal NIH 3T3 fibroblasts . Parallel experiments with polyvalent antiserum prepared against E . coli-derived v-raf (C terminus)-precipitated proteins with apparent Mr of 48 and 74 kDa, as had been described previously . Immunofluorescence flow cytometry of raf-transformed cell lines revealed intense intracytoplasmic staining . This staining was specifically inhibited by preincubation of STEGI 1 with purified raf 30-kDa protein . It should now be possible to more easily assess the role of the raf oncogene product in malignant transformation.

Gene, 1988 May 15, 65(1), 101 - 10
Transcript mapping using {35S}DNA probes, trichloroacetate solvent and dideoxy sequencing ladders: a rapid method for identification of transcriptional start points; Aldea M et al.; A simple method for RNA transcript mapping has been developed that combines the use of 35S-labeled M13 DNA probes and the presence of high concentrations of sodium trichloroacetate in the hybridization buffer . These hybridization conditions permit the use of M13 probes without purification from the template . The dideoxy sequencing ladders used for sizing the protected DNA fragments are obtained from the same M13 templates utilized to synthesize the DNA probes . The method was tested by analyzing the transcripts controlled by lac, ptr and trxA promoters . Comparison of the results with previously published data obtained with the conventional S1 nuclease mapping technique indicated that the present method is just as precise and at least 50 times more sensitive . Clones constructed for sequencing a gene of interest can be used directly to identify transcriptional start points.

J Biol Chem, 1988 May 15, 263(14), 6625 - 30
The mechanism of glucose 6-phosphate transport by Escherichia coli; Sonna LA et al.; To evaluate anion exchange as the mechanistic basis of sugar phosphate transport, natural and artificial membranes were used in studies of glucose 6-phosphate (Glc-6-P) and inorganic phosphate (Pi) accumulation by the uhpT-encoded protein (UhpT) of Escherichia coli . Experiments with intact cells demonstrated that UhpT catalyzed the neutral exchange of internal and external Pi, and work with everted as well as right-side-out membrane vesicles showed further that UhpT mediated the heterologous exchange of Pi and Glc-6-P . When loaded with Pi, but not when loaded with morpholinopropanesulfonate (MOPS), everted vesicles took up Glc-6-P to levels 100-fold above medium concentration in a reaction unaffected by the ionophores valinomycin, valinomycin plus nigericin, and carbonyl cyanide p-trifluoromethoxyphenylhydrazone . Similarly, right-side-out vesicles were capable of Glc-6-P transport, but only if a suitable internal countersubstrate was available . Thus, in MOPS-loaded vesicles, oxidative metabolism established a proton-motive force that supported proline or Pi accumulation, but transport of Glc-6-P was found only if vesicles could accumulate Pi during a preincubation . After reconstitution of UhpT into proteoliposomes it was possible to show as well that the level of accumulation of Glc-6-P (17 to 560 nmol/mg of protein) was related directly to the internal concentration of Pi . These results are most easily understood if the transport of glucose 6-phosphate in E . coli occurs by anion exchange rather than by nH+/anion support.

J Biol Chem, 1988 May 15, 263(14), 6570 - 8
DNA Polymerase III holoenzyme of Escherichia coli . IV . The holoenzyme is an asymmetric dimer with twin active sites; Maki H et al.; Pol III, a subassembly of Escherichia coli DNA polymerase III holoenzyme lacking only the auxiliary beta subunit, was purified to homogeneity by an improved procedure . This assembly consists of nine different polypeptides, likely in a 1:1 stoichiometry: a catalytic core (pol III) of alpha (132 kDa), epsilon (27 kDa), and theta (10 kDa), and six auxiliary subunits: tau (71 kDa), gamma (52 kDa), delta (35 kDa), delta' (33 kDa), chi (15 kDa), and psi (12 kDa) . The assembly behaves on gel filtration as a particle of about 800 kDa, indicating a content of two each of the subunits . A new procedure for purifying the core yielded a novel dimeric form which may provide the foundation for the dimeric nature of the more complex pol III and holoenzyme forms . Pol III readily dissociates into several subassemblies including pol III', likely a dimeric core with two tau subunits . The holoenzyme, purified by a similar procedure with ATP and Mg2+ present throughout, retained the beta subunit (37 kDa) as well as all the subunits present in pol III; the mass of the holoenzyme was estimated to be 900 kDa . The isolated initiation complex of holoenzyme with a primed template DNA and the elongation complex (formed in the presence of three deoxynucleoside triphosphates) had the same composition and stoichiometry as observed for pol III with two beta dimers in addition . An initiation complex assembled from a mixture of monomeric pol III core, gamma 2 delta delta' chi psi complex (gamma complex), beta, and tau retained the core, one beta dimer, and two tau subunits but was deficient in the gamma complex . When tau was omitted from the assembly mixture, the initiation complex contained one or two gamma complexes instead of the tau subunit . Based on these data, pol III holoenzyme is judged to be an asymmetric dimeric particle with twin pol III core active sites and two different sets of auxiliary units designed to achieve essentially concurrent replication of both leading and lagging strand templates.

J Biol Chem, 1988 May 15, 263(14), 6561 - 9
DNA polymerase III holoenzyme of Escherichia coli . III . Distinctive processive polymerases reconstituted from purified subunits; Maki S et al.; The 10 distinctive polypeptides of DNA polymerase III holoenzyme, purified as individual subunits or complexes, could be reconstituted to generate a polymerase with the high catalytic rate of the isolated intact holoenzyme . Functions and interactions of the subunits can be inferred from partial assemblies of the pol III core (alpha, epsilon, and theta subunits) with auxiliary subunits . The core possesses the polymerase and proofreading activities; the auxiliary subunits provide the core with processivity, the capacity to replicate long stretches of DNA without dissociating from the template . In a sequence of reconstruction steps, the beta subunit binds the primed template in an ATP-dependent manner through the catalytic action of a complex made up of the gamma, delta, delta', chi, and psi polypeptides . With the beta subunit in place, a processive polymerase is produced upon addition of the core . When the tau subunit is lacking, binding of polymerase to the primed template is less efficient and stable . The tau-less reconstituted polymerase is more prone to dissociation upon encountering secondary structures in the template in its path, such as a hairpin region in the single strand or a duplex region formed by a strand annealed to the template . With the tau subunit present, the interaction of the core.beta complex (the basic unit of a processive polymerase) with the primed template is strengthened . The tau-containing reconstituted polymerase can replicate DNA continuously through secondary structures in the template . The two distinctive kinds of processivity demonstrated by the tau-less and tau-containing reconstituted polymerases fit nicely into a scheme in which, organized as an asymmetric dimeric holoenzyme, the tau half is responsible for continuous synthesis of one strand, and the less stable half for discontinuous synthesis of the other.

J Biol Chem, 1988 May 15, 263(14), 6555 - 60
DNA polymerase III holoenzyme of Escherichia coli . II . A novel complex including the gamma subunit essential for processive synthesis; Maki S et al.; Processive DNA synthesis, a property of DNA polymerase III holoenzyme of Escherichia coli, was not achieved by combining the pol III core (alpha, epsilon, and theta subunits) and the beta and gamma subunits . An activity that restored processivity to these subunits was found in crude extracts and was overproduced 4-fold in cells with plasmids amplifying the tau and gamma subunits . Purified to homogeneity, the activity, assayed by reconstitution of processivity, was represented by five polypeptides which were copurified . Judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, these correspond to the known subunits gamma (52 kDa) and delta (35 kDa) and to three new polypeptides: delta' (33 kDa), chi (15 kDa), and psi (12 kDa) . The five polypeptides form a tight complex with a native molecular weight of about 200 kDa and a subunit stoichiometry of two gamma subunits to one each of the others . Processive DNA synthesis, now achieved with only three components (pol III core, beta, and the auxiliary complex), provides the opportunity to assess the functions of each and the contribution that the remaining auxiliary tau subunit makes to reconstitute a holoenzyme.

J Biol Chem, 1988 May 15, 263(14), 6547 - 54
DNA polymerase III holoenzyme of Escherichia coli . I . Purification and distinctive functions of subunits tau and gamma, the dnaZX gene products; Maki S et al.; Escherichia coli dnaZX, the gene which when mutant blocks DNA chain elongation, was cloned into a lambda PL promoter-mediated expression vector . In cells carrying this plasmid, the activity that complements a mutant dnaZ extract in replicating a primed single-stranded DNA circle was increased about 20-fold . Two polypeptides of 71 and 52 kDa were overproduced . Upon fractionation, two complementing activities were purified to homogeneity and proved to be the 71- and 52-kDa polypeptides . Immunoassays revealed their respective identities with the tau and gamma subunits of DNA polymerase III holoenzyme . The N-terminal amino acid sequences of the first 12 residues were identical in both subunits, as were their molar specific activities in dnaZ complementation . Thus, the tau subunit complements the defect in the mutant holoenzyme from the dnaZts strain as efficiently as does the gamma subunit . Inasmuch as the 71-kDa subunit (tau) can also overcome the enzymatic defect in a dnaX mutant strain, this polypeptide has dual replication functions, only one of which can be performed by the gamma subunit . Availability of pure tau and gamma subunits for study has provided the basis for proposing an asymmetry in the structure and function of a dimeric DNA polymerase III holoenzyme.

J Biol Chem, 1988 May 15, 263(14), 6647 - 55
Glucose permease of Escherichia coli . The effect of cysteine to serine mutations on the function, stability, and regulation of transport and phosphorylation; Nuoffer C et al.; The glucose permease (IIGlc/IIIGlc complex) of the bacterial phosphotransferase system mediates sugar transport across the cytoplasmic membrane concomitant with sugar phosphorylation . It contains 3 cysteine residues, of which Cys-204 and Cys-326 are localized in the hydrophobic part and Cys-421 in the hydrophilic part of the IIGlc subunit . The cysteines were replaced, one at a time, by serines, and the effect of these mutations on stability, regulation, and catalytic properties of IIGlc was investigated in vivo and in vitro . Cys-204 and Cys-326 are not required for catalytic function and are not involved in the membrane potential-dependent regulation of IIGlc activity (Robillard, G . T., and Konings, W . N . (1982) Eur . J . Biochem . 127, 597-604) . Replacement of these cysteines by serines results, however, in reduced stability of IIGlc in vivo (C204S) and in vitro (C204S and C326S), indicating that these substitutions in a hydrophobic environment can destabilize the protein structure . Cys-421 is absolutely required for transport and phosphorylation of glucose . C421S can neither be phosphorylated by phospho-IIIGlc nor catalyze the phosphoryl exchange between {14C} glucose and glucose 6-phosphate at equilibrium . C421S does not interfere with the activity of simultaneously expressed wild-type IIGlc . Unexpectedly C421S and wild-type IIGlc support growth on maltose of Escherichia coli ZSC112L (Curtis, S . J., and Epstein, W . (1975) J . Bacteriol . 122, 1189-1199), a strain which otherwise does not grow on this disaccharide as the only carbon source . C421S appears to facilitate the efflux of a growth inhibiting intermediate (glucose?) of maltose . Wild-type IIGlc catalyzes the intracellular phosphorylation of glucose derived from maltose . It is concluded that the cytoplasmic domain of IIGlc interacts with IIIGlc, the cytoplasmic subunit of the glucose permease, and also participates in phosphorylation of glucose, and that phosphorylation occurs independently of transport, although transport of glucose by wild-type IIGlc cannot occur without concomitant phosphorylation.

Carbohydr Res, 1988 May 15, 176(2), 231 - 6
Structural studies of the Escherichia coli O-149 O-antigen polysaccharide; Adeyeye A et al.; The structure of the O-antigen polysaccharide from Escherichia coli O-149 has been investigated; methylation analysis, partial hydrolysis with acid, and n.m.r . spectroscopy were the principal methods used . It is concluded that the polysaccharide is composed of trisaccharide repeating-units having the following structure . (Formula: see text) . The absolute configuration at the acetalic carbon atom of the pyruvic acid residue is S.

Gene, 1988 May 15, 65(1), 141 - 7
Molecular cloning and expression in Escherichia coli of the cDNA coding for rat lipocortin I (calpactin II); Shimizu Y et al.; Lipocortins (LC) are a family of proteins that were initially described to be induced by glucocorticosteroids and to inhibit phospholipase A2 (PLA2) . Using oligodeoxynucleotide probes corresponding to partial amino acid (aa) sequences of rat lipocortin I (LCI), we have isolated a cDNA clone for rat LCI from a cDNA library prepared from poly(A)+RNA of peritoneal cells of dexamethasone-treated rat . The cDNA insert (1355 bp) had an open reading frame of 1038 bp that encoded a 346-aa polypeptide (Mr 38,784) . The nucleotide sequence and the amino acid sequence deduced from it showed high homology with the reported sequences of human LCI . A plasmid containing the trc promoter and cDNA sequence for 346 aa residues of the rat LCI was constructed and expressed in Escherichia coli . Antibody to human LCI crossreacted with the recombinant rat LCI, and the recombinant protein had characteristics of natural rat LCI including PLA2 inhibitory activity in vitro.

J Biol Chem, 1988 May 15, 263(14), 6872 - 6
Neurospora tryptophan synthase . Characterization of the pyridoxal phosphate binding site; Pratt ML et al.; Tryptophan synthase, which catalyzes the final step of tryptophan biosynthesis, is a multifunctional protein that requires pyridoxal phosphate for two of its three distinct enzyme activities . Tryptophan synthase from Neurospora crassa, a homodimer of two 75-kDa subunits, was shown to bind 1 mol of pyridoxal phosphate/mol of subunit with a calculated dissociation constant for pyridoxal phosphate of 1.1 microM . The spectral properties of the holoenzyme, apoenzyme, and reconstituted holoenzyme were characterized and compared to those previously established for the heterotetrameric (alpha 2 beta 2) enzyme from Escherichia coli . The Schiff base formed between pyridoxal phosphate and the enzyme was readily reduced by sodium borohydride, but not sodium cyanoborohydride . The active site residue that binds pyridoxal phosphate, labeled by reduction of the Schiff base with tritium-labeled sodium borohydride, was determined to be lysine by high performance liquid chromatography analysis of the protein hydrolysate . A 5400-dalton peptide containing the reduced pyridoxal phosphate moiety was generated by cyanogen bromide treatment, purified and sequenced . The sequence is 85% homologous with the corresponding sequence obtained for yeast tryptophan synthase (Zalkin, H., and Yanofsky, C . (1982) J . Biol . Chem . 257, 1491-1500); the lysine derivatized by pyridoxal phosphate is located at the same relative position as that in the yeast and E . coli enzymes.

J Biol Chem, 1988 May 15, 263(14), 6643 - 6
Protease So from Escherichia coli preferentially degrades oxidatively damaged glutamine synthetase; Lee YS et al.; After oxidative damage (e.g . induced with iron, ascorbate, and oxygen), the inactivated glutamine synthetase is selectively hydrolyzed in extracts of Escherichia coli . We therefore tested if glutamine synthetase treated with this system is hydrolyzed preferentially by any of the known E . coli proteases . Protease So, a cytoplasmic serine protease, was found to degrade the oxidized form of glutamine synthetase to acid-soluble peptides 5-10 times faster than the native glutamine synthetase . Degradation of the oxidized glutamine synthetase was inhibited by EDTA and stimulated 5-10-fold by Mg2+, Ca2+, or Mn2+, even though casein hydrolysis by protease So is not affected by divalent cations . Apparently, these cations affect the conformation of this substrate, making it more susceptible to proteolytic attack . Protease Re, another cytoplasmic protease, also degrades preferentially the oxidized form of glutamine synthetase and seems to correspond to the glutamine synthetase-degrading activity recently described by Roseman and Levine {1987) J . Biol . Chem . 262, 2101-2110) . However, it is much less active in this reaction than protease So . No other soluble E . coli protease, including Do, Ci, Mi, Fa, Pi, or the ATP-dependent proteases Ti and La (the lon product), appears to degrade this oxidized protein . These results suggest that protease So participates in the hydrolysis of oxidatively damaged proteins and that E . coli has multiple systems for degrading different types of aberrant proteins.

J Biol Chem, 1988 May 15, 263(14), 6606 - 12
Interaction between Glu-219 and His-245 within the a subunit of F1F0-ATPase in Escherichia coli; Cain BD et al.; Oligonucleotide-directed mutagenesis was used to generate mutations in the a subunit gene (uncB) altering the glutamic acid 219 and the histidine 245 codons . Substitutions of aspartic acid, glutamine, histidine, and leucine for glutamic acid at position 219 neither alter the hydrolytic activity of membrane-bound F1 nor the association of F1 with F0 . However, the efficiency of F0-mediated proton translocation was reduced to varying degrees . Replacement of glutamic acid 219 with leucine reduced the ATP-driven proton pumping activity of intact F1F0 to undetectable levels . Roughly 5% of normal activity was observed when glutamine occupied position 219 . Surprisingly higher activity, approaching 20% of wild type levels, is seen when histidine replaced glutamic acid 219 . The aspartic acid substitution resulted in little loss of enzyme function . Substitution of glutamic acid for histidine 245 results in a reduction to about 45% of normal proton translocation . Construction of the double mutant with substitution of histidine at position 219 and glutamic acid at position 245 yields a complex with better proton translocation than with either mutant separately . The possibility that a functionally important interaction between histidine 245 and glutamic acid 219 of the a subunit may be directly involved in the proton translocation mechanism of F1F0-ATP synthase is discussed.

J Biol Chem, 1988 May 15, 263(14), 6599 - 605
Mutagenesis of the alpha subunit of the F1Fo-ATPase from Escherichia coli . Mutations at Glu-196, Pro-190, and Ser-199; Vik SB et al.; In an attempt to identify amino acid residues involved in proton translocation by the Fo sector of the Escherichia coli F1Fo-ATPase, 16 mutations at the carboxyl-terminal third of the a subunit have been isolated, and their phenotypes have been partially characterized . Thirteen mutations were constructed by "cassette" mutagenesis at two highly conserved residues, aglu196 and apro190 . Two mutations were products of oligonucleotide-directed mutagenesis of a portion of of oligonucleotide-directed mutagenesis of a portion of the uncB gene cloned into an M13 vector . One mutation was isolated after in vitro mutagenesis of the entire uncB gene in a plasmid vector with hydroxylamine . Amino acid substitutions for aglu196 (Asp, Gln, His, Asn, Lys, Ala, Ser, Pro) affect ATP-driven proton translocation and passive proton permeability by Fo to varying extents, but do not prevent growth on minimal succinate media . Amino acid substitutions of glutamine or arginine for apro190 affect F1Fo-ATPase assembly and eliminate ATP-driven proton translocation, while the substitution of asparagine at this position does not significantly affect either assembly or proton translocation . The substitution of amino acids threonine or alanine for aser199 causes no detectable phenotypic change from wild type . These and other mutations are discussed in terms of the assembly, structure, and function of the a subunit . It is concluded that aglu196 and apro190 are not obligate components of the proton channel, but that they affect proton translocation indirectly.

Biochem J, 1988 May 15, 252(1), 39 - 45
Plant holo-(acyl carrier protein) synthase; Elhussein SA et al.; 1 . An improved method was developed for the assay of plant holo-(acyl carrier protein) synthase activity, using Escherichia coli acyl-(acyl carrier protein) synthetase as a coupling enzyme . 2 . Holo-(acyl carrier protein) synthase was partially purified from spinach (Spinacia oleracea) leaves by a combination of (NH4)2SO4 fractionation and anion-exchange and gel-permeation chromatography . 3 . The partially purified enzyme had a pH optimum of 8.2 and Km values of 2 microM, 72 microM and 3 mM for apo-(acyl carrier protein), CoA and Mg2+ respectively . Synthase activity was inhibited in vitro by the reaction product 3',5'-ADP . 4 . Results from the fractionation of spinach leaf and developing castor-oil-seed (Ricinus communis) endosperm cells were consistent with a cytosolic localization of holo-(acyl carrier protein) synthase activity in plant cells.

Gene, 1988 May 15, 65(1), 93 - 9
Plasmid pACYC184 contains an ssi signal for initiation of single-strand phage DNA replication; Bahk JD et al.; Using the plaque assay system for screening the single-strand (ss) initiation determinant (ssi) sequences, we have found that 119-bp region in pACYC184, a derivative of the plasmid P15A of Escherichia coli, can direct such ss DNA initiation . This region is located downstream from the P15A origin of replication and conserves consensus sequences of the ssi signals found in the other plasmids . Signals for ss DNA initiation are defined as nucleotide sequences present on ss DNA templates and required for priming DNA synthesis . The direction of chain elongation in DNA synthesis is opposite to that of the leading strand . In this region, we found a potential stem-and-loop structure . The 119-bp DNA segment of plasmid pACYC184 cloned in f1R199 filamentous phage could direct rifampicin-resistant conversion of the ss DNA to the double-stranded replicative form.

Gene, 1988 May 15, 65(1), 129 - 33
A simple and rapid method for the selection of oligodeoxynucleotide-directed mutants; Vandeyar MA et al.; We describe an in vitro selection procedure for oligodeoxynucleotide-directed mutagenesis, which produces mutants at frequencies of greater than 90%, facilitating the identification of mutants directly by nucleotide sequencing . The method is based on the selective methylation of the mutant strand by the incorporation of 5-methyl-dCTP . Restriction endonuclease digestion of the resulting hemimethylated DNA with MspI results in the nicking of only the nonmethylated-parental strand . The parental strand is removed by treatment with exonuclease III . The mutants are recovered by transformation of a mcrAB strain of Escherichia coli with the nascent strand.

Gene, 1988 May 15, 65(1), 123 - 8
A dominant selectable marker for the construction of recombinant poxviruses; Boyle DB et al.; Mycophenolic acid has been shown to be a potent inhibitor of vaccinia virus growth . By inserting the Escherichia coli xanthine-guanine phosphoribosyl transferase gene (gpt) into the vaccinia virus genome under control of the P-7.5 promoter this inhibition was overcome . When coupled in tandem with another gene of interest, recombinant vaccinia viruses can be positively selected carrying both genes . Since the gpt gene operates as a selectable marker in most mammalian cells it will be useful as a dominant selectable marker for the construction of recombinant viruses based on other host-specific poxviruses.

J Biol Chem, 1988 May 15, 263(14), 6829 - 35
Mispair specificity of methyl-directed DNA mismatch correction in vitro; Su SS et al.; To evaluate the substrate specificity of methyl-directed mismatch repair in Escherichia coli extracts, we have constructed a set of DNA heteroduplexes, each of which contains one of the eight possible single base pair mismatches and a single hemimethylated d(GATC) site . Although all eight mismatches were located at the same position within heteroduplex molecules and were embedded within the same sequence environment, they were not corrected with equal efficiencies in vitro . G-T was corrected most efficiently, with A-C, C-T, A-A, T-T, and G-G being repaired at rates 40-80% of that of the G-T mispair . Correction of each of these six mispairs occurred in a methyl-directed manner in a reaction requiring mutH, mutL, and mutS gene products . C-C and A-G mismatches showed different behavior . C-C was an extremely poor substrate for correction while repair of A-G was anomalous . Although A-G was corrected to A-T by the mutHLS-dependent, methyl-directed pathway, repair of A-G to C-G occurred largely by a pathway that is independent of the methylation state of the heteroduplex and which does not require mutH, mutL, or mutS gene products . Similar results were obtained with a second A-G mismatch in a different sequence environment suggesting that a novel pathway may exist for processing A-G mispairs to C-G base pairs . As judged by DNase I footprint analysis, MutS protein is capable of recognizing each of the eight possible base-base mismatches . Use of this method to estimate the apparent affinity of MutS protein for each of the mispairs revealed a rough correlation between MutS affinity and efficiency of correction by the methyl-directed pathway . However, the A-C mismatch was an exception in this respect indicating that interactions other than mismatch recognition may contribute to the efficiency of repair.

Gene, 1988 May 15, 65(1), 13 - 22
Alteration of amino-terminal codons of human granulocyte-colony-stimulating factor increases expression levels and allows efficient processing by methionine aminopeptidase in Escherichia coli; Devlin PE et al.; We have improved the expression of recombinant human granulocyte-colony-stimulating factor (G-CSF), produced by either pL or trpP expression vectors in Escherichia coli, by altering the sequence at the 5' end of the G-CSF-coding region . Initial attempts to express G-CSF resulted in neither detectable G-CSF mRNA nor protein in the trpP system, and only G-CSF mRNA was detectable in the pL system . We modified both expression vectors to decrease the G + C content of the 5' end of the coding region without altering the predicted amino acid sequence . This resulted in expression of detectable G-CSF mRNA and protein in both systems . Expression reached 17% and 6.5% of the total soluble cellular protein in the pL and trpP expression systems, respectively . The N-terminal sequence of the recombinant G-CSF from the pL system was Met-Thr-Pro-Leu-Gly-Pro- . G-CSF isolated from several human cell lines (including the LD-1 cell line reported here), does not have an N-terminal methionyl residue . Deletion of the threonine codon at the beginning of the coding region for the mature G-CSF resulted in efficient removal of the N-terminal methionine residue during expression in E . coli.

J Immunol, 1988 May 15, 140(10), 3568 - 72
Nonpolymorphic regions of p190, a protein of the Plasmodium falciparum erythrocytic stage, contain both T and B cell epitopes; Sinigaglia F et al.; Two conserved regions from the genetically polymorphic p190 molecule of the malaria parasite Plasmodium falciparum have previously been expressed in Escherichia coli as separate polypeptides (190.L and 190.M) or as a single fusion protein (190.N) . In the present study we investigated whether human B and T lymphocytes recognize these conserved regions . The more amino-terminal region, 190.L (corresponding to residues 188-363 of the encoded protein sequence) reacted preferentially with sera from donors living in a malaria-endemic area . Also, EBV-transformed B cells, from a healthy donor living in a malaria-mesoendemic area, were fused with a human-mouse hybrid line (SPM4-0), yielding two hybridomas whose products recognized both 190.L and the fusion protein 190.N, but not the 190.M polypeptide . A large number of p190-specific T cell clones were obtained from PBMC of a noninfected donor, after in vitro stimulation with the recombinant fusion protein 190.N . The clones reacted with intact, parasite-derived p190, as well as either 190.L or 190.M . Four clones that recognized the more amino-terminal fragment also responded to infected E . According to these results the more amino-terminal conserved sequences of p190 have the requisites to be immunogenic in humans.

Biochim Biophys Acta, 1988 May 13, 969(3), 217 - 24
The effects of endotoxin on platelet-activating factor synthesis in cultured rat glomerular mesangial cells; Wang J et al.; Platelet-activating factor (PAF), a potent vasoactive phospholipid, may contribute to acute renal failure and septic shock accompanying endotoxemia . Rat glomerular mesangial cells in culture synthesize PAF and contract after the addition of PAF . We thus investigated the potential of mesangial cells to respond to Escherichia coli lipopolysaccharide endotoxin with enhanced PAF synthesis in vitro . The mesangial cells were incubated with {3H}acetate, substrate for lyso-PAF: acetyl-CoA acetyltransferase, and endotoxin at different concentrations for various periods of time at 37 degrees C . Lipids were extracted and PAF was isolated by thin-layer chromatography . Endotoxin stimulated PAF generation in a time- and dose-related manner . Whereas most of the PAF was associated with the cells, endotoxin more than doubled the amount of PAF released into the extracellular medium as compared to control . Furthermore, the PAF-like material obtained from endotoxin-stimulated mesangial cells irreversibly aggregated washed rabbit platelets . This effect was lost after alkaline methanolysis and was totally blocked by L-652,731, a specific PAF-receptor antagonist . Finally, the PAF-like material exerted a hypotensive effect, which was abolished by L-652,731, when infused intravenously into healthy rats . These data indicate that rat glomerular mesangial cells have the ability to synthesize PAF in response to endotoxin . This suggests that PAF, so generated within the glomerulus, may contribute to acute decrements of glomerular filtration rate in endotoxemia.

Nature, 1988 May 12, 333(6169), 140 - 5
A simple structural feature is a major determinant of the identity of a transfer RNA; Hou YM et al.; Analysis of a series of mutants of an Escherichia coli alanine transfer RNA shows that substitution of a single G-U base pair in the acceptor helix eliminates aminoacylation with alanine in vivo and in vitro . Introduction of that base pair into the analogous position of a cysteine and a phenylalanine transfer RNA confers upon each the ability to be aminoacylated with alanine . Thus, as little as a single base pair can direct an amino acid to a specific transfer RNA.

Nucleic Acids Res, 1988 May 11, 16(9), 4111 - 20
Codon preference reflects mistranslational constraints: a proposal; McPherson DT; Following the observation of lysine for arginine misincorporation at the poor choice codon arg-AGA, a comparison of codon usage patterns for highly expressed mRNA's in E . coli provides a basis for the proposal that the major codon preference is subject to mistranslational constraints . In addition, the codons are utilized, as well as arranged, to provide a hydropathically conservative amino acid as the most probable replacement resulting from a mistranslational event.

Nucleic Acids Res, 1988 May 11, 16(9), 3829 - 43
The integration host factor of Escherichia coli binds to multiple sites at plasmid R6K gamma origin and is essential for replication; Filutowicz M et al.; Examination of the effect of the himA and himD mutants of E . coli on the maintenance of plasmid R6K has revealed that the gamma origin-containing replicons cannot be established in any of the mutants deficient in the production of E . coli Integration Host Factor (IHF) . Contrary, the R6K derivatives containing other origins of the plasmid (alpha and/or beta) replicate in a host lacking functional IHF protein . We show that IHF protein binds specifically to a segment of the replication region which is essential for the activity of all three R6K origins . Mapping the IHF binding sequence with neocarzinostatin showed that the protein protects three segments of the origin: two strong binding sites reside within an AT-rich block, while the third, considerably weaker site is separated from the other two by a cluster of the seven 22 bp direct repeats . These seven repeats have been shown previously to bind the R6K-encoded initiator protein pi . We also demonstrate that the establishment of pi-origin complexes prior to IHF addition prevents the binding of the IHF protein to the gamma origin . The binding sequences of IHF and pi proteins do not overlap, therefore, we propose that the binding of pi protein alters the structure of the DNA and thereby prevents the subsequent binding of IHF protein.

Nucleic Acids Res, 1988 May 11, 16(9), 3931 - 49
Bends in SV40 DNA: use of mutagenesis to identify the critical bases involved; Milton DL et al.; Five fragments of DNA exhibiting sequence directed bends were isolated from the Simian Virus 40 genome using a two-dimensional polyacrylamide gel fractionation . The bend sites were mapped for each fragment using the circular permutation test . All five sites have multiple, short runs of A residues with helical spacing typical of other bent fragments . Base pairs important for the bends were determined for one fragment by utilizing a random, single base pair mutagenesis . Of 28 mutants with decreased or increased bends, 14 had alterations that could be interpreted to affect the spaced runs of A residues, supporting their role in bends as predicted by the ApA wedge model . One major mutation was not explainable by existing models . The remaining minor mutations may only be due to small, local DNA conformational changes in the surrounding B-DNA.

Nucleic Acids Res, 1988 May 11, 16(9), 3705 - 20
The cloning, purification and characterization of the Eco RV modification methylase; Nwosu VU et al.; The gene for the Eco RV methylase has been cloned into a plasmid under control of the strong lambda PL promoter and overexpressed in E . coli . This plasmid, pVIC1, gives reliable overexpression of the methylase at levels of about 20% of total protein . Maximum yields of soluble protein are achieved after about 6 hours of induction . If the cells are harvested later than this much of the enzyme is found in the pellet fraction following centrifugation . A two column purification scheme using phosphocellulose and Blue-Sepharose chromatography has been developed . This yielded pure methylase in amounts of 5mg per gram E . coli cell paste . The enzyme is monomeric and methylates the first deoxyadenosine residue in its recognition sequence GATATC.

Nucleic Acids Res, 1988 May 11, 16(9), 4041 - 52
Close relationship between non-viral retroposons in Drosophila melanogaster; Di Nocera PP; G elements constitute one of the several moderately repeated DNA families of the Drosophila melanogaster genome . G elements lack terminal repetitions and structurally resemble mammalian processed pseudogenes because they terminate at one end in oligo-A tracts of variable length . G elements are mostly interspersed in the chromocentric heterochromatin with other repeated DNA sequences . Nucleotide sequence analysis of G3A, a family member inserted in a non-nucleolar rDNA unit, shows that functional G elements might have coding capacity for two polypeptides; one has homology to reverse transcriptases, the other is reminiscent of RNA binding proteins derived from the cleavage of retroviral gag polyproteins . Functionally related polypeptides are similarly encoded by members of two other Drosophila repeated DNA families, the F elements and the I factors . The similarity in structural organization and the relatedness of their potential gene products favors the hypothesis that G, F, and I sequences derive from a common ancestor and result from processes based on the reverse transcription of RNA intermediates that probably differ markedly from those ensuring the maintenance and dispersion of copia-like elements.

Nucleic Acids Res, 1988 May 11, 16(9), 4009 - 23
Autolytic processing of a phosphorothioate diester bond; Buzayan JM et al.; A small satellite RNA of tobacco ringspot virus replicates in tissues infected with tobacco ringspot virus and accumulates in virus capsids, forming virus-like particles . Previous research showed that multimeric forms of this satellite RNA have tandem repeats of the "monomeric" satellite RNA sequence of 359 or 360 nucleotide residues . The multimeric RNAs undergo autolytic processing at a specific CpA phosphodiester bond, the junction, to generate the monomeric RNA . We substituted phosphorothioate diester bonds for various sets of phosphodiester bonds, in dimeric and truncated forms of the satellite RNA . The degree of reduction in autolytic cleavage varied both with the sites of substitution and the size of the RNA molecules . Analyses of a product of the autolysis reaction suggest that one phosphorothioate diester bond most strongly interferes with processing, the one introduced at the CpA junction during its synthesis from adenosine-5'-0-(1-thiotriphosphate) . However, extensive introduction of phosphorothioate diester bonds elsewhere in the molecule also decreased processing, possibly by altering conformation.

Nucleic Acids Res, 1988 May 11, 16(9), 3739 - 49
Transcriptional terminators in the caa-cal operon and cai gene; Lloubes R et al.; We have analysed by S1 nuclease mapping the in vivo termination sites of transcription of the caa-cal operon and cai gene . The termination region for caa mRNA (T1A terminator) features characteristics of a rho-independent terminator . This terminator is a convergent transcription terminator, its complementary secondary structure being present at the 3'-end of cai mRNA . The caa-cal mRNA terminator (T2A terminator) has a stable potential secondary structure and shows homology with rho-dependent terminators . In vitro transcription of caa-cal operon demonstrated that the two terminators T1A and T2A are efficient . The 3'-ends of the mRNAs which end at T1A and T2A were analysed by S1 mapping with total RNA purified from a mutant strain deficient in exoribonuclease activities, in particular RNase II . The results suggest that the potential secondary structures of T1A and T2A are sufficiently stable to prevent 3'-end degradation by RNase II . On the other hand, the T2A terminator should be efficient enough to stop transcription through the downstream DNA region involved in pColA replication.

J Immunol Methods, 1988 May 9, 109(2), 245 - 52
Cytoplasmic localization of hepatitis B core antigen in hepatitis B virus infected livers; Sansonno DE et al.; Routine use of commercially available antisera against hepatitis B core antigen (HBcAg) obtained from Escherichia coli transfected with HBV-DNA, has permitted a re-evaluation of the histochemical distribution of the antigen in liver tissue . HBcAg, classically described almost exclusively in the nucleus, was found with a very high frequency in the cytoplasm of liver cells . Our data indicate, however, that formalin fixation and paraffin embedding destroy part of HBcAg antigenicity and eliminate most of its cytoplasmic expression . HBcAg was found in 6/7 (85.7%) of HBsAg/HBeAg positive subjects, and in 2/12 (16/6%) of HBsAg/anti-HBe positive subjects; in both subgroups the cytoplasmic expression of the antigen correlated with the presence of circulating hepatitis B virus-deoxyribonucleic acid (HBV-DNA).

Biochim Biophys Acta, 1988 May 9, 940(1), 21 - 32
Effect of acceptor membrane phosphatidylcholine on the catalytic activity of bovine liver phosphatidylcholine transfer protein; Runquist EA et al.; Protein-mediated transfer of phosphatidylcholine (PC) by bovine liver phosphatidylcholine transfer protein (PC-TP) was examined using a vesicle-vesicle assay system . Donor and acceptor membranes were prepared from Escherichia coli phospholipids and limiting amounts of egg yolk PC . PC transfer between vesicles of E . coli lipid/egg PC was markedly higher than transfer of PC from vesicles of E . coli lipid/egg PC to vesicles of E . coli lipid . Kinetic parameters of the interaction between PC-TP and E . coli lipid vesicles with or without PC was investigated . The apparent dissociation constants of the complex formed between PC-TP and these vesicles were determined kinetically and from double-reciprocal plots of intrinsic PC-TP fluorescence intensity increase versus vesicle concentration . The magnitude of the dissociation constant decreased as the PC content of the vesicles increased from 0 to 5 mol% . In addition, kinetic analysis revealed that the presence of PC in acceptor vesicles increased both the association and dissociation of PC-TP from vesicles . The effect of membrane PC molecules on transfer rates was examined using bis-phosphatidylcholine, a dimeric PC molecule which is not transferred by PC-TP . Rates of PC transfer to acceptor vesicles comprised of E . coli lipid/bis-PC were virtually identical to rates observed with acceptors vesicles prepared from E . coli lipid . The results suggest that transfer of PC by PC-TP is enhanced only when insertion of protein-bound PC occurs concurrently with the extraction of a molecule of membrane PC, i.e., a concerted, one-step catalytic mechanism for phospholipid exchange.

Science, 1988 May 6, 240(4853), 793 - 6
Changing the identity of a tRNA by introducing a G-U wobble pair near the 3' acceptor end; McClain WH et al.; Although the genetic code for protein was established in the 1960's, the basis for amino acid identity of transfer RNA (tRNA) has remained unknown . To investigate the identity of a tRNA, the nucleotides at three computer-identified positions in tRNAPhe (phenylalanine tRNA) were replaced with the corresponding nucleotides from tRNAAla (alanine tRNA) . The identity of the resulting tRNA, when examined as an amber suppressor in Escherichia coli, was that of tRNAAla.

J Mol Biol, 1988 May 5, 201(1), 41 - 55
Mutational analysis of the tRNA mimicry of brome mosaic virus RNA . Sequence and structural requirements for aminoacylation and 3'-adenylation; Dreher TW et al.; The genomic RNAs of brome mosaic virus (BMV) exhibit various tRNA-like properties, including specific tyrosylation by tyrosyl-tRNA synthetases and adenylation of the 3'-CCOH derivative by tRNA nucleotidyl transferases . We have studied the effect of numerous mutations in all domains of the tRNA-like structure of BMV RNA on tyrosylation and adenylation in vitro . Surprisingly few mutations resulted in more than 50% decrease in tyrosylation rates with either wheat germ or yeast synthetases; those mutations were at the 3' terminus, the pseudoknot, and the bases of arms B and E . The results suggest an interaction of synthetase with arm A as the analog of the aminoacyl acceptor stem of tRNAs, and arm B as the analog of the anticodon arm of tRNAs, although there is no apparent interaction with the terminal loop of arm B analogous to the interaction with the anticodon in tRNAs . Mutations at several loci resulted in large losses of adenylation activity catalyzed by wheat germ and Escherichia coli nucleotidyl transferases; those loci were the pseudoknot, the bases of arms B, C and D, and at the junctions of these arms with arm A . These studies have identified mutants specifically defective in one of the tRNA-like activities, which are appropriate for investigating the role of these activities during infection in vivo.

J Biol Chem, 1988 May 5, 263(13), 6109 - 14
Site-specific substitutions of the Tyr-165 residue in the catalytic chain of aspartate transcarbamoylase promotes a T-state preference in the holoenzyme; Wales ME et al.; The amino acid residue Tyr-165C of aspartate transcarbamoylase (EC 2.1.3.2) of Escherichia coli has been proposed to be involved in the transition from the T-state to the R-state upon binding of the bisubstrate analogue N-(phosphonacetyl)-L-aspartate . Site-specific mutagenesis has been used to substitute phenylalanine for tyrosine, thus maintaining the aromatic R-group but removing the charged hydroxyl moiety . This mutation dramatically altered the aspartate requirements for the holoenzyme but did not substantially affect the homotropic or heterotropic characteristics of the oligomer . The aspartate requirements for half-maximal saturation increased from 5.5 mM at pH 7.0 for the native holoenzyme to approximately 90 mM in the mutant enzyme . Nonetheless, estimates of the kinetic cooperativity index remained similar (Hill coefficients: Tyr-165C, n = 2.1; Phe-165C, n = 2.5) . CTP inhibited both enzymes approximately 70% and ATP activated approximately 40% at the aspartate concentrations required for half-maximal saturation (5 and 90 mM, respectively) . The maximal velocity of the mutant holoenzyme is almost identical to that of the wild-type enzyme . The phenylalanine substitution does not affect the stability of the holoenzyme to heat or mercurials, and the Vmax of the catalytic trimer was 444% greater than that of the holoenzyme . Upon dissociation of the wild-type native enzyme into catalytic trimers, the Vmax increased 450% . The Km for aspartate in the separated catalytic trimer is approximately 2-fold higher than for the native catalytic trimer (16.5 versus 8 mM at pH 7.0) . It is clear from the data that although Tyr-165C is not directly involved in the active site of the enzyme, it does play a pivotal role in catalytic transitions of the holoenzyme . In addition, the homotropic and heterotropic characteristics of the enzyme do not seem to be altered by the substitution of phenylalanine for Tyr-165C in the E . coli aspartate transcarbamoylase, although other substitutions have been reported (Robey, E . H., and Schachman, H . K . (1984) J . Biol . Chem . 259, 11180-11183) which show more complex effects.

J Biol Chem, 1988 May 5, 263(13), 6012 - 5
The membrane-associated ATPase from Sulfolobus acidocaldarius is distantly related to F1-ATPase as assessed from the primary structure of its alpha-subunit; Denda K et al.; Isolation of novel membrane-associated ATPases, presumably soluble parts of the H+-ATPases, from archaebacteria has been recently reported, and their properties were found to be significantly different from the usual F1-ATPase . In order to assess the relationship of the archaebacterial ATPases to the F1-ATPases and other known ATPases, the amino acid sequence of the alpha subunit of the ATPase from Sulfolobus acidocaldarius, an acidothermophilic archaebacterium, was compared with the sequences of other ATPases . The gene encoding its alpha subunit was cloned from the genomic library of S . acidocaldarius, and the nucleotide sequence was determined . The 591-amino acid sequence deduced from the nucleotide sequence contains a small number of short stretches that shows sequence similarity to the alpha and beta subunits of F1-ATPase . However, the overall similarity is too weak to consider it to be a typical member of the F1-ATPase family when the highly conserved sequences of the F1-ATPase subunits among various organisms are taken into account . Moreover, most of these stretches overlap the consensus sequences that are commonly found in some nucleotide-binding proteins . There is no significant sequence similarity to the ion-translocating ATPases, which form phosphorylated intermediates, such as animal Na+,K+-ATPases . Thus, the S . acidocaldarius ATPase and probably other archaebacterial ATPases also appear to belong to a new group of ion-translocating ATPases that has only a distant relationship to F1-ATPase.

J Biol Chem, 1988 May 5, 263(13), 6302 - 9
Post-Golgi apparatus localization and regional expression of rat intestinal sialyltransferase detected by immunoelectron microscopy with polypeptide epitope-purified antibody; Taatjes DJ et al.; During studies on the Golgi apparatus immunolocalization of beta-galactoside alpha 2,6-sialyltransferase in intestinal cells, immunostaining of a number of post-Golgi apparatus structures including mucus droplets and plasma membrane were observed . In order to determine if this labeling was in fact due to sialyltransferase and not carbohydrate-specific antibodies in the polyclonal antiserum preparation, fusion protein to sialyltransferase was used to epitope purify polypeptide-specific antibodies . The affinity purification was performed on a column containing a beta-galactosidase-sialyltransferase fusion protein expressed in Escherichia coli . Using such antibodies we present evidence that in intestinal cells sialyltransferase is not only present in the Golgi apparatus cisternal stack but also its transtubular network and various post-Golgi apparatus structures . In absorptive enterocytes, post-Golgi apparatus vesicles, the brush border and basolateral plasma membrane, multivesicular bodies, and lysosome-like structures were labeled . In goblet cells the limiting membrane and lumen of forming and mature mucus droplets as well as the plasma membrane exhibited label for sialyltransferase . The results provide evidence for "ecto-sialyltransferase" in the plasma membranes of these cells, and suggest that most of the sialyltransferase is released from the Golgi membranes and becomes secreted with the goblet cell mucus . In addition, the polypeptide epitope-purified antibody was also used to examine regional expression of sialyltransferase in the rat intestinal epithelium . Immunolabel was restricted to the large intestine and not found in duodenum, jejunum, and ileum . Direct measurement of the enzyme activity was found to correlate with the immunoelectron microscopic data . This observation suggests that there is regional specific expression of the beta-galactoside alpha 2,6-sialyltransferase.

Biochemistry, 1988 May 3, 27(9), 3521 - 7
Interaction of bovine mitochondrial ribosomes with Escherichia coli initiation factor 3 (IF3); Denslow ND et al.; Mammalian mitochondrial ribosomes are distinguished from their bacterial and eukaryotic-cytoplasmic counterparts, as well as from mitochondrial ribosomes of lower eukaryotes, by their physical and chemical properties and their high protein content . However, they do share more functional homologies with bacterial ribosomes than with cytoplasmic ribosomes . To search for possible homologies between mammalian mitochondrial ribosomes and bacterial ribosomes at the level of initiation factor binding sites, we studied the interaction of Escherichia coli initiation factor 3 (IF3) with bovine mitochondrial ribosomes . Bacterial IF3 was found to bind to the small subunit of bovine mitochondrial ribosomes with an affinity of the same order of magnitude as that for bacterial ribosomes, suggesting that most of the functional groups contributing to the IF3 binding site in bacterial ribosomes are conserved in mitochondrial ribosomes . Increasing ionic strength affects binding to both ribosomes similarly and suggests a large electrostatic contribution to the reaction . Furthermore, bacterial IF3 inhibits the Mg2+-dependent association of mitochondrial ribosomal subunits, suggesting that the bacterial IF3 binds to mitochondrial small subunits in a functional way.

Biochemistry, 1988 May 3, 27(9), 3512 - 20
Effects of an anti-beta monoclonal antibody on the interaction of the Escherichia coli RNA polymerase with the lac and TAC promoters; Rockwell P et al.; The effects of an inhibitory monoclonal antibody (mAb) raised against the beta subunit of the Escherichia coli RNA polymerase were determined on the kinetics and structural interactions during formation of the open promoter complex (RPo) . Analysis of the kinetics of abortive initiation on linear and supercoiled templates of the lac and TAC16 promoters showed that abortive synthesis by mAb 210E8-RNA polymerase varied as a function of DNA topology . A kinetic analysis of RPl formation on the supercoiled lac UV5 promoter showed that mAb 210E8 effected a slight alteration in the isomerization rate and no effect on the initial rate of RNA polymerase binding to the promoter . The potent inhibition of initiation with linear promoters by mAb 210E8 was not apparent when the promoters were assayed in their supercoiled forms . Abortive synthesis with the TAC16 promoter was accompanied by an mAb 210E8 induced hindrance of ApUpU but not UpGpU synthesis . The data indicate that the inhibition by mAb 210E8 with the supercoiled TAC16 promoter is further alleviated when the spacer length is shifted from 16 base pairs (ApUpU formation) to 18 base pairs (UpGpU formation) . When DNase I and dimethyl sulfate were used to probe DNA structure, mAb 210E8 was found to alter polymerase interactions with the lac promoter . DNase I footprinting indicated that the structural interactions for lac P+ promoter-RNA polymerase complexes were slightly altered in the presence of mAb 210E8 . Treatment of the RNA polymerase-lac UV5 complex with dimethyl sulfate revealed an alternate mode of RNA polymerase interaction with essential guanine contacts which was intermediate between a fully protected and free promoter.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1988 May 3, 27(9), 3325 - 30
Nitrogen isotope effects on glutamate decarboxylase from Escherichia coli; Abell LM et al.; The nitrogen isotope effect on the decarboxylation of glutamic acid by glutamate decarboxylase from Escherichia coli has been measured by comparison of the isotopic composition of the amino nitrogen of the product gamma-aminobutyric acid isolated after 10-20% reaction with that of the starting glutamic acid . At pH 4.7, 37 degrees C, the isotope effect is k14/k15 = 0.9855 +/- 0.0006 when compared to unprotonated glutamic acid . Interpretation of this result requires knowledge of the equilibrium nitrogen isotope effect for Schiff base formation . This equilibrium isotope effect is k14/k15 = 0.9824 for the formation of the unprotonated Schiff base between unprotonated valine and salicylaldehyde . Analysis of the nitrogen isotope effect on decarboxylation of glutamic acid and of the previously measured carbon isotope effect on this same reaction {O'Leary, M.H., Yamada, H., & Yapp, C.J . (1981) Biochemistry 20, 1476} shows that decarboxylation and Schiff base formation are jointly rate limiting . The enzyme-bound Schiff base between glutamate and pyridoxal 5'-phosphate partitions approximately 2:1 between decarboxylation and return to the starting state . The nitrogen isotope effect also reveals that the Schiff base nitrogen is protonated in this intermediate.

Biochemistry, 1988 May 3, 27(9), 3187 - 96
Renaturase and ribonuclease H: a novel mechanism that influences transcript displacement by RNA polymerase II in vitro; Kane CM; I have previously reported an activity in HeLa cells which facilitates transcript displacement by purified mammalian RNA polymerase II in vitro . I have shown that this activity copurifies with one of two separable ribonuclease (RNase) H activities in HeLa cells . The RNase H activity in question has characteristics similar to those reported for RNase H2b from calf thymus . RNase H proteins purified from several other sources including Escherichia coli also show renaturase activity . When the renaturase/RNase H protein is present during transcription by purified RNA polymerase II, transcripts are truncated close to the 5' end, and the remainder of the transcript is displaced normally from its template by the polymerase . Since RNA polymerase II dependent transcripts in vivo normally require the presence of the 5'-triphosphate terminus for capping, the in vivo significance of RNase H as a renaturase factor is presently not understood . However, the in vitro action of renaturase/RNase H suggests that the mechanism of this reaction may involve R-loop displacement after formation of a short single-stranded region of DNA on the template strand following hydrolysis of a hybrid transcript oligonucleotide by RNase H.

Eur J Biochem, 1988 May 2, 173(3), 617 - 22
Expression of the chemically synthesized gene for ribonuclease T1 in Escherichia coli using a secretion cloning vector; Quaas R et al.; The gene for ribonuclease T1 from Aspergillus oryzae has been chemically synthesized using the segmental support technique . An Escherichia coli clone producing the ribonuclease at high levels was constructed by linking the gene downstream to the region coding for the signal peptide of the OmpA protein (a major outer membrane protein of E . coli), using the secretion cloning vector pIN-III-ompA2 . This strategy was employed in order to circumvent a possible toxic effect of the gene product on the host cell . Active ribonuclease containing four additional amino acids at the N-terminus could be isolated from the periplasmic fraction of the host . The final yield after purification was 20 mg enzyme/l liquid culture . With respect to immunological, catalytic and specific behaviour, no qualitative differences could be detected between the enzyme from the over-producing E . coli strain and ribonuclease T1 isolated from A . oryzae.

Eur J Biochem, 1988 May 2, 173(3), 537 - 46
Fur (ferric uptake regulation) protein and CAP (catabolite-activator protein) modulate transcription of fur gene in Escherichia coli; De Lorenzo V et al.; A fusion between the fur (ferric uptake regulation) gene, known to mediate negative regulation of iron absorption in Escherichia coli, and lacZ was constructed in vitro . beta-Galactosidase levels of cells harboring this fusion were under the control of sequences contained in a 185-bp DNA fragment located upstream of the fur structural gene . The fusion was prepared in multicopy (pVLN102 plasmid) and low-copy-number states, the latter constructed as a lambda phage lysogen carrying a fur'-'lacZ insert . DNase I footprinting experiments with purified Fur protein, performed on a 250-bp restriction fragment carrying the promoter region of the fusion, showed the presence of a single Fur-protected site overlapping the -10 region of a potential promoter sequence . Examination of the DNA sequences located upstream of the fur gene revealed a possible binding site for the catabolite-activator protein (CAP) . beta-Galactosidase synthesis of E . coli cells harboring the fusion were measured in fur, crp and cya genetic backgrounds and compared with the corresponding levels in wild-type strains . The data obtained indicate a moderate autoregulation of fur expression by its gene product and also a significant stimulation by the cAMP-CAP system . Transcription start sites were mapped by primer-extension experiments with total RNA obtained in vivo from cells harboring pVLN102 . The results show that transcription of the fur gene is initiated from at least two different sites separated by 6 bp, which appear to originate from two overlapping promoters sensitive to catabolic activation.

Res Vet Sci, 1988 May, 44(3), 309 - 14
Pre-weaning supplementary feed and porcine post-weaning diarrhoea; Hampson DJ et al.; Attempts were made to induce an intestinal hypersensitivity response to weaner diet by feeding pigs with small quantities of this material before weaning . In two trials using different weaner diets piglets subjected to this regimen showed no significant differences in small intestinal structure, in ability to absorb xylose, in bodyweight gain, in incidence of diarrhoea or excretion of enteropathogens after weaning compared with pigs not given any of the diet before weaning, or fed with a different diet before weaning . When post-weaning diarrhoea occurred it was associated with an earlier, more prolonged and greater proliferation of enterotoxigenic Escherichia coli in the small intestines than occurred in healthy pigs after weaning . The greater proliferation in pigs which developed diarrhoea could not be attributed either to an excessive dietary intake after weaning, or to a specific proliferation of rotaviruses.

Cancer Lett, 1988 May, 40(1), 33 - 8
In situ nick translation method reveals DNA strand scission in HeLa cells following heat treatment; Anai H et al.; DNA strand break in HeLa cells induced by heat was detected using the in situ nick translation method . The cells were incubated at 43 degrees C for various times (15, 30, 45, 60, 90 or 120 min) in Lab-Tek tissue culture chamber/slides and were fixed with ethanol/acetic acid on the slide glass . The break sites in DNA were translated artificially in the presence of Escherichia coli DNA polymerase I and 3H-labeled dTTP . The level of break sites in the DNA was visualized by autoradiographic observation of the grains . The DNA strand break appeared as early as 15 min, increased to 10.3-fold at 45 min of 43 degrees C treatment and this level related reciprocally to clonogenicity of the cell . The nick translation method thus provides a rapid in situ assay for determining heat-induced DNA damage of cultured cells, in a semi-quantitative manner.

Proc Natl Acad Sci U S A, 1988 May, 85(10), 3604 - 7
Cloning and characterization of a potentially protective antigen in lymphatic filariasis; Nilsen TW et al.; To facilitate biochemical studies of protective filarial antigens, a lambda gt11 cDNA library was constructed from Brugia malayi adult mRNA and screened with rabbit sera that recognizes a limited set of filarial antigens of approximately 25, 42, 60, and 112 kDa . Antigens of approximately equal to 25 and approximately equal to 60 kDa have been shown previously to induce enhanced clearance of microfilaremia in mice . A 154-base pair clone detected by immunological reactivity was used to isolate by hybridization a nearly full-length cDNA clone of 1.8 kilobases . Nucleotide-sequence analysis indicated that this clone was derived from a mRNA encoding a 63-kDa antigen . A fusion polypeptide containing 37 kDa of the Escherichia coli TrpE protein (anthranilate synthase) and 55 kDa of the cloned protein was recognized in immunoblot experiments with antisera raised against a partially purified preparation of the approximately equal to 60-kDa protective filarial antigen . These data relate the cloned antigen to a potentially protective antigen in lymphatic filariasis.

Proc Natl Acad Sci U S A, 1988 May, 85(10), 3377 - 81
A Xenopus ribosomal protein S6 kinase has two apparent kinase domains that are each similar to distinct protein kinases; Jones SW et al.; We report the molecular cloning of cDNAs for S6 kinase II (S6KII) mRNAs present in Xenopus ovarian tissue . Two cDNAs were isolated by hybridization to oligonucleotide probes designed to encode tryptic peptides isolated from S6KII . The two cDNAs show 91% sequence similarity to each other . These two cDNAs predict proteins of 733 (S6KII alpha) and 629 (S6KII beta) amino acids that show 95% sequence similarity over the 629 amino acids where they are colinear . Amino acids 44-733 of S6KII alpha were expressed in Escherichia coli and the recombinant protein was used to raise antiserum in rabbits . This antiserum reacted with authentic S6KII prepared from Xenopus eggs . This interaction was specifically blocked by the recombinant protein from E . coli . The sequences of S6KII alpha and -beta predict four tryptic peptides whose sequences are identical to four peptides isolated from a tryptic digest of S6KII . The S6KII proteins have a very unusual structure when compared with previously studied protein kinases . They contain two apparent kinase domains, each similar to distinct protein kinases . The amino-terminal 366 amino acids show high sequence similarity to the regions of protein kinase C, the catalytic subunit of cAMP-dependent protein kinase, and cGMP-dependent protein kinase that contain the sites for ATP binding and are believed to be the catalytic centers for phosphotransferase activity . The remainder of the S6 kinase molecule shows high sequence similarity to the ATP-binding and presumed catalytic domain of the catalytic subunit of phosphorylase b kinase.

Carcinogenesis, 1988 May, 9(5), 837 - 42
The initiator tRNA acceptance assay as a short-term test for carcinogens . 1 . A standardized procedure; Hradec J; A short-term test for carcinogens has been developed based on the interaction of chemical carcinogens with tRNA(FMet) in vitro . Transfer RNA from rat or rabbit liver is pre-treated with compounds to be tested in the presence of microsomal enzymes and NADPH . Re-isolated tRNA is then charged with L-methionine by aminoacyl-tRNA synthetases from E . coli B . Carcinogens induce a stimulation of tRNA charging whereas chemically similar non-carcinogenic compounds do not show this effect . Experiments with model substances N-methyl-N'-nitro-N-nitrosoguanidine (strong carcinogen) and aflatoxin G1 (weak carcinogen) revealed some differences in dose effect relationships . It is advisable to test unknown compounds at three different concentrations (10(-5), 10(-7) and 10(-9) mg/ml) with at least two different quantities of microsomal enzymes . Tests on greater than 150 different compounds performed so far indicate that the evaluation of results as % of stimulation (when compared with the control value obtained with the charging of tRNA treated with the solvent only) may allow a quantitative discrimination between weak and intermediate, and strong carcinogens . The procedure is rapid, well reproducible and relatively inexpensive and may be used to complement the other short-term tests for carcinogenicity.

Arch Biochem Biophys, 1988 May 1, 262(2), 481 - 90
Control of leucine transport in yeast by periplasmic binding proteins; Wainer SR et al.; The concentrative inward transport of leucine in Saccharomyces carlsbergensis involves two transport systems (S1 and S2); S1 is a system of high affinity and low translocation velocity, and S2 is a system of low affinity and high translocation velocity . The inward transport process of the amino acid is discriminated into two kinetically defined steps: first, binding to periplasmic proteins and second, translocation across the plasmalemma . When cells were incubated with glucose to increase the metabolic energy charge, we observed that JTmax (maximum flux that each system can exhibit for the translocation step) increased for both systems . This increase in JTmax is due to variations in the parameters defining the initial step (Ks (apparent dissociation constant) and N (concentration of binding sites)): for S1, N1 increases and for S2, KS2 diminishes . Dissipation of the electrochemical proton gradient produced an increase of KS1 and a decrease of N2, resulting in a decrease of JTmax in both systems . Instead, osmotic shock decreases N1 and N2, which suggests that periplasmic components were removed, resulting also in a decrease of JTmax in both systems . These results are consistent with the proposition that the total unidirectional flux of the amino acid proceeds by means of a system of multiple components, with the simultaneous operation of two independent transport processes . We propose that the initial interaction of leucine with components of the cellular envelope might be the essential step for the subsequent translocation of the amino acid across the permeability barrier.

Virology, 1988 May, 164(1), 141 - 6
Molecular cloning and sequencing of the gene (M2) encoding the major virion structural protein (mu 1-mu 1C) of serotypes 1 and 3 of mammalian reovirus; Tarlow O et al.; Full-length c-DNA copies of the M2 gene from the Lang strain of type 1 and the Dearing strain of type 3 reovirus have been cloned in the Escherichia coli plasmid pAT153 . DNA sequencing of these clones showed that the type 3 gene was 2207 nucleotides long and the single long open reading frame encoded a primary translation product (mu 1) of 709 amino acids with a molecular weight of 76,000 . The type 1 gene was three nucleotides shorter at 2204 with the deletions occurring near the center of the coding sequence so that the primary translation product of this gene was one amino acid shorter at 708 . Sequence homology between the two genes had an overall value of 85%, rising to 95% when only the noncoding sequences were compared . The 334 nucleotide changes between the two genes were distributed throughout the sequence with no apparent areas of concentration . Comparison of the predicted amino acid sequences showed that there were 24 differences between the two giving a homology of 96.6% at the protein level . The amino acid changes of which only 9 were nonconservative were again spread fairly evenly throughout the coding sequence although there was one small patch of 5 changes in a stretch of 10 amino acids near the carboxyl terminus . The post-translational cleavage to convert mu 1 to the major virion protein mu 1C is revealed as involving the removal of 42 amino acids exclusively from the amino terminus of mu 1 . Simple addition of trypsin-sensitive cleavage sites or predicted secondary structure failed to show the cause of the large difference known to exist in the protease sensitivities of virions carrying these two proteins.

Mutat Res, 1988 May, 199(1), 145 - 58
Examination of vectors with two dominant, selectable genes for DNA repair and mutation studies in mammalian cells; Debenham PG et al.; A series of vectors with two dominant selectable genes was constructed for repair and mutation studies following transfer into mammalian cells . The recombinant genes (SV-gpt and HSVtk-neo) were placed in different relative orientations and positions in the vectors . These variables were shown to affect transformation frequency of cells by the vectors especially where one of the genes had a relatively weak expression, modelled by truncating the promoter of the HSVtk-neo gene . The use of two-gene vectors to assess DNA repair was investigated by cutting the SV-gpt gene with a restriction endonuclease and monitoring correct rejoining by selecting for gene activity after transfer into various cell types . In such experiments, selection was first applied for the undamaged HSVtk-neo gene to eliminate transfer artefacts, followed by counterselection for the activity of the damaged SV-gpt gene . The measured frequency of correct rejoining of the damaged gene was found to vary both with the vector construct and with the recipient cell species (Chinese hamster V79 or human transformed fibroblasts) . Despite this variation, correct rejoining was found to be consistently lower in radiosensitive (ataxia telangiectasia) human cells than in wild-type human cells, irrespective of the vector construct . In these experiments, some of the transformed cell colonies showed 'sectoring' on exposure to the counterselection, suggesting a slow determination of the fate of transferred DNA . For mutation studies a V79 cell clone carrying a single copy of one of these two-gene vectors was identified and shown to be stably integrated . Mutations of the SV-gpt gene in these cells were isolated while maintaining selection for the HSVtk-neo gene, to attempt to limit mutational loss of the total integrated sequence and provide at least one identifiable junction for analysis of deletion events . Spontaneous and X-ray-induced mutants were identified with a variety of genetic changes, as shown by Southern analysis, from presumed point mutations to deletions and rearrangements of the vector sequence . Rescue of integrated two-gene vector sequences from transformed cells, by recloning in E . coli, was shown to be feasible; thus alterations in transferred DNA can be analysed in detail.

Am J Hum Genet, 1988 May, 42(5), 726 - 34
Resolution of a missense mutant in human genomic DNA by denaturing gradient gel electrophoresis and direct sequencing using in vitro DNA amplification: HPRT Munich; Cariello NF et al.; The combination of denaturing gradient gel electrophoresis (DGGE) and in vitro DNA amplification has allowed us to (1) localize a DNA mutation to a given 100-bp region of the human genome and (2) rapidly sequence the DNA without cloning . DGGE showed that a mutation had occurred, but the technique revealed little about the nature or position of that mutation . The region of the genome containing the mutation was amplified by the polymerase chain-reaction technique, providing DNA of sufficient quality and quantity for direct sequencing . Amplification was performed with a 32P end-labeled primer that allowed direct Maxam-Gilbert sequencing of the amplified product without cloning . HPRTMunich was found to contain a single-base-pair substitution, a C-to-A transversion at base-pair position 397 . We report the generation of a 169-bp, wild-type DNA probe that encompasses most of exon 3 of the human hypoxanthine guanine phosphoribosyltransferase (HPRT) gene and contains a low-temperature melting domain of approximately 100 bp . HPRTMunich, an HPRT mutant isolated from a patient with gout, has a single amino acid substitution; the corresponding DNA sequence alteration must lie within the low-temperature melting domain of exon 3 . We report the separation of HPRTMunich from the wild-type sequence using DGGE . In addition to base-pair substitutions, DGGE is also sensitive to the methylation state of the molecule . The cDNA for HPRT was cloned into a vector and propagated in Escherichia coli dam+ and dam- strains; thus, methylated and unmethylated HPRT cDNA was obtained.(ABSTRACT TRUNCATED AT 250 WORDS)

Am J Vet Res, 1988 May, 49(5), 674 - 7
Duration of immunity and efficacy of an oil emulsion Escherichia coli bacterin in cattle; Collins NF et al.; An oil emulsion Escherichia coli bacterin administered in 1- and 2-dose vaccination regimens was evaluated in beef cattle . Serologic responses to the K99 pilus antigen were monitored, and suckling offspring from vaccinated and nonvaccinated cows were inoculated with virulent, K99-positive, enterotoxigenic Escherichia coli . The degree of protection and duration of immunity conferred were determined in 2 respective studies . In the first study (study A), titers of pregnant cattle were determined from time of vaccination through calving (a 6- to 20-week period) . Titers of 24 cows vaccinated with a single 2-ml dose of bacterin were compared with those of 24 cows given a 2-dose regimen and with those of 23 nonvaccinated cattle (contemporary controls) . Inoculum consisting of 1.2 X 10(12) viable enterotoxigenic E coli/dose administered to nursing calves from these dams yielded 0% mortality (0 deaths/20 calves) in calves from 1-dose vaccinates, 6% mortality (1 death/18 calves) in calves from 2-dose vaccinates, and 37% mortality (7 deaths/19 calves) in calves from nonvaccinated dams . Study B was an extended evaluation conducted in cattle that were kept in the study up to 87 weeks from initial vaccination until calving . Serologic titers to the K99 pilus antigen were compared in 1-dose, 2-dose, and nonvaccinated cattle in groups of 8, 6, and 6, respectively . Calves from these dams were inoculated with 8.1 X 10(11) viable enterotoxigenic E coli/dose, which resulted in 0% mortality (0 deaths/5 calves) in calves from 1-dose vaccinates, 0% mortality (0 deaths/5 calves) in calves from 2-dose vaccinates, and 80% mortality (5 deaths/6 calves) in calves from nonvaccinated dams.

Am J Vet Res, 1988 May, 49(5), 613 - 7
Pathologic changes and tissue gentamicin concentrations after intravenous gentamicin administration in clinically normal and endotoxemic cats; Jernigan AD et al.; Hematologic and serum biochemical values, tissue gentamicin concentrations, and renal pathologic changes were determined in clinically normal and endotoxemic cats given 3 mg of gentamicin/kg of body weight, IV . Endotoxemia was induced by IV administration of 0.5 microgram of Escherichia coli endotoxin/kg of body weight . In experiment 1, 6 cats were given endotoxin . After rectal temperature increased at least 1 degree C, cats were given gentamicin . Blood samples were collected before and at 1 and 3 hours after administration of gentamicin . With the exception of severe leukopenia, other hematologic changes or changes in serum biochemical values were not observed . In experiment 2, 24 cats were allotted to 4 groups and were given gentamicin, endotoxin, gentamicin plus endotoxin, or neither substance . Three hours later, cats were euthanatized, and tissue and body fluid specimens were obtained and were assayed for gentamicin concentration . Kidney specimens were examined microscopically . Endotoxemic cats had more gentamicin in the renal medulla than did control cats, but none of the cats had detectable renal lesions . The possible nephrotoxic synergism between gentamicin and severe endotoxemia and the lack of major differences in gentamicin concentration in extrarenal tissues indicated that the dosage of gentamicin in endotoxemic cats does not have to exceed the dosage recommended for clinically normal cats . A single dose of gentamicin administered IV did not cause renal damage in mildly endotoxemic cats, but nephrotoxicity ascribed to multiple doses of gentamicin in more severely endotoxemic cats needs to be evaluated.

Am J Vet Res, 1988 May, 49(5), 603 - 7
Pharmacokinetics of gentamicin in cats given Escherichia coli endotoxin; Jernigan AD et al.; Nineteen cats were given 3 mg of gentamicin sulfate/kg of body weight by rapid IV, SC, or IM injection for baseline values . Serum concentration of gentamicin vs time data were analyzed using a noncompartmental model based on statistical moment theory . One week later, each cat was given 0.5 microgram of Escherichia coli endotoxin/kg, IV . After cats had an increase in rectal temperature of at least 1 C, 3 mg of gentamicin/kg was administered by the same route used the previous week . Serum concentration of gentamicin vs time data were analyzed, and pharmacokinetic values were compared with base-line values . For IV studies, the half-life (t1/2) of gentamicin and the mean residence time were significantly different (P less than 0.05) compared with base line, whereas the total body clearance and apparent volume of distribution at steady state were not . The harmonic mean +/- pseudo SD for the t1/2 of gentamicin after IV administration was 76.8 +/- 12.6 minutes for base line and was 65.2 +/- 12.2 minutes in the same cats given endotoxin . The t1/2 of gentamicin after SC administration was 74.6 +/- 6.2 minutes for base line and was 65.2 +/- 13.6 minutes in the same cats given endotoxin . After IM administration, the t1/2 of gentamicin was 60.3 +/- 10 minutes for base line and was 59.7 +/- 13.6 minutes in the same cats given endotoxin . After IV administration of gentamicin, the arithmetic mean +/- SD for the mean residence time was 102.4 +/- 16.1 minutes for base line vs 79.2 +/- 18.4 minutes in the same cats given endotoxin.(ABSTRACT TRUNCATED AT 250 WORDS)

Vet Pathol, 1988 May, 25(3), 205 - 10
Comparison in gnotobiotic pigs of lesions caused by verotoxigenic and non-verotoxigenic Escherichia coli; Hall GA et al.; To compare the pathogenesis of calf and rabbit strains of E . coli, gnotobiotic pigs were infected with 10(10) colony forming units (cfu) of verotoxigenic strain RDEC-1 or S102-9, or a non-verotoxigenic E . coli (X114/83) . Pigs were killed 4 days later, and intestinal tissue was fixed and examined by light, scanning, and transmission electron microscopy . Strains S102-9 and RDEC-1 caused diarrhea, attached to enterocytes, and effaced microvilli, confirming that the calf and rabbit strains possessed similar mechanisms of pathogenicity . Non-verotoxigenic strain X114/83 did not cause diarrhea, but in 5/5 piglets it was detected in histological sections adherent to enterocyte surfaces . Exfoliated enterocytes were seen in 4/5 . Bacteria attached to enterocytes by "cups" and "pedestals," with effacement of microvilli, were seen by electron microscopy in 1/5 piglets . It was concluded that strain S102-9 appears to be an animal equivalent of human enterohemorrhagic E . coli, that verotoxin is not essential in the pathogenesis of attaching and effacing lesions, and that the lesions induced by S102-9 are more severe in gnotobiotic pigs than in gnotobiotic or conventional calves.

J Appl Physiol, 1988 May, 64(5), 2026 - 32
Effects of nitroprusside on lung mechanics and hemodynamics after endotoxemia in awake sheep; Wright P et al.; We examined the effects of intravenous sodium nitroprusside (NP) infusion on pulmonary arterial pressure (Ppa), pulmonary vascular resistance (PVR), dynamic compliance (Cdyn), resistance to airflow across the lungs (RL), and alveolar-arterial O2 pressure gradient (PAO2-PaO2) (room air) after endotoxemia in awake sheep . NP infused 2.5 h after endotoxin administration immediately reduced mean Ppa from 30 +/- 3 to 17 +/- 3 (SE) cmH2O, PVR from 6.3 +/- 0.7 to 4.8 +/- 0.5 cmH2O.l-1.min, and RL from 340 +/- 48 of base line to 205 +/- 73% and increased Cdyn from 54 +/- 5 of base line to 80 +/- 14% without affecting PAO2--PaO2 . Ppa and lung mechanics returned immediately to preinfusion levels when NP was stopped . In vitro experiments with NP showed a dose-dependent relaxation of preconstricted pulmonary artery and vein, carbachol-preconstricted sheep tracheal strips, and bronchial rings . We conclude that NP reverses pulmonary hypertension and lung mechanics abnormalities after endotoxin and that this is due to effects of NP on airway and vascular smooth muscle . The return of these abnormalities after NP cessation suggests the continued presence of vascular and airway-constricting factors late after endotoxin . The lack of effect of NP on blood oxygenation suggests that deleterious effects on hypoxic vasoconstriction are offset by improved lung mechanics.

J Clin Microbiol, 1988 May, 26(5), 995 - 9
Comparison of two GM1-erythrocyte assays to detect heat-labile Escherichia coli enterotoxin in stool specimens; Germani Y et al.; Two erythrocyte immunoassay techniques to detect the presence of Escherichia coli heat-labile enterotoxin (LTh) in stool supernatants and cell-free culture supernatants were compared . In the competitive assay, GM1 ganglioside was coated onto V-shaped-well microdilution plates and enterotoxin was coupled to sheep erythrocytes . As little as 0.8 ng of LTh per ml was detected by this method, which was based on the competition between the LTh of the test sample and the sensitized erythrocytes . The second assay made use of chimera antibody prepared by coupling polyclonal anti-LTh antibody to a monoclonal antibody specific for sheep erythrocytes . In this case, LTh, which was specifically bound to a GM1 ganglioside-coated plate, was detected by successively adding the chimera antibody and sheep erythrocytes . The limit of detection of the chimera antibody erythrocyte immunoassay was 0.2 ng/ml . Stool samples were collected from 167 infants hospitalized for diarrhea in the hospital of Noumea, New Caledonia . False-negative reactions due to proteases present in the stool samples were avoided by the addition of phenylmethylsulfonyl fluoride.

Biull Eksp Biol Med, 1988 May, 105(5), 573 - 6
{Mitogenic effect of Legionella pneumophila: the ratio of proliferating T- and B-cells changes after immunization}; Pronin AV et al.; The splenic lymphocyte proliferative response of guinea-pigs immunized with L . pneumophila antigen (ALP) was studied . The immune animals were shown to express enhanced reactivity in response to E . coli lipopolysaccharide, as compared to the controls, while the response to concanavalin A was markedly decreased . After 3 days in the culture ALP induced a significant nonspecific lymphocyte transformation that was expressed to a similar degree in both experimental and control group . However, after a 5-day incubation ALP induced a specific proliferative response of the immunized guinea-pig splenocytes . The experiments on fractionation by passing through the column with nylon wool revealed that T cells are activated in non-immunized guinea-pigs . The results obtained prove that T cells mediate the immune response to ALP in guinea-pigs.

Gan To Kagaku Ryoho, 1988 May, 15(5), 1815 - 25
{Recent advances in L-asparaginase studies}; Taguchi T; We surveyed the progress of L-asparaginase studies for last ten years or so, on the progress and the present situation for its clinical application . At present, L-asparaginase is recognized as one of basic drugs for improvement of remission induction mainly in infant ALL and is being positively incorporated in the treatment of high-risk diseases such as intractable ALL and the myelogenous recurrence . L-asparaginase, an E . coli-derived protein preparation, has various side effects except bone marrow suppression . Immune responses caused by E . coli-derived protein preparation is accordingly a characteristic side effect of the L-asparaginase . For the purpose of avoiding this side effect, studies on immobilized enzymes and enzyme derivatives devoid of antigenicity are under progress.

Proc Natl Acad Sci U S A, 1988 May, 85(10), 3535 - 9
Saturation mutagenesis of a major histocompatibility complex protein domain: identification of a single conserved amino acid important for allorecognition; Murray R et al.; The interactive association between T lymphocytes and their target cells is an important system of cell-cell interactions . Major histocompatibility complex class I molecules are the cell surface structures recognized by cytolytic T lymphocytes . To define the molecular structures recognized by cytotoxic T lymphocytes, we have saturated the 270-base-pair alpha 1 exon of the H-2Dp gene with point mutations, rapidly producing a "library" of 2.5 x 10(3) independent mutants . The library contains enough recombinant clones (each clone encoding approximately one amino acid replacement mutation) to predict a mutation at each nucleotide position of the alpha 1 exon . The functional analysis of the first five transfected gene products tested has shown that mutation of a conserved tyrosine at position 27 to asparagine destroys recognition of the H-2Dp gene product by polyclonal alloreactive cytotoxic T lymphocytes . Recognition of the same mutant molecule by three monoclonal antibodies and H-2-restricted lymphocytic choriomenengitis virus-specific cytotoxic T lymphocytes is unaffected.

Proc Natl Acad Sci U S A, 1988 May, 85(10), 3391 - 5
A viral cleavage site cassette: identification of amino acid sequences required for tobacco etch virus polyprotein processing; Carrington JC et al.; Mature viral-encoded proteins of tobacco etch virus (TEV) arise by proteolytic processing of a large precursor . The proteinase responsible for most of these cleavages is a viral-encoded 49-kDa protein . All known or predicted cleavage sites in the TEV polyprotein are flanked by the conserved sequence motif Glu-Xaa-Xaa-Tyr-Xaa-Gln-Ser or Gly, with the scissile bond located between the Gln-Ser or Gly dipeptide . By using cell-free systems to manipulate and express cloned cDNA sequences, a 25-amino acid segment containing a putative proteolytic cleavage site of the TEV polyprotein has been introduced into the TEV capsid protein sequence . This recombinant protein is cleaved by the 49-kDa proteinase at the introduced cleavage site, thus demonstrating portability of a functional cleavage site . The role of the conserved amino acid sequence in determining substrate activity was tested by construction of engineered proteins that contained part or all of this motif . A protein that harbored an insertion of the conserved 7-amino acid segment was cleaved by the 49-kDa TEV proteinase . Cleavage of the synthetic precursor was shown to occur accurately between the expected Gln-Ser dipeptide by microsequence analysis . Proteins containing insertions that generated only the Gln-Ser, or only the serine moiety of the conserved sequence, were insensitive to the 49-kDa proteinase.

Proc Natl Acad Sci U S A, 1988 May, 85(10), 3363 - 6
The cytoplasmic domain of Escherichia coli leader peptidase is a "translocation poison" sequence; von Heijne G et al.; Leader peptidase is an integral, transmembrane protein of the plasma membrane of Escherichia coli . Its membrane assembly requires its internal, uncleaved signal sequence, its large periplasmic carboxyl-terminal region, and an apolar domain that is known as a "hydrophobic helper." We now show that the polar cytoplasmic domain of leader peptidase is a unique membrane assembly element, which we term a "translocation poison" sequence . This sequence is defined by its ability to block the action of a signal sequence that either precedes or follows it . To our knowledge, this is the first entirely polar topogenic element . Deletion analysis shows that the role of the leader peptidase hydrophobic helper sequence in its membrane assembly is to overcome the block to assembly caused by the poison sequence.

Can J Surg, 1988 May, 31(3), 169 - 71
Treatment of experimental peritonitis with intraperitoneal povidone-iodine solution; Oguz M et al.; Intraperitoneal lavage with povidone-iodine solution has been reported by some to be beneficial in the treatment of peritonitis and by others to cause local and toxic side effects . In this study, 200 white mice, divided into four groups of 50, were subjected to bacterial peritonitis . The first group had no treatment; peritoneal lavage was carried out using povidone-iodine solution in the second group and a 0.9% sodium chloride solution in the third . In the fourth group, antibiotics (clindamycin and gentamicin) were instilled intraperitoneally without peritoneal lavage . The povidone-iodine solution had no beneficial effect, the death rate after 1 week (76%) being similar to that in the control group (78%) and much higher than that in mice treated with sodium chloride lavage (38%) and antibiotics without lavage (16%) . A second series of experiments was, therefore, carried out to investigate the toxic effect of povidone-iodine solution intraperitoneally on mice without peritonitis; the solution was found to be toxic.

Arch Biochem Biophys, 1988 May 1, 262(2), 455 - 70
Proteolysis as a probe of ligand-associated conformational changes in rat carbamyl phosphate synthetase I; Marshall M et al.; Elastase, V8 protease, subtilisin, trypsin, and chymotrypsin all cleaved the 1462-residue polypeptide of rat carbamyl phosphate synthetase I in segment C 160-180 residues from the COOH-end . Its activator N-acetylglutamate (AcGlu) increased the rate of cleavage approximately ninefold, presumably by binding preferentially to the conformation in which C is exposed . ATP/Mg2+ prevented proteolysis both +/- AcGlu . Kd,app for AcGlu (66 microM) and ATP (4.2 microM with AcGlu and 5 mM Mg2+) was estimated from the pseudo-first-order rate constants for inactivation caused by cleavage with elastase at C . Chymotrypsin and trypsin also hydrolyzed the enzyme, independent of AcGlu, at site D within less than 20 residues of the COOH-end . D was protected by ATP only in the presence of AcGlu and K+, and enzyme hydrolyzed exclusively at D had greater than 30-fold higher Km's for AcGlu and ATP . Digestion by trypsin at a third site (B) approximately 530 residues upstream from C appeared to occur subsequent to hydrolysis at C . Slow cleavage by elastase at an additional site (A) to give 360- and 1100-residue peptides was unaffected by AcGlu and ATP, and caused only modest loss of activity . These peptides were isolated by chromatography on DEAE-cellulose . Assignment of the smaller one to the NH2-end on the basis of its cysteine content places site A in the junction between the segments homologous to the small glutaminase and large synthetase subunits of Escherichia coli carbamyl phosphate synthetase II . Neither peptide alone was active; maximal regain of activity (approximately 25%) occurred on combining them in equimolar proportions . The sizes of the peptides produced by further digestion of the site A digest gave the approximate locations of the other sites . Sites A (Ala-417) and B (Arg-787) have recently been identified by NH2-terminal sequencing (S . G . Powers-Lee and K . Corina (1986) J . Biol . Chem . 261, 15349-15352) . Reasons for the low value of KAcGlu,app are examined, and protection by ATP is discussed in relation to previous models for the conformational equilibria of the enzyme.

Surgery, 1988 May, 103(5), 547 - 52
Spontaneous and polyclonal Ig secretion by circulating B cells after surgery; Di Padova F et al.; Abnormalities of the immune response are commonly observed after surgery . In many cases, they are part of a physiologic rather than of a pathologic response to trauma . In this study we show that after elective surgery in otherwise healthy subjects the B cell compartment is deeply affected, as documented by the appearance, 7 days after the intervention, of circulating lymphoblastoid B cells spontaneously secreting in vitro IgG and IgA antibodies . Analogous lymphoblastoid B cells have been described after in vivo immunization and represent a sensitive marker of the B cell response against the immunizing antigen . To better understand the origin of the reaction, we have analyzed the specificity of the antibodies secreted in culture supernatants . We show that the antibody response is polyclonal, since low titers of antibodies against several different bacterial antigens--such as tetanus toxoid, pneumococcal capsular polysaccharides (PCPs), and the lipopolysaccharides (LPSs) of several enteropathogenic strains of Escherichia coli--are detected . This response seems to reflect the previous immunologic experience of the single patient and to be caused by antigens released from traumatized tissues or absorbed through breaches in skin or mucous membranes.

Proc Natl Acad Sci U S A, 1988 May, 85(9), 3039 - 43
A second DNA methyltransferase repair enzyme in Escherichia coli; Rebeck GW et al.; The Escherichia coli ada-alkB operon encodes a 39-kDa protein (Ada) that is a DNA-repair methyltransferase and a 27-kDa protein (AlkB) of unknown function . By DNA blot hybridization analysis we show that the alkylation-sensitive E . coli mutant BS23 {Sedgwick, B . & Lindahl, T . (1982) J . Mol . Biol . 154, 169-175} is a deletion mutant lacking the entire ada-alkB operon . Despite the absence of the ada gene and its product, the cells contain detectable levels of a DNA-repair methyltransferase activity . We conclude that the methyltransferase in BS23 cells is the product of a gene other than ada . A similar activity was detected in extracts of an ada-10::Tn10 insertion mutant of E . coli AB1157 . This DNA methyltransferase has a molecular mass of about 19 kDa and transfers the methyl groups from O6-methylguanine and O4-methylthymine in DNA, but not those from methyl phosphotriester lesions . This enzyme was not induced by low doses of alkylating agent and is expressed at low levels in ada+ and a number of ada- E . coli strains.

Mutat Res, 1988 May, 199(1), 123 - 30
Evidence for a specific regulation of recA gene transcription in Escherichia coli; Villaverde A et al.; The kinetics of the recA, sfiA and umuDC genes transcription were studied during a double SOS-inducing treatment in Escherichia coli cells using several strains carrying lacZ gene fusions . A transient inhibition in recA, but not in sfiA or umuDC promoted beta-galactosidase synthesis was detected after successive UV-irradiations . Results obtained with a recA--lacZ fusion introduced in several DNA-repair mutants demonstrated that neither a lower LexA inactivation nor a decrease in the production of the inducing signal are the events through which the successive UV-irradiation promoted the arrest of recA transcription . On the contrary, a specific UV-dose-dependent delay appears to be the reason for the inhibition of the recA gene transcription in cells irradiated twice.

J Med Chem, 1988 May, 31(5), 1021 - 6
Asymmetrical bisintercalators as potential antitumor agents; Leon P et al.; Ditercalinium and its analogues are dimeric molecules made up of two identical 7H-pyrido{4,3-c}carbazole rings linked by symmetrical linking chains . These dimers elicit antitumor properties through a new mechanism of action . Recently, a relationship was found between their antitumor properties and their cytotoxic effect on the polA Escherichia coli mutant strain, suggesting that 7H-pyrido{4,3-c}carbazole dimers might induce a DNA deformation that could be recognized by the E . coli SOS repair system . Thus, the role of symmetry in ditercalinium analogues for their DNA binding, antitumor properties, and bacterial toxicity is investigated in the present study, by introducing asymmetric parameters in their structures . Dimers were either synthesized with an asymmetrical rigid linking chain or made up of two chemically different chromophores, i.e., acridine and 7H-pyrido{4,3-c}carbazole . The asymmetrical dimers remain able to bisintercalate into DNA with high affinities, but a dramatic loss in their antitumor potency is observed . On the other hand, these asymmetrical dimers are cytotoxic for polA E . coli mutants, like their symmetrical analogues . These results show that the symmetry plays a crucial role for the antitumor potency in the 7H-pyrido{4,3-c}carbazole dimers series.

J Bacteriol, 1988 May, 170(5), 2414 - 7
Microcin B17, a novel tool for preparation of maxicells: identification of polypeptides encoded by the IncFII minireplicon pMccB17; Mayo O et al.; The DNA replication inhibitor peptide microcin B17 is shown to be a useful tool for preparing Escherichia coli maxicells . To illustrate its usefulness, we have identified polypeptides synthesized from pMccB17 and R100 IncFII miniplasmids . After comparing the respective polypeptides and the miniplasmid restriction maps, we concluded that these plasmids share extensive homology in the basic replicon but are different for an adjacent region (parD) that is involved in plasmid stability and maintenance.

J Bacteriol, 1988 May, 170(5), 2392 - 4
Genetic and phenotypic analyses indicating occurrence of the recN262 and radB101 mutations at the same locus in Escherichia coli; Sargentini NJ et al.; The radB101 and recN262 mutations showed essentially identical phenotypes when compared in isogenic Escherichia coli strains for their effects on gamma and UV radiation survival and on conjugal recombination in a uvrA recB recC sbcB sbcC strain . Complementation tests involving attempts to reconstitute a radB+ recN+ strain by transductions between radB101 and recN262 donors and recipients, and tests involving plasmids carrying recN+ and recN::Tn1000 inserts, indicated that the radB and recN genes are identical . We suggest that the radB101 mutation now be referred to as recN2001.

J Bacteriol, 1988 May, 170(5), 2267 - 75
Export of hybrid proteins FhuA'-'LacZ and FhuA'-'PhoA to the cell envelope of Escherichia coli K-12; Coulton JW et al.; The fhuA gene of Escherichia coli K-12 encodes an outer membrane protein that acts as the ferrichrome-iron(III) receptor . To determine the export signals and sorting information within FhuA, gene fusions of fhuA'-'lacZ and fhuA'-'phoA were constructed . Although a FhuA'-'LacZ hybrid protein was detected in the Triton X-100-insoluble fraction of the cell envelope, direct immunoelectron microscopic observation showed that this protein remained in the cytoplasm . FhuA'-'PhoA hybrid proteins were all exported across the cytoplasmic membrane . Those hybrids containing up to 88 amino acids of FhuA (FhuA88) fused to PhoA were released along with other periplasmic proteins . Hybrids containing 180 or more amino acids of FhuA (FhuA180) fused to PhoA were associated with the outer membrane . It is proposed that some information inherent in the sequences between FhuA88 and FhuA180 confers stable association with the outer membrane.

J Bacteriol, 1988 May, 170(5), 2197 - 201
murH, a new genetic locus in Escherichia coli involved in cell wall peptidoglycan biosynthesis; Dai D et al.; A temperature-sensitive mutant of Escherichia coli defective in peptidoglycan synthesis was characterized . The incorporation of radiolabeled meso-diaminopimelate into peptidoglycan by the mutant was inhibited at the restrictive growth temperature, resulting in autolysis . The defective step appeared to be part of the terminal stage in peptidoglycan synthesis involving the incorporation of disaccharide peptide units into the wall peptidoglycan . The mutation was assigned to a new locus, designated murH, at 99.2 min on the E . coli linkage map.

J Bacteriol, 1988 May, 170(5), 2051 - 5
Synthesis of an Escherichia coli protein carrying a signal peptide mutation causes depolarization of the cytoplasmic membrane potential; Pollitt NS et al.; A deletion mutation (lpp delta 9 delta 13 delta 14) in the signal peptide of the major outer membrane lipoprotein of Escherichia coli (Lpp) was found to cause severe effects on cell physiology, resulting in cessation of growth within 10 min of induction of lpp delta 9 delta 13 delta 14 expression and rapid cell death . Further investigation revealed that lpp delta 9 delta 13 delta 14 expression caused slow processing of several other exported proteins . The origin of this effect was traced to depolarization of the electrochemical potential across the cytoplasmic membrane, which is known to be required for efficient protein export . Analysis of the processing rate of the mutant, either prior to complete depolarization or in a suppressor strain in which depolarization does not occur, indicates that the mutant protein was capable of secretion at a rate which, while less than that of the wild type, was reasonably rapid compared with the rates of other E . coli secreted proteins . The existence of this type of signal peptide mutation suggests that the cell may have a mechanism to avoid membrane damage from secretory proteins carrying membrane-active signal peptides which is bypassed by the lpp delta 9 delta 13 delta 14 mutant.

J Bacteriol, 1988 May, 170(5), 2040 - 4
Attachment site of the genetic element e14; Brody H et al.; The Escherichia coli K-12 genetic element, e14, contains a 216-base-pair region that is homologous to a portion of the host chromosome . This region serves as the integration site for the element . The 216-base-pair homology is interrupted by 28 mismatches distributed through the sequence . The actual integrative crossover occurs within the first 11 base pairs from one end of the region . To test factors which affect e14 site-specific recombination, we cloned the attachment sites of free e14 and the host chromosome into the same plasmid . The cloned attachment sites recombined intramolecularly in a process that required the presence of a chromosomal copy of e14 in the host cell as well as the induction of SOS . Recombination events that mimicked both integration and excision occurred under the same conditions and to roughly the same extent.

Int J Radiat Biol Relat Stud Phys Chem Med, 1988 May, 53(5), 829 - 37
Enhanced survival of gamma-irradiated Escherichia coli following pretreatment with dithiothreitol; Smith ST et al.; Survival of three strains of Escherichia coli K12 was studied with respect to radiation protection by dithiothreitol (DTT) . The three strains compared were AB2462 recA, AB2470 rec21 and their DNA repair-competent prototype, AB1157 . The strains were incubated in 10 mmol dm-3 DTT for 60 min and allowed an expression period for SOS functions to appear which may have been induced by DTT . Following the expression period the DTT-incubated cells and incubated control cells were irradiated . When AB1157 cells were pretreated with chloramphenicol (200 micrograms cm-3) for a period of 30 min prior to addition of the induction media no increase in survival was seen . When catalase (0.1 mg cm-3) was added to the AB1157 cells prior to the induction media a decrease in the degree of induction was noted with an enhancement ratio (ER) of 0.893 (ER-1 = 1.12) . Furthermore, DTT-treated AB2462 and AB2470 demonstrated no increase in survival when compared to control cells . In radiation experiments on either strain of E . coli with or without DTT present during irradiation, the following were observed: (1) survival of AB1157 was enhanced with a dose modification factor (DMF) of 1.7 with DTT present and 1.3 with pretreatment; (2) the rec mutants showed no change in survival at any dose with a DMF of approximately 1.0 . Results indicate that, using our protocol, inducible repair is of more importance than free radical scavenging by DTT . Furthermore, DTT-treated AB2462 demonstrated no increase in survival when compared to control cells . In radiation experiments on either strain of E . coli with and without DTT present during irradiation, the following were observed: (1) survival of AB1157 was enhanced with a DMF of 1.7 with DTT present during irradiation and 1.3 with only pretreatment; (2) the recA and recB mutants showed no change in cell survival at any dose with a DMF of approximately 1.0 . Results indicate that, using our pretreatment protocol, inducible repair is of more importance in protection than free radical scavenging by DTT.

Infect Immun, 1988 May, 56(5), 1237 - 41
Heterologous expression of the 65-kilodalton antigen of Mycobacterium leprae and murine T-cell responses to the gene product; Lamb FI et al.; The gene encoding the immunodominant 65-kilodalton antigen of Mycobacterium leprae was subcloned from a lambda gt11 clone into the high-copy-number plasmid pUC8 . Escherichia coli containing these recombinants produced large amounts of the antigen, which was purified by polyacrylamide gel electrophoresis in the presence of urea . The ability of E . coli to recognize the mycobacterial promoter was confirmed by constructing additional clones in which the gene is flanked by transcriptional terminators from phage fd . A similar approach was used to demonstrate the expression of this gene in Streptomyces lividans . Mice immunized with killed M . leprae showed cell-mediated immune reactivity to the purified 65-kilodalton protein which stimulated both in vitro lymphoproliferative and in vivo delayed-type hypersensitivity responses.

Biol Chem Hoppe Seyler, 1988 May, 369 Suppl, 199 - 204
Structural analogies between adhesive proteins and cysteine proteinase inhibitors; Keil-Dlouha V et al.; Structural analogies were found between adhesive proteins, fibronectin and laminin, and a group of cysteine proteinase inhibitors . The sequences analogous with inhibitors occur in both adhesive molecules in the vicinity of their cell-binding sites: they comprise residues 1112-1212 in fibronectin and residues 1538-1639 of B1 chain in laminin . Both adhesive molecules display the highest degree of analogy with stefin-type inhibitors . The structural analogies also exist between fibronectin, laminin and P21, the c-Ha-ras gene protein produced by Escherichia coli.

Am Rev Respir Dis, 1988 May, 137(5), 1180 - 4
Interleukin-1-beta gene expression in human monocytes and alveolar macrophages from normal subjects and patients with sarcoidosis; Kern JA et al.; To assess the ability of human alveolar macrophages to produce interleukin-1-beta (IL1-beta), we examined IL1-beta mRNA accumulation in autologous monocytes and alveolar macrophages from normal volunteers . Escherichia coli lipopolysaccharide stimulation of monocytes induced rapid IL1-beta mRNA accumulation, reaching a maximum at 2 to 4 h and declining thereafter . Alveolar macrophages, however, accumulated much less mRNA than did monocytes . This difference could not be explained by differences in kinetics of IL1-beta gene expression between the 2 cell types, isolation techniques, or alveolar macrophage lidocaine exposure . This suggests that differences in transcription of the IL1-beta gene exist between these 2 cell types . Aging is a possible factor important in some functional differences between these 2 cell types . To determine if this difference in the capacity to express the IL1-beta gene might be a function of cell maturity, monocytes were aged in vitro for 7 days . After this culture period, monocytes had a marked decrease in the ability to accumulate IL1-beta mRNA, suggesting that cell aging may be one mechanism involved in producing these transcriptional differences . Because IL1-beta has also been implicated in the inflammatory and fibrotic responses in pulmonary sarcoidosis, 4 patients with newly diagnosed sarcoidosis underwent bronchoalveolar lavage, and IL1-beta mRNA accumulation was compared in their alveolar macrophages and blood monocytes . Comparing normal alveolar macrophages to those from patients with sarcoidosis showed no differences in the kinetics of IL1-beta mRNA expression or in the LPS-induced levels of IL1-beta mRNA accumulation . In addition, augmented levels of IL1-beta transcript were not noted in unstimulated sarcoid alveolar macrophages.(ABSTRACT TRUNCATED AT 250 WORDS)

Mol Immunol, 1988 May, 25(5), 429 - 37
Cloning, sequence and expression of bovine interleukin 1 alpha and interleukin 1 beta complementary DNAs; Maliszewski CR et al.; Interleukin 1 (IL-1) is a cytokine which mediates a variety of immunoregulatory and inflammatory activities . Using human IL-1 alpha and IL-1 beta probes, cDNAs for the corresponding bovine genes were isolated from an alveolar macrophage library . The open reading frames of the bovine IL-1 alpha and IL-1 beta cDNAs encode proteins of 268 and 266 amino acids, respectively, each with a predicted mol . wt of approx . 31,000 . Both forms of bovine IL-1 exhibit a high degree of sequence homology with IL-1 gene products from other mammalian species . Based upon comparisons with human IL-1 amino acid sequences, the post-translationally processed, mature forms of bovine IL-1 would occur as 17-18,000 mol . wt proteins . Sequences encoding mature bovine IL-1 alpha and IL-1 beta were inserted into E . coli expression plasmids and biologically active proteins were synthesized as judged by the ability of the recombinant proteins to induce proliferation of bovine thymocytes . Both IL-1 alpha and IL-1 beta exist as single genomic copies . In addition, bovine IL-1 beta mRNA is approx . 10-fold more abundant than IL-1 alpha mRNA in stimulated alveolar macrophages.

Biochimie, 1988 May, 70(5), 611 - 8
Mutations in ribosomal proteins L7/L12 perturb EF-G and EF-Tu functions; Bilgin N et al.; In vitro cycling rates of E . coli ribosomes and of elongation factors EF-Tu and EF-G have been obtained and these are compatible with translation rates in vivo . We show that the rate of translocation is faster than 50 s-1 and therefore that the EF-G function is not a rate limiting step in protein synthesis . The in vivo phenotype of some L7/L12 mutants could be accounted for by perturbed EF-Tu as well as EF-G functions . The S12 mutants that we studied were, in contrast, only perturbed in their EF-Tu function, while their EF-G interaction was not impaired in relation to wild type ribosomes.

Biochimie, 1988 May, 70(5), 597 - 603
Affinity labeling at the A-site of Escherichia coli ribosomes by a non-hydrolyzable gamma-amide analog of GTP; Babkina GT et al.; gamma-Amides of GTP and affinity and photoaffinity derivatives of gamma-amides of GTP: gamma-anilide of GTP, gamma-(4-azido)anilide of GTP, gamma-{N-(4-azidobenzyl)-N-methyl}amide of GTP, gamma{4-N-(2-chloroethyl)-N-methylaminobenzyl}amide of GTP and gamma-{4-N-(2-oxoethyl)-N-methylaminobenzyl}amide of GTP substituted efficiently for GTP in the EF-Tu-dependent transfer of aminoacyl-tRNA to the ribosome but, in contrast to GTP, they were not hydrolyzed in this process . They represent a new class of non-hydrolyzable GTP analogs with preserved gamma-phosphodiester bond . The radioactive analog of GTP: gamma-{4-N-(2-chloroethyl)-N-methylamino{14C}benzyl}amide of GTP was used as an affinity labeling probe for the identification of components of the GTPase center formed in the EF-Tu-dependent transfer reaction of aminoacyl-tRNA to the ribosomal A-site . Within a six-component complex of poly(U)-programmed E . coli ribosomes with elongation factor Tu, Phe-tRNA(Phe) (at the A-site), tRNA(Phe) (at the P-site) and the {14C}GTP analog, mainly the ribosomal 23S RNA and to a lesser extent the ribosomal proteins L17, L21, S16, S21 and the ribosomal 16S RNA were labeled by the reagent . No significant modification of EF-Tu was detected.

Mutagenesis, 1988 May, 3(3), 179 - 91
Molecular dosimetry of alkylating agents: quantitative comparison of genetic effects on the basis of DNA adduct formation; van Zeeland AA; This is a review of the work on molecular dosimetry carried out by several laboratories within the third Environmental Programme of the Commission of the European Communities . The molecular dosimetry project is a coordinated research project in which several laboratories cooperated on various aspects of quantitative comparative mutagenesis . This involved (i) development of assay systems for measurement of DNA adducts using radioactively labelled mutagens; (ii) development of immunological assays for detection and quantification of DNA adducts; (iii) isolation and characterization of monoclonal antibodies against DNA adducts; (iv) quantitative comparison of genetic effects of the model compounds within one assay system; and (v) extrapolation of genetic effects of the model compounds from in vitro systems to whole animal studies.

Curr Genet, 1988 May, 13(5), 377 - 82
Transformation of Penicillium chrysogenum with a dominant selectable marker; Bull JH et al.; We have cloned a mutant oligomycin resistance allele of the mitochondrial ATP synthase subunit 9 gene from the filamentous fungus Penicillium chrysogenum . The gene was isolated using the equivalent gene from Aspergillus nidulans as a hybridisation probe . Using the cloned gene it is possible to select for oligomycin resistance in P . chrysogenum transformation experiments . This transformation system was used to introduce further copies of the P . chrysogenum isopenicillin N synthetase gene, which were stably maintained without selection . An assessment of the frequency with which homologous integration occurs was also made . With this system, it should prove possible to transform any strain of P . chrysogenum.

Oncogene Res, 1988 May, 2(4), 325 - 33
Guanine nucleotide binding properties of purified v-Ki-ras p21 protein produced in Escherichia coli; Hara M et al.; We have purified the v-Ki-ras p21 protein by expression in Escherichia coli of a plasmid encoding 189 amino acids of the Kirsten murine sarcoma virus oncogene . The purified p21 (over 95%) exhibited a tenfold greater affinity for GTP (1.6 x 10(-7) M) than for GDP (1.1 x 10(-6) M) . The preferential binding of v-Ki-ras p21 towards GTP was attributed to the faster dissociation rate of GDP from the protein . The affinity of GDP to v-Ki-ras p21 decreased with the depletion of Mg2+ while that of GTP did not change significantly . The dissociation constant rate of the p21-GDP complex was estimated as 2 x 10(-3) s-1 in the presence of Mg2+ and increased to 2 x 10(-2) s-1 after the removal of Mg2+ . These results are discussed with respect to the role of GDP-GTP exchange in the regulation of p21 function.

Mol Microbiol, 1988 May, 2(3), 405 - 12
Nucleotide sequence of the mannitol (mtl) operon in Escherichia coli; Davis T et al.; The nucleotide sequence of the known portions of the mannitol operon in Escherichia coli (mtlOPAD) has been determined . Both the operator-promoter region and the intercistronic region between the mtlA and mtlD genes (encoding the mannitol-specific Enzyme II of the phosphotransferase system and mannitol-1-phosphate dehydrogenase, respectively) show parallels with corresponding regions of the glucitol (gut) operon, but neither the mtlA nor the mtlD gene products show obvious homology with the corresponding gene products of the glucitol operon . Five potential cyclic AMP receptor protein binding sites were identified in the mtlOP region, all showing near identity with the consensus sequence . Four regions of dyad symmetry (four to seven bases in length), serving as potential repressor binding sites, overlap with the potential cyclic AMP receptor protein binding sites . Repetitive extragenic palindromic (REP) sequences, forming stem-loop structures in the intercistronic region between mtlA and mtlD and following the mtlD gene were identified . Probable terminator sequences were not found in any of these three regulatory regions . Mannitol-1-phosphate dehydrogenase exhibits two overlapping, potential NAD+ binding sites near the N-terminus of the protein . Computer techniques were used to analyse the mtlD gene and its product.

Proc Natl Acad Sci U S A, 1988 May, 85(10), 3367 - 71
Stabilization of an associated folding intermediate of bovine growth hormone by site-directed mutagenesis; Brems DN et al.; By using oligonucleotide-directed mutagenesis, Lys-112 of bovine growth hormone (bGH) was changed to leucine, and its resulting effect on folding was studied . Equilibrium denaturation curves for the mutant protein exhibit biphasic or nonsymmetrical transitions by a variety of spectroscopic and hydrodynamic techniques, whereas the wild-type protein at the same concentration exhibits symmetrical transitions . The mutant protein refolds slower (by a factor of 30) and more readily precipitates upon refolding than the wild-type protein . These folding characteristics of the mutant protein are demonstrated to be a result of stabilization of an associated folding intermediate . A 38-amino acid fragment (96-133) derived from the mutant protein is helical, likely amphipathic, and more stabilized by increasing peptide concentration than is the corresponding helical peptide from the wild-type protein . The increased stability of the associated intermediate and the increased helicity of the peptide from the mutant protein are explained by preferential intermolecular interactions between helices due to enhanced hydrophobic attraction by their amphipathic surfaces.

J Immunol, 1988 May 1, 140(9), 2893 - 8
Differential activation of T cell clones stimulated by macrophages exposed to antigen complexed with monoclonal antibodies . A possible influence of paratope specificity on the mode of antigen processing; Manca F et al.; We have used the enhanced uptake by FcR-bearing cells observed when Ag is administered as an immune complex to investigate the possible impact of specific antibodies on processing and presentation of antigen by accessory cells . The Ag Escherichia coli beta-galactosidase alone or bound to different mAb was incubated with peritoneal macrophages . These were subsequently exposed to a battery of Ag-specific T hybridoma clones . The resulting production of IL-2 was taken as a measure of effective presentation . The results of 43 mAb-T clone combinations showed a potentiation of presentation of Ag at substimulatory concentration in the majority of the cases, indicating that each mAb is conducive to FcR-mediated uptake by macrophages, and that each T clone can be stimulated by properly presented Ag . In contrast, nine combinations yielded a lower response, two of them falling to baseline values . We attribute these results, which corroborate our previous evidence of directional help in the beta-galactosidase system, to a modulation in enzymatic processing of Ag and its subsequent presentation imposed by the paratope of the mAb binding to the relevant epitope.

J Bacteriol, 1988 May, 170(5), 2236 - 9
Characterization of a novel L-serine transport system in Escherichia coli; Hama H et al.; A novel transport system for L-serine was found in Escherichia coli cells grown on medium containing amino acid mixture . This novel system is distinguishable from the known three transport systems for L-serine, namely, the serine-threonine system, one of the leucine-isoleucine-valine systems, and the glycine-alanine system . Uptake of L-serine via this novel system was inhibited by none of the amino acids tested, indicating that it is highly specific for L-serine . This system was induced by L-leucine, but not by L-serine . The Km for L-serine was 50 microM, and the Vmax was 23 nmol/min per mg of cell protein . Transport of L-serine via this system was strongly inhibited by KCN, an inhibitor of the respiratory chain, or by carbonyl cyanide m-chlorophenylhydrazone, an H+ conductor . Uptake of H+ was induced by L-serine influx . These results indicate that an H+-serine cotransport mechanism is operative in this novel L-serine transport system.

J Bacteriol, 1988 May, 170(5), 2212 - 20
Association of degradation and secretion of three chimeric polypeptides in Escherichia coli; Gentz R et al.; We investigated the stability of fusion proteins composed of the signal peptide of the heat-labile enterotoxin of Escherichia coli and three polypeptides: the bacterial cytoplasmic chloramphenicol acetyltransferase, the mouse dihydrofolate reductase, and human immune interferon . We demonstrate that these proteins are rapidly degraded as a result of being targeted to the secretion apparatus of E . coli, with the extent of degradation varying among the three fusion proteins . Four lines of experimental evidence are presented in support of this suggestion . First, the chimeric polypeptides containing a functional signal peptide were detected in low amounts in vivo . When a mutation was introduced in the signal peptide, resulting in lack of recognition by the secretion apparatus, the chimeric proteins accumulated at high levels in the cytoplasm of the cell . Second, both the wild-type and mutant polypeptides accumulated in a purified and reconstituted in vitro translation system from E . coli and were equally susceptible to digestion by an exogenous protease . Third, the chimeric polypeptides lacking the signal peptide accumulated in a stable form in vivo . Fourth, the precursors of the proteins containing a functional signal peptide accumulated in a secA ts mutant at the restrictive temperature when secretion was blocked, suggesting that degradation is tightly linked to the secretion apparatus.

J Bacteriol, 1988 May, 170(5), 2208 - 11
Effect of substitution of glycine for arginine at position 146 of the A1 subunit on biological activity of Escherichia coli heat-labile enterotoxin; Okamoto K et al.; The ADP-ribosyltransferase activity of polypeptide A1 of cholera toxin and that of Escherichia coli heat-labile enterotoxin (LT) are primarily responsible for the toxic activities of these toxins . Since the amino acid sequences of the two A1 polypeptides are very similar, their functional mechanisms are considered to be the same . Arg-146 of polypeptide A1 is thought to be involved in the active site, because this amino acid of cholera toxin has been identified as the site of self-ADP-ribosylation . However, the exact role of Arg-146 and the significance of self-ADP-ribosylation in toxicity remain unclear . We substituted Arg-146 of polypeptide A1 of LT with Gly by oligonucleotide-directed mutagenesis and examined the biological property of the resultant mutant LT . The substitution changed the mobility of subunit A on sodium dodecyl sulfate-polyacrylamide gel but did not reduce the vascular permeability activity of LT . This result indicates that Arg-146 is not absolutely required for toxic activity and that LT can express its toxic activity without self-ADP-ribosylation at Arg-146.

Pol J Pharmacol Pharm, 1988 May-Jun, 40(3), 265 - 72
Pyrogenic fever and blood plasma glucocorticoids after nonsteroid anti-inflammatory drugs; Switala M et al.; Rabbits were injected with the lipopolysaccharide from E . coli (LPS) and received orally nonsteroid anti-inflammatory drugs (NSAIDs): acetylsalicylic acid, indomethacin, mefenamic acid, ibuprofen, aminophenazone, metamizole sodium, and phenylbutazone . These NSAIDs exerted antipyretic action without inhibiting the increase in the level of plasma glucocorticoids induced by LPS . This finding indicates the lack of correlation between the pyrogenic action of bacterial pyrogen and pyrogenic increase in the plasma glucocorticoid level . The investigated NSAIDs when given alone to normothermic rabbits differently affected the plasma glucocorticoid level: acetylsalicylic acid, indomethacin and ibuprofen depressed the plasma level of these hormones, mefenamic acid and phenylbutazone elevated it, and aminophenazone and metamizole sodium did not alter it significantly.

Acta Pathol Jpn, 1988 May, 38(5), 541 - 7
Shwartzman reaction in the brain induced by fractions of Fusobacterium necrophorum and Escherichia coli lipopolysaccharide in rabbits; Nakajima Y; Multifocal fibrin thrombosis and suppurative meningitis in the central nervous system was induced by intracerebral inoculation with a cytoplasmic or supernatant fraction of Fusobacterium necrophorum followed by intravenous inoculation with Escherichia coli lipopolysaccharide endotoxin in rabbits . Formation of fibrin thrombosis was reduced by heparin administration . Single intracerebral inoculation with the cytoplasmic or supernatant fraction caused suppurative meningitis . Nervous lesions were often associated with fibrinoid degeneration of the blood vessels . Bacterial lipopolysaccharide endotoxin did not induce apparent meningitis or fibrin thrombosis . The formation of fibrin thrombosis in the central nervous system might be attributable to the Shwartzman reaction.

Biol Chem Hoppe Seyler, 1988 May, 369 Suppl, 163 - 7
Expression of rat and mouse cathepsin B precursors in Escherichia coli; Mort JS et al.; Vectors for the expression of mouse preprocathepsin B and rat procathepsin B, under the control of a modified lac operon regulatory region, in plasmid pKK233-2 were constructed . In rapidly growing cells induction of preprocathepsin B was cytotoxic . Immune precipitable cathepsin B, compatible in size with that expected for the unglycosylated proenzyme, was produced in both constructs . However, pepsin digestion of extracts did not produce any cathepsin B activity, indicating either that the recombinant forms are not correctly folded or were produced at such low levels that activity could not be detected.

Biol Chem Hoppe Seyler, 1988 May, 369 Suppl, 153 - 6
The elastase-inhibitor eglin has no effect in an ovine model of endotoxemia; Redl H et al.; Small doses of endotoxin have been shown to induce pulmonary microvascular injury in sheep, possibly by the action of granulocytes . Eglin, a potent inhibitor of neutrophil elastase, was tested in an ovine model of endotoxemia . The experiment was performed in 12 unanesthetized chronically instrumented sheep with a lung lymph preparation . Endotoxin (S . abort . equii) was infused at 24 ng/(kg x h) with application of 20 mg/kg eglin 1 h before endotoxin in the treatment group and followed by 5 mg/(kg x h) . No significant improvement due to the treatment was seen for either cardiovascular status (pulmonary and systemic vascular resistance) or permeability changes in the lung (lymph flow and lymph/plasma protein ratio), although sufficient eglin concentrations were achieved in plasma and lymph . The lack of an effect of eglin might be because higher concentrations are needed to block elastase-like activity of ovine granulocytes or because of a minor role for neutrophil elastase in this shock model.

J Gen Microbiol, 1988 May, 134 ( Pt 5), 1355 - 64
Biochemical analysis of spontaneous fepA mutants of Escherichia coli; Elish ME et al.; The fepA gene of Escherichia coli encodes the outer-membrane receptor protein for ferrienterobactin . Previous genetic studies indicated that fepA mutations occur frequently and suggested that most of the mutations were deletions . In this work seven spontaneous fepA mutations were analyzed by enzyme assay (enterobactin synthase and enterobactin esterase) and by DNA hybridization studies . In two strains, UT500 and UT700, the mutations were confined to the fepA gene . In the remaining mutants, the mutations were large deletions; in several cases, 27 kb or more of DNA had been lost . The deletions, all of which eliminated approximately the left half of the enterobactin gene cluster, extended from the vicinity of the fepC gene counterclockwise into the chromosome . A minimum of three clockwise endpoints were identified and at least two counterclockwise endpoints were detected . The variation in endpoints among the deletions argues against the involvement of a normal transposon in their formation . Also, unexpected homology was found between enterobactin gene cluster DNA and lacPOZ and pSC101.

J Gen Microbiol, 1988 May, 134 ( Pt 5), 1315 - 21
HEp-2 adhesion and the expression of a 94 kDa outer-membrane protein by strains of Escherichia coli belonging to enteropathogenic serogroups; Chart H et al.; Sixty strains of Escherichia coli belonging to enteropathogenic serogroups (EPEC) were examined for the ability to adhere to HEp-2 cells, the possession of the genes encoding EPEC adherence factor (EAF) and the ability to express an outer-membrane protein (OMP) of 94 kDa thought to be involved in bacterial adhesion to eukaryotic cells . An absolute correlation was found between HEp-2 adhesion and the possession of the genes encoding EAF . An OMP of 94 kDa was observed in the SDS-PAGE profile of most adhesive strains . In some strains this protein was prone to proteolytic degradation . An antiserum raised to a HEp-2 adhesive strain of EPEC did not react with the 94 kDa OMP of all EPEC which were EAF-positive and HEp-2 adhesive, indicating some interstrain antigenic variation of this protein . Although this 94 kDa protein was surface-exposed, specific antibodies binding to the 94 kDa protein in situ in the outer membrane did not interfere with adhesion of EPEC to HEp-2 cells . Therefore, these studies question the value of this protein as a potential vaccine component.

J Gen Microbiol, 1988 May, 134 ( Pt 5), 1283 - 8
Colicin K decreases the density of intramembrane particles (IMP) in the cell membrane of Escherichia coli; Smarda J; Toxic effects of both main colicin types, i.e . of porin and nuclease types, involve the direct contact of their molecules with the plasma membrane of sensitive cells . In the present study, it was tested whether this contact provokes a lateral or vertical movement of intramembrane protein particles (IMP) or a direct cleavage of the proteins . IMP were visualized by freeze-fracturing and electron microscopy on the protoplasmic fracture face (PF) of colicin-treated cells of Escherichia coli . Possible changes in distribution and in density of IMP due to treatment with colicins E1-E7 and K were followed . As a control, the bacteria were equilibrated at 0 degrees C before quenching, which caused a reversible formation of smooth areas and a decrease in the mean density of IMP on the PF . Colicins E1-E7 had no clear-cut effect on the disposition of IMP . Only colicin K decreased the IMP density, by 10% in E . coli strain 58-161 and by 17% in strain C6; the distribution of IMP remained homogeneous . Trypsin reactivation of colicin-K-inactivated bacteria was not reflected by restoration of the original density of IMP; on the contrary, it led to a further decrease, of 1-13%, in IMP density, presumably by proteolytic cleavage . Varying densities of IMP in different strains of the same bacterial species (under standard conditions) were confirmed.

J Gen Microbiol, 1988 May, 134 ( Pt 5), 1141 - 6
Cloning and expression in Escherichia coli of a recA-like gene from the acidophilic autotroph Thiobacillus ferrooxidans; Ramesar RS et al.; A recombinant plasmid, pRSR100, containing the functional analogue of the Escherichia coli recA gene was isolated from a genomic library of Thiobacillus ferrooxidans ATCC 33020 . The plasmid complemented defects in DNA repair and homologous recombination in E . coli recA mutant strains . Antiserum raised against E . coli RecA protein reacted with the native but defective E . coli HB101 RecA protein; it did not react with protein extracts from the recA deletion mutant E . coli JK696, but it reacted with two protein bands in extracts of E . coli JK696(pRSR100) . A single band with an apparent Mr equal to the higher-Mr band in E . coli JK696(pRSR100) was detected in T . ferrooxidans cell extracts with the E . coli RecA antiserum.

Mol Biol (Mosk), 1988 May-Jun, 22(3), 767 - 79
{Characteristics of the context shift in the frequency of synonymic codons in Escherichia coli}; Borodovskii MIu et al.; We have demonstrated, that coding regions of E . coli DNA exhibit the non-random shifts of codon usage frequency depending both on the type of the 3' nucleotide adjacent to the codon and the degree of gene expression . Analysis of primary data--statistics of tetranucleotide occurrences--was performed by the techniques of contingency tables . The results of the investigation allowed us to suggest that the phenomenon observed is connected with the influence of the 3'-adjacent nucleotide on the level of missens-errors and another types of inaccuracy during translation . The specific advantages of such mutations in the third position of the codon are based on the adaptation of the codon to the 3' context in order to increase the efficiency of translation.

Mol Biol (Mosk), 1988 May-Jun, 22(3), 726 - 30
{Display of 8-hydroxy-GTP substrate properties of UTP in the reaction of polynucleotide synthesis catalyzed by RNA-polymerase from Escherichia coli in the presence of poly{d(AT).d(AT)} template}; Bruskov VI et al.; 8-oxy-GTP was obtained via reaction of GTP with ascorbic acid and addition of hydrogen peroxide . 8-oxy-GTP is recognized and displays substrate properties of UTP on substitution of 8-oxy-GTP for UTP in polynucleotide synthesis catalyzed by E . coli RNA polymerase on a poly{d(A-T)}.poly{d(A-T)} template . Such incorporation does not take place at equimolar quantities of GTP and 8-Br-GTP . The incorporation of 8-oxy-GTP instead of UTP, is 2.5-3 times higher upon replacement of Mg2+ by Mn2+ ions . The dinucleotide ApU serving as an initiator rises the incorporation level of 8-oxy-GTP both for Mg2+ and Mn2+ ions . 8-oxy-GTP slightly inhibits poly{r(A-U)} synthesis, but UTP strongly inhibits the incorporation of 8-oxy-GTP . {alpha-32P} 8-oxy-GTP is incorporated mainly instead of UTP, but it can be incorporated also during the substitution of 8-oxy-GTP for ATP.

Mol Biol (Mosk), 1988 May-Jun, 22(3), 670 - 9
{A modified method of isolation of "tight" 70S ribosomes from Escherichia coli highly active at different stages of the elongation cycle}; Makhno VI et al.; The functional activity of the wide-spread "tight" 70S ribosomes is usually equal to 55-80% . We show here that the inactive fraction of this type of ribosomes is virtually blocked by residual endogenous RNA's . These RNA's are shown to be removable by introducing an additional stage in the isolation procedure including: 1 . short heating (15 min, 37 degrees C) of "tight" 70S under dissociation conditions, i . e . in a buffer containing 3 mM MgCl2 and 200 mM NH4Cl; 2 . washing off endogenous RNA's on a sucrose density gradient in the same buffer; 3 . final selection of purified "tight" 70S on the sucrose gradient containing 5 mM MgCl2 and 50 mM NH4Cl . "Tight" 70S ribosomes isolated by such a procedure are 90-100% active with respect to tRNA binding (including the factor-dependent one), peptide bond synthesis and translocation.

Mol Biol (Mosk), 1988 May-Jun, 22(3), 624 - 7
{RNA-recognizing function of the DNA-recognizing structure helix-turn-helix in ribosomal proteins S4 and S7 of Escherichia coli and plant chloroplasts}; Shestopalov BV; The segments which satisfy the necessary requirements for DNA-recognizing structure helix-turn-helix coding have been found in amino acid sequences of RNA-recognizing E . coli ribosomal proteins S4 and S7 . It has appeared that among these segments there are those that in chloroplasts S4 and S7 homologues satisfy these necessary requirements also: S4-97-121 and 108-132 (Ec), 91-115 and 102-126 (Zea mays); S7-109-133 (Ec) 110-134 (Euglena gracilis, Glycine max) . Such segments have been confronted with known experimental data on 16S rRNA-binding region localization . It has appeared: 1) the segment 97-121 of S4 is localized in RNA-recognizing fragment 46-124 and one of two RNA-binding regions of this fragment containing Lys-120--belongs to the predicted DNA-recognizing structure; the segment 109-133 of S7 includes the only RNA-binding region of S7-113-117 . It is concluded that E . coli ribosomal proteins S4 and S7 and their homologues from plant chloroplasts have a DNA-recognizing structure helix-turn-helix performing the RNA-recognizing function.

Mikrobiologiia, 1988 May-Jun, 57(3), 499 - 502
{Effect of polyamines on the thermal resistance of Escherichia coli cells in heat shock}; Amosov FG et al.; Polyamines intensify the effect of a heat shock on Escherichia coli M-17 cells . The lethal effect of polyamines rises in the spermidine--spermine series as their concentration and the duration of heat action is increased . The effect of polyamines on the cells subjected to a heat shock is not associated with the activity of amine oxidases and, apparently, does not destabilize the membranes . As was demonstrated using electron microscopy, the cells undergo morphological changes in the presence of polyamines in the course of a heat shock; in particular, electron-dense regions appear in the nucleoid zone, presumably, due to DNA conformational rearrangements during the shock in the presence of polyamines.

J Biochem (Tokyo), 1988 May, 103(5), 797 - 804
Rat cytosolic aspartate aminotransferase: molecular cloning of cDNA and expression in Escherichia coli; Horio Y et al.; cDNA clones for rat cytosolic aspartate aminotransferase (cAspAT, L-aspartate:2-oxoglutarate aminotransferase) {EC 2.6.1.1} were isolated from a rat cDNA library, and the primary structure of the gene for cAspAT was deduced from its cDNA sequence . Rat cAspAT consists of 412 amino acids and its molecular weight is 46,295 . The deduced amino acid sequence of rat cAspAT was compared with the sequences of AspATs from other species . The degree of sequence identities of rat/mouse cAspAT, rat/pig cAspAT, rat/chicken cAspAT, rat/pig mAspAT, and rat/Escherichia coli AspAT were 97.1, 89.6, 81.7, 48.1, and 41.2%, respectively . A coding region of rat cAspAT cDNA was inserted into E . coli expression vector pUC9, and enzymatically active cAspAT was expressed as a beta-galactosidase-cAspAT hybrid protein . This hybrid protein represented about 18% of the soluble proteins in E . coli and its kinetic properties were comparable with those of cAspAT preparations purified from rat liver.

Ann Inst Pasteur Microbiol, 1988 May-Jun, 139(3), 315 - 23
Non-radioactive oligonucleotide probe for detection of clinical enterotoxigenic Escherichia coli isolates of bovine origin; Kumar A et al.; Oligonucleotides (32 or 34 mer) corresponding to enterotoxigenic Escherichia coli STIa (ST-P) toxin gene were tailed with Bio-11-dUTP using terminal deoxynucleotidyl transferase . Plasmids from clinical E . coli isolates were prepared by modified rapid alkaline lysis procedure and dot-spotted . Biotinylated oligonucleotide probes were hybridized to detect the STIa toxin gene . The results were in agreement with that obtained by radioactive oligonucleotide probes . Of 135 clinical isolates (sampled from 6 different regions of France), only 7 (5.2%) were found to be STIa+ . These 7 isolates were the only ones to be found positive for the K99 adhesive pili antigen . Both the probes were specific to the STIa toxin gene and failed to detect the closely related STIb (ST-H) toxin gene . Possibilities of their wide usage in clinical labs are discussed.

Hinyokika Kiyo, 1988 May, 34(5), 811 - 3
{Single day treatment in acute cystitis}; Yamaguchi K et al.; The effect of a single day treatment with 600 mg norfloxacin 600 mg ofloxacin or 1,920 mg trimethoprim-sulfamethoxazol was determined on 114 patients with acute cystitis . The overall clinical efficacy was excellent in 101 patients (89%), moderate in 9 patients (8%) and poor in 4 patients (3%) . Recurrence was observed in 8 cases (8%) within 6 weeks after the treatment . The effectiveness rate and the recurrence rate were inferior in those caused by S . epidermidis compared with those caused by E . coli.

Vet Microbiol, 1988 May, 17(1), 83 - 90
Double-sandwich enzyme-linked immunosorbent assay for determination of Escherichia coli heat-labile porcine enterotoxins; Picard B et al.; A "double-sandwich" ELISA for the detection and measurement of a heat-labile enterotoxin produced by porcine enterotoxigenic strains of Escherichia coli (LTp) is described . In contrast with other heat-labile toxins, LTp did not bind to agarose gels and exhibited a very low affinity for GM1 in the classical GM1-ELISA technique . The similarity of LTp with cholera toxin was confirmed by immunoblotting . This property allowed the binding of LTp to rabbit IgG anti-cholera toxin antibodies (covalently linked to polystyrene plates) and sheep anti-cholera toxin serum . The immunocomplex was revealed by anti-sheep immunoglobulin antibodies conjugated with peroxidase . Application of the "double-sandwich" ELISA to the quantitation of toxin production by two strains, which differ only in the presence or the absence of the K88ab antigen, showed that the Ent+, K88+ strain produced significantly less toxin than the Ent+, K88- derivative.

Tsitologiia, 1988 May, 30(5), 606 - 10
{Instability of immunoglobulin expression during differentiation induction in the human lymphoblastoid cell line RPMI-6410t}; Seregina TM et al.; The induction of immunoglobulin heavy chain classes switch from IgM to IgG was demonstrated in vitro in cells of RPMI-6410t line . The IgG+-sublines, formed as a result of the switch are characterized by instability of IgG synthesis . After removal of the inductor from the growth environment, IgG+ cells gradually reduce the level of secreted IgG . Such a transition to the functional rest state is likely to be connected with the convertible Ig-gene activity suppression in IgG+ cells, since after their activation by LPS IgG-secretion is partly or completely restored . The IgG+-sublines obtained may serve a convenient model for investigating the Ig-gene expression regulation in differentiated human B-cells.

Prikl Biokhim Mikrobiol, 1988 May-Jun, 24(3), 400 - 4
{Isolation of spheroplasts from Escherichia coli 85 for aspartate-ammonia-lyase localization}; Shcherbakova VN et al.; Conditions were determined for preparation of spheroplasts from E . coli under the action of lysozyme in the presence of EDTA . The preparation took from 10 to 15 min . The degree of conversion to spheroplasts was monitored spectrophotometrically at 660 nm . The spheroplasts formed were unstable in Tris-HCl buffer and immediately lysed, but they were more stable in 1 M sucrose . At lysozyme concentrations above 40 micrograms/ml of the reaction mixture, the cells lysed to a greater extent . The distribution of aspartate ammonia-lyase activity between the precipitate of the spheroplasts and the supernatant allowed the authors to suggest that aspartase should be located in the cytoplasm.

Mutagenesis, 1988 May, 3(3), 257 - 61
Sensitivity of polA mutants of Escherichia coli K-12 to ozone and radiations; Parduez SN et al.; Different polA mutants of Escherichia coli K-12 were exposed to ozone, X-rays and UV radiation, in order to compare the role of the various enzymatic activities of DNA polymerase I in the repair of damage caused by these agents . As was the case with radiations, the polymerase-deficient mutants were the most sensitive to ozone, followed by the 5'-3' exonuclease-deficient mutants . The 3'-5' exonuclease activity of pol I appeared to play a minor role in the repair of damage induced by all these agents.

J Microsc, 1988 May, 150 ( Pt 2), 99 - 115
Classification of images of biomolecular assemblies: a study of ribosomes and ribosomal subunits of Escherichia coli; Frank J et al.; Images of macromolecules obtained in the electron microscope are subjected to correspondence analysis . The structure inherent in the data in the resulting low-dimensional factor space is characterized by a mixed classification method which combines the dynamic clouds clustering technique with hierarchical ascendant classification (HAC) . For our data, the rejection of marginal clusters obtained by dynamic clouds clustering appears as a crucial prerequisite for a stable performance of HAC . The method is applied to two sets of 204 and 177 images that show the 70S ribosome of Escherichia coli, in the range of overlap views as defined by A . Verschoor and co-workers, and to two sets of 480 and 496 images of the 50S subunit of E . coli depleted of L7/L12 proteins in the well-defined crown view . Reproducible classes are obtained, which are characterized by images reconstituted from factorial coordinates . These classes appear to be related to different orientations on the specimen grid (in the case of the 70S particle) and to different conformational states (50S subunit).

Mol Gen Genet, 1988 May, 212(2), 378 - 81
Effect of DNA sequence changes on UV mutability of a purine anticodon triplet of glyU cloned on M13 phage; Ciesla Z et al.; Mutant forms of the glyU (glycyl tRNA) gene cloned in M13mp8 were subjected to uninduced targeted UV mutagenesis; i.e . phage particles were irradiated and used to infect unirradiated umuC+ or irradiated umuC mutant cells . The irradiated phage carried GAG at the anticodon triplet and transitions to GAA were scored . The uninduced targeted mutation rate was reduced by altering the sequence of the gene in the vicinity of the target purine (Pu) residue . In particular a triplet of pyrimidines (PyPyPy) 5' to the target G was changed to PyPuPy in order to prevent formation of cyclcobutane and 6-4 pyrimidine dimers close to the target . On this basis we suggest a mechanism for one type of uninduced regionally targeted UV mutagenesis.

Circ Shock, 1988 May, 25(1), 9 - 20
Energy metabolism during chronic endotoxin infusion in the rat, with special reference to free fatty acid turnover; Goran MI et al.; We have studied various aspects of whole-body energy metabolism during chronic endotoxin infusion in the rat . In particular, we studied free fatty acid kinetics using bolus injections of 14C-palmitate . The general response to 7 days of chronic endotoxin infusion could be divided into an initial 3- to 4-day period of illness with loss of appetite, followed by rapid recovery . Using saline-infused, pair-fed controls, the following observations were made throughout the 7-day period of endotoxin infusion: 1) Plasma free fatty acids, glycerol and 3-hydroxybutyrate concentrations were reduced; 2) plasma glucose concentrations were elevated; 3) free fatty acid production rates were unaffected; 4) metabolic clearance of free fatty acids was unaffected on day 1 but was significantly greater on days 3 and 7; 5) haematocrits and plasma volumes were elevated . The results suggest that the observed changes in circulating free fatty acid concentrations are more likely to be a consequence of haemodynamic alterations than of metabolic alterations.

Circ Shock, 1988 May, 25(1), 1 - 7
Further characterization of a model of chronic endotoxemia in the rat: adenine nucleotide content in liver; Deaciuc IV et al.; Male Sprague-Dawley rats (320-380 g) were treated with Escherichia coli endotoxin (ET) either acutely (3 and 4.5 mg/100 g b.w.,) or chronically (0.1 mg/100 g b.w./24 h, i.v . from subcutaneously implanted osmotic pumps) . Control rats received only sterile saline . At 6 h after injection and after 30, 54, 126, and 198 h of continuous ET infusion, the animals were sacrificed, and adenine nucleotide content in liver was assayed . Acute ET treatment with either dose produced a slight decrease in ATP content (15% and 11% with 3 mg and 4.5 mg ET/100 g b.w., respectively) associated with an increase of ADP and AMP content and a fall of the energy charge (by 9.2% and 6.4%, with the two ET doses, respectively) . Chronic ET treatment did not affect either of the measured parameters . However, in both saline- and ET-infused rats a decrease of ATP content paralleled by an increased level of ADP and AMP and by a fall of energy charge were seen when compared to acutely saline-injected rats . Such changes can be attributed to the surgical trauma associated with pump implantation . It is concluded that the energy status of the liver during endotoxemia cannot account for metabolic disturbances associated with this pathophysiological state.

Mol Microbiol, 1988 May, 2(3), 377 - 83
Repression of the aroF promoter by the TyrR repressor in Escherichia coli K-12: role of the 'upstream' operator site; Cobbett CS; Three sequences are required for complete repression of the aroF promoter by the TyrR repressor protein . Two of these operator sites lie adjacent to each other and overlap the -35 region of the aroF promoter while the third lies about 70 base pairs upstream of the promoter . An aroF-cat (chloramphenicol acetyltransferase) gene fusion has been used to assay the effect of DNA insertions that alter the distance between the two promoter-proximal and the third, distal, operator sites on the repression of the aroF promoter in vivo . The distal site contributes to the repression of the promoter up to a distance of about 400 base pairs and its effect is not dependent on its separation from the first and second sites by an integral number of turns of the DNA helix.

Chem Res Toxicol, 1988 May-Jun, 1(3), 160 - 8
Construction of an Escherichia coli vector containing the major DNA adduct of activated benzo{a}pyrene at a defined site; Benasutti M et al.; The mutagenic and carcinogenic substance benzo{a}pyrene reacts with DNA following activation to its corresponding 7,8-diol 9,10-epoxide (BPDE), and the major DNA adduct (BP-N2-Gua) is formed when the C(10)-position of BPDE reacts with the N2-position of guanine . It is unknown if this adduct is a premutagenic lesion in vivo . Herein, the construction and characterization of an M13mp19-based, E . coli vector that contains BP-N2-Gua located in the unique PstI restriction endonuclease recognition site at nucleotide position 6249 in the (-)-strand is described (designated, BP-N2-Gua-M13mp19) . First, the oligonucleotide 5'-TGCA-3' was reacted with BPDE and a product (5'-T(BP-N2)GCA-3') was isolated by HPLC that, when enzymatically digested to deoxynucleosides, yielded an adduct that comigrated on HPLC with an authentic BP-N2-Gua deoxynucleoside standard . Second, the 5'-hydroxyl group of 5'-T-(BP-N2)GCA-3' was phosphorylated with ATP and T4 polynucleotide kinase, and the product (5'-pT(BP-N2)GCA-3') was purified by HPLC . This product is stable when heated at 80 degrees C at both neutral and alkaline pH . Third, M13mp19 was manipulated such that the sequence 5'-pTGCA-3' was selectively removed from the (-)-strand in its unique PstI recognition site, and 5'-pT(BP-N2)GCA-3' was ligated into this gap with T4 DNA ligase and ATP . The product of this reaction (BP-N2-Gua-M13mp19) was shown to be insensitive to cleavage by PstI, which suggests that a modification is located in the PstI recognition site . The most likely modification is the adduct BP-N2-Gua.

Mol Cell Biol, 1988 May, 8(5), 2211 - 3
Cloning of methylated transforming DNA from Neurospora crassa in Escherichia coli; Orbach MJ et al.; An arg-2 mutant of Neurospora crassa was transformed to prototrophy with a pBR322-N . crassa genomic DNA library . Repeated attempts to recover the integrated transforming DNA or segments thereof by digestion, ligation, and transformation of Escherichia coli, with selection for the plasmid marker ampicillin resistance, were unsuccessful . Analyses of a N . crassa transformant demonstrated that the introduced DNA was heavily methylated at cytosine residues . This methylation was shown to be responsible for our inability to recover transformants in standard strains of E . coli; transformants were readily obtained in a strain which is deficient in the two methylcytosine restriction systems . Restriction of methylated DNA in E . coli may explain the general failure to recover vector or transforming sequences from N . crassa transformants.

Virology, 1988 May, 164(1), 75 - 80
Isolation of mutations of the phage Mu ner gene; Konig U et al.; A method was devised which allows the easy detection of mutations within the ner gene of Mu DNA . This method is based upon the observation that a transcriptional gene A-galK fusion containing the complete ner gene and the cts62 allele does not express the galK gene in an Escherichia coli strain lacking functional integration host factor under inducing conditions (white colonies on MacConkey galactose plates at 42 degrees) In contrast, a gene ner-galK fusion which lacks part of the ner gene exhibits GalK activity (red colonies) on MacConkey galactose plates at 42 degrees . After mutagenesis of a plasmid carrying a transcriptional gene A-galK fusion, putative ner mutants could be identified on indicator plates . Cloning experiments locate the mutation(s) to the right of the HindIII site which is situated within the early promoter of Mu DNA . One of the mutants was sequenced and revealed two substitutions: one within the-10 region of the early promoter, and another near the end of the ner gene . The former lesion was shown to be pleiotropic.

Crit Care Med, 1988 May, 16(5), 521 - 30
Evidence for a role of endorphins in the cardiovascular pathophysiology of primate shock; Gurll NJ et al.; Using the opiate receptor antagonist naloxone, we tested the hypothesis that endorphins act on opiate receptors to cause cardiovascular depression in primate shock . Mean arterial pressure (MAP), cardiac output, and left ventricular contractility (LV dP/dtmax) were measured in 34 anesthetized cynomolgus monkeys . Hemorrhagic shock was induced by bleeding into a heparinized reservoir to achieve (t = 0) and maintain MAP at 45 mm Hg . At t = 60 min, the reservoir was clamped and the animals were treated with 2 mg/kg plus 2 mg/kg.h naloxone (n = 5) or 0.9% NaCl as a control (n = 5) . There were no significant differences in the cardiovascular responses to naloxone and saline when acid-base balance and core body temperature were not controlled . Pressor responses to naloxone, however, were present in proportion to arterial pH and body temperature . When these factors were controlled, naloxone (n = 6) significantly increased MAP and LV dP/dtmax by 48% and 83%, respectively, whereas saline (n = 6) had no significant effect . Blood was reinfused at t = 120 min, and survival rate at 72 h was significantly (p = .01) higher with naloxone (3/6) than saline controls (0/6) . In the endotoxic shock model, cynomolgus monkeys were treated with 2 mg/kg plus 2 mg/kg.h naloxone (n = 6) or 0.9% NaCl (n = 6) when MAP reached 75 mm Hg or its nadir 60 to 90 min after Escherichia coli endotoxin, 5 mg/kg iv . Naloxone significantly increased MAP and LV dP/dtmax by 24% and 22%, respectively, whereas saline had no effect . Survival rate at 48 h was significantly (p = .01) higher with naloxone (6/6) than saline (1/6) . Plasma beta-endorphin and beta-lipotropin concentrations rose three to five-fold in both shock models and were not affected by treatment . We conclude that endorphins are activated in primate shock and act on opiate receptors to contribute to the cardiovascular depression found with hemorrhage and endotoxemia.

Microb Pathog, 1988 May, 4(5), 335 - 44
Cloning of the adenylate cyclase genetic determinant of Bordetella pertussis and its expression in Escherichia coli and B . pertussis; Brownlie RM et al.; A recombinant plasmid, pRMB1, identified from a gene library of B . pertussis, restored adenylate cyclase (AC) and haemolysin (HLY) activities to B . pertussis BP348 (a Tn5-insertion mutant deficient in both these activities) . B . pertussis BP348 was considerably less virulent than wild type strains of B . pertussis when 3-week-old mice were challenged intranasally; possession of pRMB1 restored virulence . Neither AC nor HLY activities were expressed in E . coli harbouring pRMB1 . However, expression of calmodulin-responsive AC was obtained in E . coli when restriction fragments of pRMB1 were subcloned into other vectors; expression depended on the lac and tac promoters of these vectors . The enzyme was not readily solubilized from urea extracts of E . coli and required sonication for efficient release . One plasmid conferred a specific AC enzymic activity to E . coli which was greater than that for wild type B . pertussis strains . Unlike extracts of B . pertussis, extracts from E . coli expressing enzymic AC activity, did not elevate cAMP levels in S49 lymphoma cells . A second plasmid, pRMB2, was identified from the gene library, which contained a trans-acting regulatory determinant required for expression of AC, HLY and other virulence-associated factors in B . pertussis.

Ann Inst Pasteur Microbiol, 1988 May-Jun, 139(3), 295 - 306
Production, purification and partial characterization of a new adhesive factor (F42) produced by enterotoxigenic Escherichia coli isolated from pigs; Leite DS et al.; Production of the F42 adhesive factor by porcine enterotoxigenic Escherichia coli (ETEC) grown on minimal solid medium was glucose-dependent . The addition of alanine and sodium acetate to this medium repressed the expression of this antigen whose production was also inhibited when the pH of the growing medium was lower than 7.4 . The antigen was extracted from F42-positive ETEC grown in minimal liquid medium supplemented with 0.5% glucose . The cells were suspended in buffered 1 M NaCl and heated at 60 degrees C . The supernatant was then treated with ammonium sulphate and the resulting precipitate treated with deoxycholate followed by chromatography of the deoxycholate-soluble material on Sepharose-4B . The molecular weight of F42 purified antigen was near 31,000 daltons and its pI 3.2, as determined by polyacrylamide gel electrophoresis and isoelectric focusing, respectively . Immunoelectrophoretic studies showed that the purified F42 antigen presented a slight anodic migration and was recognized only by its homologous antiserum.

Res Vet Sci, 1988 May, 44(3), 394 - 5
Use of a monoclonal antibody to detect the F41 fimbrial adhesion of enterotoxigenic Escherichia coli; Thorns CJ et al.; A monoclonal antibody that identifies a specific epitope on the F41 fimbrial adhesin was used in coagglutination and enzyme-linked immunosorbent assay (ELISA) tests, to identify successfully strains of Escherichia coli expressing the F41 adhesin . The antibody also bound to frozen sections of ileum from piglets infected with an F41 positive E coli demonstrating that the epitope is expressed in vivo.

J Urol, 1988 May, 139(5), 900 - 3
P-fimbriae receptors in patients with chronic pyelonephritis; Jacobson SH et al.; The adherence of fluorescein isothiocyanate-labeled P-fimbriated Escherichia coli to uroepithelial cells from 19 women with chronic pyelonephritis was determined with the fluorescence-activated cell sorting technique . The application of this method has made it possible to study bacterial binding to a large number of cells . Renal function was determined in all patients and the recurrences of P-fimbriated Escherichia coli bacteriuria, cystitis and acute pyelonephritis during a 3-year followup were studied . We found a significant correlation between the P-fimbriae receptor accessibility on uroepithelial cells and glomerular filtration rate (r equals -0.75, p less than 0.001) . Uroepithelial cells from the patients with chronic pyelonephritis and renal insufficiency had a higher binding capacity of P-fimbriated Escherichia coli than uroepithelial cells from patients with a normal glomerular filtration rate . There was no correlation between kidney function and the availability of P-fimbriae receptors in a control group of patients with polycystic kidney disease.

J Bacteriol, 1988 May, 170(5), 2283 - 6
Arg-220 of the PstA protein is required for phosphate transport through the phosphate-specific transport system in Escherichia coli but not for alkaline phosphatase repression; Cox GB et al.; The pstA gene encodes an integral membrane protein of the phosphate-specific transport system of Escherichia coli . The nucleotide change in the previously described pstA2 allele was found to be a G----A substitution at position 276 of the nucleotide sequence, resulting in the premature termination of translation . Three mutations in the pstA gene were produced by site-directed mutagenesis . The amino acid substitutions resulting from the three site-directed mutations were Arg-170----Gln, Glu-173----Gln, and Arg-220----Gln . These amino acid residues were selected because a previous PstA protein structure prediction placed them within the membrane . The Arg-220----Gln mutation resulted in the loss of phosphate transport through the phosphate-specific transport system, but the alkaline phosphatase activity remained repressed . Neither the Arg-170----Gln nor the Glu-173----Gln mutation affected phosphate transport . The results are discussed in relation to a proposed structure of the PstA protein.

Infect Immun, 1988 May, 56(5), 1364 - 70
Protective antigens against enterotoxigenic Escherichia coli O101:K99,F41 in the infant mouse diarrhea model; Duchet-Suchaux M; Protective antigens present in whole cells of bovine enterotoxigenic Escherichia coli strains were tested in an infant mouse diarrhea model . Reduction of mortality rates of infant mice suckling vaccinated mothers was measured 1, 2, and 5 days after oral challenge with strain B41 (O101:K99,F41:H-) . Vaccines consisted of strains of serogroup O101, O9, O8, or O20 and of variants bearing or not bearing antigens K99 and F41 . K99 and F41 expressions were checked by slide agglutination with K99 and F41 antisera and by mannose-resistant microhemagglutination with horse, sheep, and guinea pig erythrocytes . Absence of production of K99 antibodies following hyperimmunization with K99-negative variants was established in rabbit antisera . Strains bearing K99 alone induced less protection of infant mice 5 days after challenge than strains bearing F41 alone or both K99 and F41 . Absence of expression of K99 in the vaccinal strains resulted in either a slight decrease (strains bearing additional F41) or complete abolishment (strains bearing K99 alone) of protection . Failure of F41 synthesis by O101 strains or variants resulted in no protection 5 days after challenge . F41 probably also supplied most of the protection induced by the O9 strain . When negative for both K99 and F41, strains of serogroup O101 still provided protection 1 and 2 days after challenge . This protection was also induced by strain H510a, the reference for O101 antigen . Thus, O antigen contributed to the best vaccinal protection, in addition to K99 and F41.

Infect Immun, 1988 May, 56(5), 1334 - 40
Identification of a human erythrocyte receptor for colonization factor antigen I pili expressed by H10407 enterotoxigenic Escherichia coli; Pieroni P et al.; We have identified a receptor for colonization factor antigen I (CFA/I) pili in human erythrocyte membranes . Erythrocyte binding assays, using whole organisms, suggested that the CFA/I receptor was a glycoprotein containing important sialic acid moieties . Subsequently, human erythrocyte membranes were extracted with lithium diiodosalicylate to obtain a soluble glycoprotein fraction from which to isolate receptors . The extracted material caused agglutination of the CFA/I+ but not the CFA/I- organisms at a protein concentration of 0.5 mg/ml . The CFA/I receptor was identified in iodinated extract by an affinity isolation procedure, using whole bacterial cells . Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography of the washed, extract-coated H10407 CFA/I+ organisms revealed a band with an apparent molecular weight of 26,000 which was present in the original extract but was not observed on extract-coated H10407 CFA/I- bacteria . The addition of purified CFA/I pili reduced binding of the 26,000-molecular-weight receptor to CFA/I+ bacteria . The CFA/I-specific receptor species also bound to wheat germ agglutinin-agarose . This observation supported the suggestion that the CFA/I receptor identified in this report is a sialoglycoprotein.

Infect Immun, 1988 May, 56(5), 1288 - 94
Characterization of P fimbriae on O1, O7, O75, rough, and nontypable strains of Escherichia coli; Pere A et al.; P fimbriae of 37 uropathogenic Escherichia coli O1:K1, O7:K1, O22, O75, rough:K1, and nontypable strains were characterized by immunoprecipitation with 14 fimbria-specific rabbit antisera . The fimbrial composition of these strains, as reflected by the apparent molecular weights of the fimbrial peptides, was correlated with the O serogroup of the strains, but serological cross-reactivity of P fimbriae of different E . coli serogroups was frequently observed . The genetic clonal relationships of the strains were analyzed by determining the electrophoretic types, based on 18 chromosomally encoded enzymes . Among the O1:K1 strains, the same P-fimbrial variants occurred on strains that were either closely related or very distinct in their electrophoretic types, indicating that the P fimbriae have evolved in association with the O and K antigens . In contrast, certain O7:K1 and R:K1 strains as well as some O22 and O75 strains were genotypically identical and shared similar P-fimbrial variants, which differed serologically from those of other E . coli serogroups . Our results show that, despite the structural variability seen in electrophoretic analysis of P fimbriae of different serogroups, many P-fimbrial variants share common antigenic determinants that are recognized by rabbit antisera . Based on immunoprecipitation analyses, three anti-P-fimbria sera have now been identified that react with P fimbriae of 82 of 84 uropathogenic E . coli strains characterized in Finland.

Infect Immun, 1988 May, 56(5), 1057 - 60
A hemagglutinin of uropathogenic Escherichia coli recognizes the Dr blood group antigen; Nowicki B et al.; A receptor moiety and blood group substance recognized by the O75X adhesin was studied . Well-defined erythrocytes representing different blood group systems and bacterial derivatives carrying plasmid pBJN406 encoding the adhesin were used in a direct hemagglutination assay . We showed that Dr blood group antigen, a component of the IFC blood group complex, is the receptor for the O75X fimbrialike adhesin (Dr hemagglutinin) of uropathogenic Escherichia coli . The molecule recognized by the Dr hemagglutinin on Dr blood group substance is a chloramphenicol-like structure . The inhibitory effect of the active compounds indicates that a tyrosine-containing molecule could be a natural receptor for the Dr hemagglutinin . Dr blood group substance was found in tubular basement membrane and Bowman's capsule of the human kidney . Specific attachment of a Dr hemagglutinin-positive bacterial strain to the kidney substructures was inhibited by chloramphenicol.

Infect Immun, 1988 May, 56(5), 1023 - 9
Hyperadhesive mutant of type 1-fimbriated Escherichia coli associated with formation of FimH organelles (fimbriosomes); Abraham SN et al.; The relationships of the genes and gene-products mediating D-mannose-specific attachment of type 1 fimbriae of Escherichia coli to eucaryotic cells were investigated by deletion mutation analysis of recombinant plasmid pSH2, which carries the genetic information for the synthesis and expression of functional type 1 fimbriae . Mutant pUT2004 was derived by a deletion remote from the structural gene encoding the 17-kilodalton (kDa) subunit protein of type 1 fimbriae . Phenotypically, the mutant demonstrated an eightfold-higher mannose-specific hemagglutination titer than the parent strain . On electron microscopy, the mutant strain expressed the same number of fimbriae as the parent strain . However, numerous 10-nm-diameter rounded structures (fimbriosomes) were observed both closely associated with fimbriae and in the culture medium . Fimbriosomes isolated from the medium agglutinated guinea pig erythrocytes in a mannose-sensitive manner . Dissociation of the fimbriosomes yielded a single 29-kDa protein, as demonstrated by sodium dodecyl sulfate gel electrophoresis . Antibodies raised against fimbriosomes reacted with a 29-kDa protein on immunoelectroblots of dissociated type 1 fimbriae and also blocked the adherence of other strains of type 1 fimbriated E . coli to eucaryotic cells . These findings suggest that the enhanced adhesive properties of the mutant pUT2004 strain are associated with overproduction of the 29-kDa FimH in the form of fimbriosomes which contain the determinant of the D-mannose-sensitive adhesion of type 1 fimbriae.

Plasmid, 1988 May, 19(3), 222 - 30
Structural instability of a bifunctional plasmid pZG1 and single-stranded DNA formation in Streptomyces; Pigac J et al.; Structural instability of a hybrid plasmid pZG1, consisting of Escherichia coli pBR322 and Streptomyces pIJ350 plasmids, has been studied in Streptomyces . After transformation of S . lividans 1326 and S . rimsus R6 protoplasts with pZG1, transformants harbored the intact pZG1 and various deleted plasmid forms . The pattern of deleted plasmids varied with the transformant colony age and changed upon subcultivation . The presence of intact pZG1 in at least a part of the Streptomyces colonies indicated that the plasmid was capable of replicating in the transformants and that deletion events occurred after at least one round of replication . Less instability of pZG1 in S . rimosus R6 was observed when this strain was transformed with the DNA isolated from the same strain . pZG1 and its various derivatives were found in S . lividans 1326 and S . rimosus R6 as double- and single-stranded DNA molecules . Structural instability of pZG1 could therefore be due at least in part to the presence of single-stranded DNA.

Mol Biol (Mosk), 1988 May-Jun, 22(3), 645 - 58
{Structural and functional organization of the transposon Tn2555 carrying saccharose utilization genes}; Doroshenko VG et al.; Transposon Tn2555 was isolated from a clinical E . coli strain carries the genes for sucrose utilization . Previously it was shown that Tn2555 is very unstable and undergoes structural rearrangements with a high frequency . Several deletion derivatives of Tn2555 and one with an inversion of the internal segment were found . They form the Tn2555 transposon family . This paper describes further structural and functional analysis of Tn2555 . In the course of the experiments on pBR325 (Mob-) mobilization by conjugative RP4 derivatives, containing Tn2555 family elements, it was found, that all of them induce cointegrate formation . Some of these cointegrates were able to dissociate in rec+ and recA E . coli cells . Restriction endonuclease analysis of the resulting plasmids have shown, that among them were the end products of the Tn2555 transposition from RP4 to pBR325 . Besides, the pBR325 derivatives, containing a discrete DNA segment of approximately 800 b.p., originating from Tn2555, were found . The segment can transpose from pBR325 to RP4 indicating that it is an insertion sequence . This new IS-element was designated IS286 . The size and the genetic properties of IS286 resemble those of the IS1 element . However restriction analysis and Southern hybridization data show no significant homology between IS286 and IS1 . It was found that the Tn2555 family elements are flanked by directly repeated IS286 . One of them (Tn2555.3) contains an additional copy of IS286 in its internal region.

J Biochem (Tokyo), 1988 May, 103(5), 766 - 72
Chemical modifications of histidyl and tyrosyl residues of inorganic pyrophosphatase from Escherichia coli; Samejima T et al.; Chemical modifications by photooxidation in the presence of rose bengal (RB) and with tetranitromethane (TNM) were carried out to elucidate the amino acid residues involved in the active site of inorganic pyrophosphatase (pyrophosphate phosphohydrolase) {EC 3.6.1.1} from Escherichia coli Q13 . The photooxidation caused almost complete inactivation, which followed pseudo-first-order kinetics depending on pH and concentration of RB . The presence of Mg2+ or complex between Mg2+ and substrate or substrate analogues, imidodiphosphate and sodium methylenediphosphate, gave partial protection against the photoinactivation, whereas the substrate alone showed no protective effect . The enzyme was almost completely inactivated by chemical modification with TNM, depending upon the concentration of TNM . The amino acid analyses and enzyme activity measurements revealed that 2 histidyl residues among 5 photooxidized residues and 2 tyrosyl residues per subunit were essential for the enzyme activity . The circular dichroism (CD) spectra in the far ultraviolet region showed no significant alteration during these two modifications, indicating that the polypeptide chain backbone of the enzyme remained unaltered . However, the modifications altered considerably the CD bands in the near ultraviolet region and the fluorescence spectra, indicating that subtle change in conformation had occurred in the vicinity of the active site in the enzyme molecule . These results strongly suggest that histidyl and tyrosyl residues may be involved in the active site or be located in the vicinity of the active site and seem to participate in the mechanism of stability against heat inactivation.

Vet Microbiol, 1988 May, 17(1), 29 - 43
Mapping of porcine parvovirus DNA and development of a diagnostic DNA probe; Krell PJ et al.; Dimeric and monomeric replicative forms of DNA of porcine parvovirus (PPV) strain NADL-2 were isolated and examined by restriction enzyme analysis and reciprocal Southern blot hybridization during development of a DNA probe for PPV . Genomic single stranded PPV DNA was 5.0 kb long, and results substantiated the rolling-hairpin model of parvovirus DNA replication with the primer sequence located in the 3' terminal hairpin loop . An additional finding was the generation of a 4.7 kb species of viral DNA which was considered to be a 0.3 kb deletion variant of genomic PPV DNA . A 3.0 kb DNA fragment obtained by Pst I/Hind III digestion of monomer replicative form DNA was cloned into a plasmid vector, pUC 19 . The cloned fragment, recovered from transformed Escherichia coli strain TB1 and labelled with {32P} dCTP, was evaluated by dot hybridization as a probe for PPV in infected cell cultures . The probe was specific for PPV infected cells, and was 100 times more sensitive than the standard hemagglutination test.

Prikl Biokhim Mikrobiol, 1988 May-Jun, 24(3), 310 - 8
{Isolation and various properties of nucleoside monophosphate kinases from Escherichia coli}; Rikhter VA et al.; A technique is proposed for isolation of nucleosidemonophosphate kinases--AMP-kinase (EC 2.7.4.11), GMP-kinase (EC 2.7.4.8), CMP-kinase (EC 2.7.4.14), UMP-kinase (EC 2.7.4.14) and TMP-kinase (EC 2.7.4.9)--from E . coli MRE-600 . It involves cell destroying, precipitation of nucleic acids with polyethyleneimine, fractionation with ammonium sulphate followed by chromatography on different carriers (DEAE-Toyopearl-650 M, Matrex gel Blue A, Matrex gel Red A) . The technique enables all the five enzymes to be obtained separately and without contaminations with nucleotide dephosphorylating enzymes . For all the enzymes the pH optimum was found to range from 6.5 to 8.0, and Mg2+ ions were found to be the best activator for all the enzymes studied . The substrate specificity was investigated with respect to acceptors and donors of the phosphate groups . The enzymes showed strict specificity to the heterocyclic base of the acceptor phosphate group . AMP-, GMP- and CMP-kinases phosphorylated the corresponding deoxynucleoside monophosphates less effectively than ribonucleoside monophosphates . ATP was found to be the most effective phosphate donor for all the enzymes under study.

Bioorg Khim, 1988 May, 14(5), 694 - 6
{A method for inducing the point C----T transitions in DNA sequences corresponding to the ends of restriction sites}; Medvedev OA et al.; A new method for inducing of C----T substitutions into cytosine-containing restriction sites is developed . The method, based on the selective modification of cytosine residues in DNA sticky ends by sodium bisulfite, was illustrated by induction of a base substitution (C----T at the BamHI site of pBR322 plasmid DNA.

Biochem Cell Biol, 1988 May, 66(5), 425 - 35
Characterization of CDPdiacylglycerol hydrolase in mitochondrial and microsomal fractions from rat lung; Mok A et al.; CDPdiacylglycerol pyrophosphatase (E.C . 3.6.1.26) activity has been examined in rat lung mitochondrial and microsomal fractions . While the mitochondrial hydrolase exhibited a broad pH optimum from pH 6-8, the microsomal activity decreased rapidly above pH 6.5 . Apparent Km values of 36.2 and 23.6 microM and Vmax values of 311 and 197 pmol.min-1.mg protein-1 were observed for the mitochondrial and microsomal preparations, respectively . Addition of parachloromercuriphenylsulphonic acid led to a marked inhibition of the microsomal fraction but slightly stimulated the mitochondrial activity at low concentrations . Mercuric ions were inhibitory with both fractions . Although biosynthetic reactions utilizing CDPdiacylglycerol require divalent cations, addition of Mg2+, Mn2+, Ca2+, Zn2+, Co2+, and Cu2+ all inhibited the catabolic CDPdiacylglycerol hydrolase activity in both fractions . EDTA and EGTA also produced an inhibitory effect, especially with the mitochondrial fraction . Although addition of either adenine or cytidine nucleotides led to a decrease in activity with both fractions, the marked susceptibility to AMP previously reported for this enzyme in Escherichia coli membranes, guinea pig brain lysosomes, and pig liver mitochondria was not observed . These results indicate that rat lung mitochondria and microsomes contain specific CDPdiacylglycerol hydrolase activities, which could influence the rate of formation of phosphatidylinositol and phosphatidylglycerol for pulmonary surfactant.

Mol Gen Genet, 1988 May, 212(2), 390 - 2
Plasmids with the uidA reporter gene for the detection of promoters and transcription signals; Bardonnet N et al.; We have constructed promoter-probe plasmids, pNB4 and pNB5, based on the promoterless gene for beta-glucuronidase (uidA) of Escherichia coli . Unique restriction sites for EcoRI, SacI, KpnI, SmaI, XmaI, XbaI, SalI, SphI and HindIII in pNB4 and for HindIII, PstI and BglII in pNB5 were included upstream of the uidA structural gene . The usefulness of these plasmids was demonstrated by cloning the promoter-operator region of the E . coli uxaB gene . We observed that expression of the uxaB-uidA operon fusion followed the transcription-regulating properties of the uxaB promoter . Another construct, pNB2, can be used to detect operator and terminator signals . Recipient cells transformed with such recombinant plasmids can be revealed by growth on medium containing a chromogenic beta-glucuronidase substrate.

Mol Gen Genet, 1988 May, 212(2), 375 - 7
Expression of Tn5-derived kanamycin resistance in the fungus Phycomyces blakesleeanus; Arnau J et al.; A plasmid, carrying the Tn5 gene for kanamycin resistance lacking its own promoter, has successfully been used in the selection of DNA sequences of the fungus Phycomyces blakesleeanus having promoter activity in Escherichia coli . Many of these sequences were also effective in promoting resistance to kanamycin when the corresponding chimeric plasmids were introduced in the fungus via spheroplast transformation . The selected phenotype was easily propagated through vegetative spores and behaved as a stable character since it was not appreciably lost in the absence of selection.

Mol Gen Genet, 1988 May, 212(2), 317 - 24
Effect of recF, recJ, recN, recO and ruv mutations on ultraviolet survival and genetic recombination in a recD strain of Escherichia coli K12; Lloyd RG et al.; DNA repair and recombination were investigated in a recD mutant of Escherichia coli which lacked the nuclease activity of the RecBCD enzyme . The resistance of this mutant to ultraviolet (UV) light was shown to be a function of recJ . A recD recJ double mutant was found to be more sensitive to UV radiation than a recB mutant, whereas recD and recJ single mutants were resistant . Recombination in conjugational crosses with Hfr donors was also reduced in recD recJ strains, but the effect was modest in comparison with the sensitivity to UV . Within certain limits, mutations in recF, recN, recO, lexA and ruv did not affect sensitivity to UV and recombination in a recD mutant any more than in a recD+ strain . The possibility that recD and recJ provide overlapping activities, either of which can promote DNA repair and recombination in the absence of the other, is discussed.

Mol Gen Genet, 1988 May, 212(2), 265 - 70
Transcription of the target is required for IS102 mediated deletions; Bernardi F et al.; We have constructed several plasmids to test the specificity of target selection for IS102 associated deletions . We had previously shown that the cIII region of phage lambda is a target for IS102 mediated deletions and this region was therefore introduced in pSC101, where IS102 resides, in such a way that it was silent, transcribed or fully expressed . The deletion selection was provided by the galactokinase system . The results we have obtained show that transcription of the target region is required for deletion formation . The frequency of deletions in this target depends on its position along the transcript and is also influenced by translation . Implications of these results on the regional specificity of transposition are discussed.

Mol Gen Genet, 1988 May, 212(2), 199 - 202
Identification, cloning, nucleotide sequence and chromosomal map location of hns, the structural gene for Escherichia coli DNA-binding protein H-NS; Pon CL et al.; Beginning with a synthetic oligonucleotide probe derived from its amino acid sequence, we have identified, cloned and sequenced the hns gene encoding H-NS, an abundant Escherichia coli 15 kDa DNA-binding protein with a possible histone-like function . The amino acid sequence of the protein deduced from the nucleotide sequence is in full agreement with that determined for H-NS . By comparison of the restriction map of the cloned gene and of its neighboring regions with the physical map of E . coli K12 as well as by hybridization of the hns gene with restriction fragments derived from the total chromosome, we have located the hns gene oriented counterclockwise at 6.1 min on the E . coli chromosome, just before an IS30 insertion element.

Mol Microbiol, 1988 May, 2(3), 419 - 25
Aerobic expression of the nar operon of Escherichia coli in a fnr mutant; Bonnefoy V et al.; Mutations allowing aerobic expression of the anaerobically controlled nar operon have been located in the autoregulated fnr gene . Cloning and sequencing of the mutant fnrd20 allele, and fnr mRNA quantitation by dot blot assay, revealed that the mutation was the result of an IS5 insertion into the control region of fnr that enhanced transcription of the fnr gene at least ten-fold . Examination of the regulatory region of the negatively autoregulated fnr gene indicated that it shared homologous sequences with the positively Fnr-controlled frd and nar operons . The increase in fnr transcription in the fnrd20 mutated allele could be partly the result of loss of autoregulation, since the IS5 separated the Fnr target site from the '-35' region of the promoter.

Mol Microbiol, 1988 May, 2(3), 363 - 70
Nucleotide sequencing and expression of the fadL gene involved in long-chain fatty acid transport in Escherichia coli; Said B et al.; The fadL gene of Escherichia coli codes for an outer membrane protein involved in long-chain fatty acid transport . Its product was purified from outer membrane proteins . We determined the nucleotide sequence of a 2.8-kb chromosomal DNA segment that contains the fadL gene . The fadL gene consists of a 1149-nucleotide coding region and contains a highly hydrophobic polypeptide of 383 amino acids with a calculated molecular weight of 42,380 . We have used S1-mapping analysis to identify the transcription initiation site . A region exhibiting extensive dyad symmetry and perfect homology to the catabolite activator protein binding site was detected.

DNA, 1988 May, 7(4), 275 - 86
Human gene encoding the 78,000-dalton glucose-regulated protein and its pseudogene: structure, conservation, and regulation; Ting J et al.; The isolation and characterization of a human functional GRP78 gene and a processed pseudogene are described . We present the complete primary structure of the human GRP78 gene, which spans over 5 kb and consists of eight exons . Sequence comparisons reveal that the GRP78 gene shares unusual homology among the human, rat, and hamster in the protein-coding and 3' untranslated regions . In addition, short domains highly conserved with HSP70 isolated from human, Drosophila, Xenopus, yeast, and E . coli DNA are identified within the hydrophobic regions of GRP78 . The intronless pseudogene resembles that of a processed gene . It is flanked by a short direct repeat and is embedded within an AT-rich genomic region . The highly active promoter from the functional human GRP78 gene contains a TATA box, five CCAAT sequences, and two potential binding sites for the transcriptional factor Sp1 . It consists of a distal domain that enhances basal level expression and a proximal domain essential for responses to calcium ionophore and for a temperature-sensitive mutation which induce the GRP78 gene . Both domains are highly conserved between the rat and the human GRP78 promoters.

DNA, 1988 May, 7(4), 243 - 51
Synthesis and expression of a gene for a mini type II dihydrofolate reductase; Vermersch PS et al.; The Type II dihydrofolate reductases (DHFR) are resistant to the folate analogs, trimethoprim and methotrexate . The monomer is very small (MW 9,000) and has no structural homology with other known DHFR types . A dhfr structural gene was synthesized which incorporates many unique restriction sites (Nco I, Nhe I, Pvu I, Hind III, Sma I, Bgl II, Xho I, and Ban I) within the coding sequence . This gene encodes a small DHFR (68 amino acids) which is 10 amino acids shorter at the amino-terminus than natural Type II DHFRs . The last 60 residues of the synthetically encoded protein are identical in sequence to R388 DHFR . The enzyme is functional and relatively stable, as evidenced by trimethoprim resistance conferred to cells expressing the synthetic gene . The gene was cloned onto a high-copy-number plasmid, pPV7SYN5, in which a trp-lac promoter drives transcription of both the dhfr gene and the primer for plasmid replication (RNA II) . High levels of the small DHFR are accumulated in stationary phase cultures of MC1061(p3) containing pPV7SYN5 without the addition of IPTG.

Antimicrob Agents Chemother, 1988 May, 32(5), 740 - 6
Effect of aminoglycoside concentration on reaction rates of aminoglycoside-modifying enzymes; Bongaerts GP et al.; Reaction rates of several reference aminoglycoside-modifying enzymes were studied at various substrate concentrations . The resulting concentration-response curves showed wide variation in threshold concentration, in curve slope, in enzyme saturation, and in substrate inhibition . Together, the curves of a defined aminoglycoside panel yielded more specific information for each individual aminoglycoside-modifying enzyme tested than did conventional substrate profiles obtained at a single substrate concentration.

Am J Vet Res, 1988 May, 49(5), 687 - 92
Transposon mutagenesis in Bordetella avium; Leyh RD et al.; Transposon mutagenesis was used to introduce specific mutations in the chromosome of Bordetella avium . The transposon Tn5 was transferred by conjugation to B avium from Escherichia coli strain SM10 by the suicide plasmid vector pSUP1011 . A defined minimal medium for growth of B avium was developed for scoring transposon-containing mutants for specific nutritional requirements . Approximately 1% of all mutants tested were auxotrophic in some form, and these organisms were grouped into 13 classes . Also, 2 classes of hemagglutination-negative mutants were isolated . Stability of Tn5 insertion was examined, and frequencies of reversion less than 10(-7) were observed, indicating stable mutations.

J Appl Physiol, 1988 May, 64(5), 2100 - 7
Dexamethasone blocks increased leukotriene B4 production during endotoxin-induced lung injury; Olson NC et al.; We hypothesized that leukotriene B4 (LTB4) might be produced during endotoxemia in pigs and, if so, might play a role in the pathophysiology of acute respiratory failure . Escherichia coli endotoxin (055-B5) was infused intravenously into anesthetized pigs at 5 micrograms/kg the 1st h, followed by 2 micrograms.kg-1.h-1 for 3 h . Endotoxemic pigs were treated with dexamethasone (DEX, iv) 18 h (5 mg/kg) and 1 h (5 mg/kg) before onset of endotoxemia . During phases I (i.e., 0-2 h) and II (i.e., 2-4 h), endotoxin decreased cardiac index, caused granulocytopenia, and increased mean pulmonary arterial pressure, pulmonary vascular resistance, alveolar-arterial O2 gradient, and hematocrit . During phase II, plasma LTB4 levels were increased (as determined by radioimmunoassay, reverse-phase high-performance liquid chromatography, and ultraviolet spectroscopy) . Endotoxin increased the levels of LTB4 and albumin in bronchoalveolar lavage fluid (BALF) . DEX blocked or greatly attenuated the endotoxin-induced hemodynamic abnormalities and blocked the increases in plasma and BALF LTB4 levels . We conclude that LTB4 is produced during porcine endotoxemia and could possibly play a role in the pathophysiology of endotoxin-induced lung injury in anesthetized pigs.

Mol Cell Biol, 1988 May, 8(5), 2097 - 104
Establishment of composite DNA derived from L factor as a plasmid in mouse embryonal carcinoma (F9) cells; Nishimori K et al.; We have recently reported a mammalian cell plasmid (L factor) whose structure is related to that of polyomavirus (T . Kusano, H . Uehara, H . Saito, K . Segawa, and M . Oishi, Proc . Natl . Acad . Sci . USA 84:1789-1793, 1987) . When composite DNA constructed from L factor and a foreign gene was introduced into mouse embryonal carcinoma (F9) cells by transfection, the DNA was reestablished in the cells as a plasmid . The reestablished plasmid DNA in F9 cells could be rescued in Escherichia coli . The plasmid-bearing cells underwent normal in vitro differentiation in response to retinoic acid . The efficiency of plasmid establishment of the L-factor-derived DNA and transcriptional and transient replicational activities were compared with those of similar composite DNA constructed from polyomavirus and an embryonal carcinoma mutant of polyomavirus which is permissive in F9 cells . The results suggest an inverse relationship between the efficiency of the plasmid establishment and the activity of gene expression controlled by the intrinsic enhancer-promoter of the DNA.

Oral Surg Oral Med Oral Pathol, 1988 May, 65(5), 545 - 50
Oral squamous papillomas: detection of HPV DNA by in situ hybridization; Eversole LR et al.; Oral squamous papillomas were segregated from other papillary lesions on the basis of histopathologic features . Twenty representative papillomas were evaluated for the presence of papillomavirus genus-specific antigen with the use of an immunoperoxidase technique . These same tumors were analyzed for human papillomavirus (HPV) types 2, 4, 6, and 11 with biotinylated full-length double-stranded DNA probes by in situ hybridization . Only one case exhibited papillomavirus antigen reactivity . Alternatively, seven of twenty cases (35%) yielded positive results for HPV 6 or 11 DNA; one papilloma exhibited a dual infection with both HPV 2 and 6 when assayed under conditions of high-stringency hybridization . It is concluded that some oral squamous papillomas harbor HPV genotypes akin to those encountered in genital tract condylomas . Viral DNA can be detected in the absence of capsid antigen immunoreactivity, thereby obviating the use of antigen detection assays for determining the presence or absence of virus.

Proc Natl Acad Sci U S A, 1988 May, 85(10), 3484 - 8
Isolation of the gene encoding the Hin recombinational enhancer binding protein; Johnson RC et al.; In vitro DNA inversion mediated by the protein Hin requires the presence of a recombinational enhancer sequence located in cis relative to the recombination sites and a protein, Fis, which binds to the enhancer . We have cloned and determined the primary sequence of the gene encoding Fis . The deduced amino acid sequence of Fis indicates that the protein is 98 amino acids long and contains a potential helix-turn-helix DNA binding motif at its carboxyl terminus . The gene encoding Fis maps at 72 min on the Escherichia coli chromosome . The construction of mutant strains of E . coli that lack a functional fis gene demonstrates that Fis is not essential for cell growth under laboratory conditions but is required for high rates of Hin-mediated site-specific inversion in vivo.

Virology, 1988 May, 164(1), 156 - 64
Detection of a murine coronavirus nonstructural protein encoded in a downstream open reading frame; Leibowitz JL et al.; Mouse hepatitis virus (MHV) gene 5 contains two open reading frames . We have expressed the second open reading frame of this gene (gene 5 ORF 2) in an Escherichia coli expression system . This system utilized a plasmid which contained the promoter and the first 36 codons of the recA gene fused in frame with the MHV gene 5 ORF 2, which is fused in turn to the beta-galactosidase gene . The protein product of this gene fusion was used to raise antibody to gene 5 ORF 2 . The specificity of the antibody was verified by immunoprecipitation of the in vitro transcribed and translated protein product of gene 5 ORF 2 . The second reading frame of MHV gene 5 was shown to be expressed during the course of infection by immunocytochemistry and radioimmunoprecipitation using the antibody raised against the E . coli fusion protein and by two-dimensional gel electrophoresis.

Mutat Res, 1988 May, 199(1), 1 - 9
Vector-mediated DNA double-strand break repair analysis in normal, and radiation-sensitive, Chinese hamster V79 cells; Debenham PG et al.; DNA double-strand break repair was assessed in 2 new radiation-sensitive V79 hamster cell lines (irs1 and irs2) by their ability to rejoin restriction endonuclease cuts in a transferred selectable SV40--E . coli gpt recombinant gene . The studied gene was carried in the vector pPMH16 which also contained a second selectable HSVtk-neo recombinant gene which acted as a control for DNA transformation . The parental V79 cells showed correct rejoining of KpnI and EcoRV double-strand breaks in approximately 18% and 36% of transformants respectively (correcting for the expression of undamaged gpt in neo+ transformants) . irs1 shows a significantly reduced (approximately 3-fold) ability to rejoin correctly such double-strand scissions . However, irs2 rejoined such lesions as correctly as the V79 cells . The data are discussed in the context of the assay and the possible repair deficiencies of these radiosensitive mutant cells.

J Bacteriol, 1988 May, 170(5), 2163 - 73
Isolation and characterization of noncleavable (Ind-) mutants of the LexA repressor of Escherichia coli K-12; Lin LL et al.; The LexA repressor of Escherichia coli represses a set of genes that are expressed in the response to DNA damage . After inducing treatments, the repressor is inactivated in vivo by a specific cleavage reaction which requires an activated form of RecA protein . In vitro, specific cleavage requires activated RecA at neutral pH and proceeds spontaneously at alkaline pH . We have isolated and characterized a set of lexA mutants that are deficient in in vivo RecA-mediated cleavage but retain significant repressor function . Forty-six independent mutants, generated by hydroxylamine and formic acid mutagenesis, were isolated by a screen involving the use of operon fusions . DNA sequence analysis identified 20 different mutations . In a recA mutant, all but four of the mutant proteins functioned as repressor as well as wild-type LexA . In a strain carrying a constitutively active recA allele, recA730, all the mutant proteins repressed a sulA::lacZ fusion more efficiently than the wild-type repressor, presumably because they were cleaved poorly or not at all by the activated RecA protein . These 20 mutations resulted in amino acid substitutions in 12 positions, most of which are conserved between LexA and four other cleavable proteins . All the mutations were located in the hinge region or C-terminal domain of the protein, portions of LexA previously implicated in the specific cleavage reactions . Furthermore, these mutations were clustered in three regions, around the cleavage site (Ala-84-Gly-85) and in blocks of conserved amino acids around two residues, Ser-119 and Lys-156, which are believed essential for the cleavage reactions . These three regions of the protein thus appear to play important roles in the cleavage reaction.

J Bacteriol, 1988 May, 170(5), 2136 - 42
Isolation and characterization of methyl viologen-sensitive mutants of Escherichia coli K-12; Morimyo M; Escherichia coli mutants sensitive to methyl viologen (MV), an active oxygen propagator, were isolated . Among them, the new genes mvrA and mvrB were mapped at 7 and 28 min on the E . coli linkage map, respectively . MV toxicity was exerted only in the presence of oxygen and was suppressed by the radical scavenger uric acid but not by the hydroxyl radical scavenger mannitol . The mvr mutants were sensitive only to MV and had a normal repair capacity for the MV-damaged DNA . From these results, these mutants were assumed to be related to the elimination of MV-specific toxic species . Gene mvrA was cloned into vector pBR322 and its sequence was determined . The mvrA gene, which was predicted to range in size from 600 to 900 base pairs (bp) by transposon Tn1000 insertion analysis, was identified to be 807 bp, with an approximately 60-bp promoter sequence carrying consensus sequences for the -35 region, the -10 region, and a ribosome-binding site . The MvrA protein deduced from the DNA sequence was 29.7 kilodaltons, which was in good agreement with the 29 kilodaltons of the MvrA protein identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis after a maxicell labeling experiment.

J Bacteriol, 1988 May, 170(5), 2106 - 12
Isolation and properties of minB, a complex genetic locus involved in correct placement of the division site in Escherichia coli; de Boer PA et al.; Mutation of Escherichia coli minicell locus (minB) results in aberrant placement of the division septum . In this paper we report the isolation and characterization of the minB locus . Replacement of the chromosomal minB+ allele by cloned minB sequences containing transposon insertions resulted in the minicell phenotype, indicating that minB+ function is required to maintain the normal division pattern . Paradoxically, overexpression of the locus also resulted in the minicell phenotype . The locus codes for several peptides whose expression is coordinately affected by transposon mutations that also eliminate minB+ function . A subset of the minB peptides is sufficient to prevent minicell formation in minB1 mutants or to induce minicell formation when overproduced in wild-type strains, implicating these peptides in the normal process of localization of the division site . The results indicate that minB is a complex locus whose expression must be maintained within certain limits to maintain the normal pattern of localization of the division septum.

J Bacteriol, 1988 May, 170(5), 2089 - 94
Physical and biochemical characterization of cloned sbcB and xonA mutations from Escherichia coli K-12; Phillips GJ et al.; In Escherichia coli K-12, sbcB/xonA is the structural gene for exonuclease I, an enzyme that hydrolyzes single-stranded DNA to mononucleotides in the 3'-to-5' direction . This enzyme has been implicated in the DNA repair and recombination pathways mediated by the recB and recC gene products (exonuclease V) . We have cloned several sbcB/xonA mutant alleles in bacterial plasmids and have partially characterized the cloned genes and their protein products . Two of the mutations (xonA2 and xonA6) retain no detectable exonucleolytic activity on single-stranded DNA . The xonA6 allele was shown to harbor an insertion of an IS30-related genetic element near the 3' end of the gene . Two other mutations, sbcB15 and xonA8, exhibited significantly reduced levels of exonuclease I activity as compared to the cloned wild-type gene . A correlation was observed between levels of exonuclease I activity and the ability of the sbcB/xonA mutations to suppress UV sensitivity in recB and recC strains . Also, recombinant plasmids bearing either the sbcB15 or xonA6 allele exhibited a high degree of instability during growth of their bacterial hosts . The results suggest that the sbcB/xonA gene product is a bi- or multifunctional protein that interacts with single-stranded DNA and possibly with other proteins in the suppression of genetic recombination and DNA-repair deficiencies in recB and recC mutants.

J Bacteriol, 1988 May, 170(5), 2005 - 11
Translational control of exported proteins that results from OmpC porin overexpression; Click EM et al.; The regulation of synthesis and export of outer membrane proteins of Escherichia coli was examined by overexpressing ompC in multicopy either from its own promoter or from an inducible promoter in an expression vector . Overexpression of OmpC protein resulted in a nearly complete inhibition of synthesis of the OmpA and LamB outer membrane proteins but had no effect on synthesis of the periplasmic maltose-binding protein . Immunoprecipitation of labeled proteins showed no evidence of accumulation of uncleaved precursor forms of OmpA or maltose-binding protein following induction of OmpC overexpression . The inhibition of OmpA and LamB was tightly coupled to OmpC overexpression and occurred very rapidly, reaching a high level within 2 min after induction . OmpC overexpression did not cause a significant decrease in expression of a LamB-LacZ hybrid protein produced from a lamB-lacZ fusion in which the fusion joint was at the second amino acid of the LamB signal sequence . There was no significant decrease in rate of synthesis of ompA mRNA as measured by filter hybridization of pulse-labeled RNA . These results indicate that the inhibition is at the level of translation . We propose that cells are able to monitor expression of exported proteins by sensing occupancy of some limiting component in the export machinery and use this to regulate translation of these proteins.

J Virol, 1988 May, 62(5), 1486 - 94
A herpes simplex virus mutant in which glycoprotein D sequences are replaced by beta-galactosidase sequences binds to but is unable to penetrate into cells; Ligas MW et al.; Herpes simplex virus (HSV) glycoprotein gD is a major component of the virion envelope and is thought to play an important role in the initial stages of viral infection and stimulates the production of high titers of neutralizing antibodies . We assumed that gD plays an essential role in virus replication, and so to complement viruses with mutations in the gD gene we constructed a cell line, denoted VD60, which is capable of expressing high levels of gD after infection with HSV . A recombinant virus, designated F-gD beta, in which sequences encoding gD and a nonessential glycoprotein, gI, were replaced by Escherichia coli beta-galactosidase sequences, was selected on the basis that it produced blue plaques on VD60 cell monolayers under agarose overlays containing 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-Gal) . F-gD beta was able to replicate normally on complementing VD60 cells . However, F-gD beta was unable to form plaques on noncomplementing Vero cells . Virions lacking gD were produced in normal amounts by Vero cells infected with F-gD beta, and the virus particles were distributed throughout the cytoplasm and on the cell surface, suggesting that gD is not essential for HSV envelopment and egress . Virions lacking gD were able to bind to cells, but were unable to initiate synthesis of viral early polypeptides . Plaque production of F-gD beta particles lacking gD was enhanced by polyethylene glycol treatment, suggesting that gD is essential for penetration of HSV into cells . Other HSV glycoproteins have been implicated in the entry of virus into cells, and thus this process appears to involve multiple interactions at the cell surface.

Infect Immun, 1988 May, 56(5), 1158 - 61
Type II heat-labile enterotoxin-producing Escherichia coli isolated from animals and humans; Seriwatana J et al.; Heat-labile enterotoxin (LT)-producing Escherichia coli strains, as identified by the Y1 adrenal cell assay, were examined with a DNA probe coding for type I and type II LTs . Of 236 LT-producing E . coli isolates, 60% hybridized with LT-I, 17% hybridized with LT-II, and 23% did not hybridize with either probe and no longer produced LT as determined by the Y1 adrenal cell assay . These isolates presumably lost plasmids coding for LT-I during storage . A total of 75% of LT-producing E . coli isolates (27 of 36) from cows, 64% of LT-producing E . coli isolates (7 of 11) from buffalo, 31% of LT-producing E . coli isolates (4 of 13) from beef obtained in markets, and 2% of LT-producing E . coli isolates (3 of 168) from humans contained genes coding for LT-II . Genes coding for LT-II were not found in 50 LT-I-producing and heat-stable enterotoxin-producing E . coli isolates from 11 children with diarrhea and 44 LT-nonproducing and heat-stable enterotoxin-producing E . coli isolates from 12 other children with diarrhea . A total of 9% of LT-II-producing E . coli isolates (3 of 34) from cows and buffalo hybridized with DNA probes for genes coding for verocytotoxin 2 (VT2), and 18% (6 of 34) hybridized with a DNA probe coding for enterohemorrhagic E . coli (EHEC) adhesin fimbriae . E . coli SA-53, the original isolate in which LT-II was found, contained genes coding for VT2 and EHEC adhesin fimbriae . Five VT-producing, LT-II-producing E . coli isolates that hybridized with the EHEC probe did not contain DNA sequences coding for VT1 or VT2 . LT-II-producing E . coli strains were frequently isolated from cattle and buffalo but were rarely isolated from humans.

Infect Immun, 1988 May, 56(5), 1135 - 43
Isolation and characterization of recombinant Escherichia coli clones secreting a 24-kilodalton antigen of Treponema pallidum; Hsu PL et al.; Escherichia coli clones containing Treponema pallidum DNA in the pUC8 vector and secreting a 24-kilodalton antigen of T . pallidum have been isolated . Both syphilitic human and syphilis-immune rabbit sera reacted with the recombinant p24 antigen, indicating that an equivalent protein in T . pallidum is capable of eliciting antibody responses during natural infections . The p24 antigen of T . pallidum was identified by using two-dimensional gel electrophoresis and immunoblotting with monospecific anti-p24 serum . We tentatively concluded that this cloned antigen is a secreted protein or a labile or minor component of T . pallidum because (i) p24 was secreted by the recombinant E . coli cells; (ii) recombinant p24 in E . coli cells was processed into several smaller species with molecular masses ranging from 12 to 20 kilodaltons, which correlate well with the masses of secreted antigens described by others; and (iii) p24 protein appeared to be highly antigenic during natural infections, but only a very small amount of this antigen was associated with or retained by the purified organisms . The possible role of the p24 protein in determining the growth characteristics of T . pallidum is suggested by the ability of recombinant p24 to induce growth changes in E . coli cells . All E . coli colonies expressing the p24 polypeptide exhibited a flat and rough colony morphology and a filamentous growth pattern that were different from those of other E . coli cells . The DNA sequence coding for the p24 polypeptide is located on a 1.7-kilobase-pair BamHI fragment of the T . pallidum genomic DNA and is absent in the nonpathogenic Treponema phagedenis DNA . However, any possible relationship between the p24 antigen and the virulence of T . pallidum remains to be determined . In preliminary studies, rabbits immunized with the purified p24 were not protected from the infection with live T . pallidum organisms.

EMBO J, 1988 May, 7(5), 1509 - 13
The Escherichia coli 30S ribosomal subunit; an optimized three-dimensional fit between the ribosomal proteins and the 16S RNA; Schuler D et al.; We have generated a computerized fit between the 3-dimensional map of the E.coli 30S ribosomal proteins, as determined by neutron scattering, and the recently published 3-dimensional model for the 16S RNA . To achieve this, the framework of coordinates for RNA-protein cross-link sites on the phosphate backbone in the RNA model was related to the corresponding framework of coordinates for the mass centres of the proteins by a least squares fitting procedure . The resulting structure, displayed on a computer graphics system, gives the first complete picture of the E.coli 30S ribosomal subunit showing both the proteins and the double-helical regions of the RNA . The root mean square distance between cross-link sites and protein centres is 32 A . The position of the mass centre of the combined double-helical regions was calculated from the model and compared with the position of the mass centre of the complete set of proteins . The two centres are displaced relative to one another by 20 A in the model structure, in good agreement with the experimental value of 25 A found by neutron scattering.

EMBO J, 1988 May, 7(5), 1503 - 7
Reading frame switch caused by base-pair formation between the 3' end of 16S rRNA and the mRNA during elongation of protein synthesis in Escherichia coli; Weiss RB et al.; Watson-Crick base pairing is shown to occur between the mRNA and nucleotides near the 3' end of 16S rRNA during the elongation phase of protein synthesis in Escherichia coli . This base-pairing is similar to the mRNA-rRNA interaction formed during initiation of protein synthesis between the Shine and Dalgarno (S-D) nucleotides of ribosome binding sites and their complements in the 1540-1535 region of 16S rRNA . mRNA-rRNA hybrid formation during elongation had been postulated to explain the dependence of an efficient ribosomal frameshift on S-D nucleotides precisely spaced 5' on the mRNA from the frameshift site . Here we show that disruption of the postulated base pairs by single nucleotide substitutions, either in the S-D sequence required for shifting or in nucleotide 1538 of 16S rRNA, decrease the amount of shifting, and that this defect is corrected by restoring complementary base pairing . This result implies that the 3' end of 16S rRNA scans the mRNA very close to the decoding sites during elongation.

Mol Gen Genet, 1988 May, 212(2), 203 - 6
Heat stress in the presence of low RNA polymerase activity increases chromosome copy number of Escherichia coli; Guzman EC et al.; A temperature shift-up accompanied by a reduction in RNA polymerase activity in Escherichia coli causes an increased rate of initiation leading to a 1.7- to 2.2-fold increase in chromosome copy number . A temperature shift-up without a reduction in polymerase activity induces only a transient non-scheduled initiation of chromosome replication caused by heat shock with no detectable effect on chromosome copy number.

J Clin Microbiol, 1988 May, 26(5), 879 - 84
Virulence factors in Escherichia coli strains isolated from Swedish piglets with diarrhea; Soderlind O et al.; Parenteral vaccination of sows against Escherichia coli diarrhea in their newborn piglets has become more common during the last decade in Sweden, and the vaccination has generally had positive effects . For more than 20 years we have investigated E . coli strains isolated from piglets and weaned pigs with enteric disorders, noting the presence of O groups, enterotoxins, and adhesins . There has been a continuous change in the frequency of these virulence factors . The present study was performed during 1983 and 1984 to follow this change, since such information is essential for the proper choice of vaccines . A total of 856 E . coli strains were obtained from 683 herds divided into three age groups: 1 to 6 days old, 1 to 6 weeks old, and weaned pigs . O group 149 still dominated in the last two age groups, while O group 101 was, for the first time, the most frequent O group in neonatal piglets . All but four O149 strains carried the K88 antigen, which was found in only one other strain (O group 8) . K99 antigen was most often found in O groups 101 and 64, and among all the K99 strains ST mouse was the most common (44 of 57), followed by ST mouse-ST pig strains (12 of 57) . The 987P antigen was demonstrated in 26 strains belonging to O groups 141 and OX46 and nontypable strains . Two strains belonging to O group 101 were positive for F41 antigen; one of them also carried the K99 antigen . Among all non-O149 strains, ST mouse was the most common type of enterotoxigenic E . coli ( n = 88), followed in decreasing order by ST mouse-ST pig strains ( n = 69) and ST pig strains ( n = 33) . In 114 strains producing enterotoxins no adhesive factor was found . Thus, vaccination of the Swedish sow population for more than 5 years with vaccines containing O149 and K88 antigens has apparently changed the pattern of enterotoxigenic E . coli in neonatal diarrhea . The frequency of O149:K88 strains has been reduced, and O101:K99:ST mouse strains now dominate . However, O149 strains remain the dominant O group in piglets older than 1 week . In spite of all our diagnostic efforts, no virulence factors were detected in about one third of the piglets and weaned pigs with enteric disorders.

J Clin Microbiol, 1988 May, 26(5), 850 - 6
Immune response to Escherichia coli alpha-hemolysin in patients; Seetharama S et al.; The serum antibody response of patients infected with alpha-hemolysin (AH)-producing Escherichia coli was measured by three immunoassays: tube neutralization, microneutralization, and enzyme-linked immunosorbent assay . All three assay results showed good correlation with each other . The mean anti-AH titer in patients with E . coli infection was higher than the mean titer in noninfected patients . The hemolysin-neutralizing activity was immunoglobulin G . The amount of lipopolysaccharide (LPS) antibody did not correlate with the amount of AH antibody . LPS antibody measured by enzyme-linked immunosorbent assay was predominantly of the immunoglobulin G class . Adsorption of LPS antibody by E . coli H79 LPS did not affect anti-AH titers, indicating that LPS and AH have different antigenic determinants . AHs prepared from several different E . coli strains had identical or similar antigenic determinants at the active site . Hemolysin proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis were stained identically by human sera with AH antibody and by a mouse monoclonal AH antibody.

Biull Eksp Biol Med, 1988 May, 105(5), 577 - 8
{Use of recombinant proteins carrying antigenic determinants of the envelope proteins of AIDS virus in the diagnosis of acquired immunodeficiency syndrome}; Bukrinskii MI et al.; The testing of sera from patients with AIDS and AIDS-related complex, using ELISA with recombinant env protein produced by E . coli, gave a 94% coincidence with the results obtained on "Organon" test-system . Sera samples that gave different results in two tests lacked antibodies to env-encoded proteins, as revealed by immunoblotting assay . The application of the above test for the estimation of the disease prognosis is discussed.

Arthritis Rheum, 1988 May, 31(5), 616 - 22
A recombinant autoantigen derived from the human (U1) small nuclear RNP-specific 68-kd protein . Expression in Escherichia coli and serodiagnostic application; Netter HJ et al.; A human liver complementary DNA expression library was screened using sera from patients with high titers of autoantibodies, to search for clones expressing major autoantigens that are relevant in connective tissue diseases . One of the clones isolated expressed a major epitope(s) that was immunoreactive with anti-U1 RNP sera, as shown by several techniques . Affinity-purified autoantibodies from the cloned RNP protein specifically recognized the 68-kd U1 RNP protein of HeLa cell nuclear extracts . All sera containing anti-U1 RNP antibodies detected by immunodiffusion, counterimmunoelectrophoresis, or immunoblotting also recognized the cloned RNP protein . The RNP antigen-expressing bacterial colonies and the partially purified cloned RNP fusion protein have been applied to fast and sensitive immunologic assays for the detection and quantification of anti-U1 RNP antibodies.

Proc Natl Acad Sci U S A, 1988 May, 85(10), 3372 - 6
The alpha and beta chains of avian retrovirus reverse transcriptase independently expressed in Escherichia coli: characterization of enzymatic activities; Soltis DA et al.; Reverse transcriptase of the avian sarcoma and leukosis retroviruses is a heterodimer composed of a 63-kDa alpha and a 95-kDa beta polypeptide chain, both of which are encoded in the pol gene and are produced by proteolytic processing of a larger precursor . We previously constructed a bacterial expression clone of the entire pol coding region that produces a protein 4 kDa larger than the mature viral beta subunit . By use of this clone and synthetic oligonucleotides to introduce stop codons, two derivatives have been constructed: one that directs synthesis of a protein equivalent to the mature beta subunit and the other that directs synthesis of a protein equivalent to alpha subunit . Predicted amino acid sequences of these proteins differ from their viral counterparts only by an initiator methionine that was added to the N termini for expression in Escherichia coli . Both bacterially expressed proteins exhibit reverse transcriptase activity and appear to function as homodimers . The properties of these proteins resemble those of the viral reverse transcriptase heterodimer; however, the bacterially produced alpha dimer protein could be distinguished from the other proteins by its increased sensitivity to heat inactivation, which also has been reported for the corresponding viral product . These results show that correct folding and expression of enzymatic function does not require formation of a precursor . The alpha and beta clones provide a convenient source of individual pol gene products for further evaluation of their roles in the synthesis and integration of retroviral DNA.

Mutat Res, 1988 May, 193(3), 229 - 36
Survival and mutagenic effects of 5-azacytidine in Escherichia coli; Lal D et al.; Survival and mutagenesis caused by 5-azacytidine was studied in Escherichia coli . Survival was partially lexA- and recA-dependent and was decreased by the presence of a DNA (cytosine-5)methyltransferase . The dcm, MspI, and EcoRII methyltransferase genes all decreased survival . There was no direct relationship between amount of methylase enzyme present and cell survival, but only plasmids containing a methylase gene sensitized cells to 5-azacytidine . Survival was not affected by uvrA, uvrB or umuCD mutations . Induction of sulA::lacZ fusions by 5-azacytidine was inhibited in strains containing elevated levels of DNA methylase . Cells resistant to 5-azacytidine when they contained a plasmid specifying the EcoRII methylase were sensitive if the plasmid specified the complete EcoRII restriction-modification system . The mechanism of cell death in these situations is therefore different . Mutation of the rpoB gene by 5-azacytidine was studied . The mutation rate was decreased by the presence of recA and lexA mutations . Mutation in umuCD had little effect on the mutation rate . The recA430 mutation, which does not support SOS-dependent mutagenesis induced by UV light, does support 5-azacytidine induced mutagenesis . The presence of DNA (cytosine-5)methyltransferase had no effect on the mutation rate caused by 5-azacytidine treatment . The mutagenic and lethal lesions caused by 5-azacytidine in the absence of methylase therefore differ from the lethal lesions that occur in the presence of methylase . The former could be due to the opening of the 5-azacytosine ring in DNA . Cell death in the presence of methylase could be due to tight binding of methylase to azacytosine containing DNA as well as inhibition of induction of the SOS response.

J Immunol, 1988 May 1, 140(9), 3212 - 8
Characteristics and epitope mapping of a cloned human autoantigen La; Sturgess AD et al.; The La (SS-B) polypeptide is a ribonucleoprotein against which high titer antinuclear antibodies (ANA) react in the human autoimmune disease primary Sjogren's syndrome . To identify the autoepitopes with which the ANA anti-La (anti-SS-B) reacts, we isolated a 1.4-kb cDNA clone for La from a lambda gt10 library made from a human Burkitt's cell line . This clone contained an open reading frame of 1065 bp, encoding a 40.1-kDa polypeptide that corresponded to the carboxyl-terminal end of the La protein . The predicted polypeptide sequence of the recombinant protein was highly charged and unrelated to any previously published sequence . We also compared this clone to a previously published cDNA sequence for La and demonstrated significant differences, particularly that the open reading frame in our cDNA continued for 926 additional bases 3' to a putative termination codon in the previously reported sequence . The recombinant La protein was expressed in Escherichia coli and tested for reactivity with 200 sera containing ANA of various specificities . Only the sera containing anti-La antibodies reacted with the cloned La . By expressing subclones of the La cDNA as fusion proteins with beta-galactosidase, we have localized at least one epitope for the binding of anti-La antibodies to the carboxyl-terminal 103 amino acids of the La protein . No anti-La binding could be demonstrated to the region of the La protein that had previously been predicted to contain an autoepitope for the binding of anti-La (SS-B) antibodies . Studies of cloned autoepitopes could provide important clues to the role ANA play in disease and lead to targeted intervention in the treatment of primary Sjogren's syndrome.

J Bacteriol, 1988 May, 170(5), 2083 - 8
DNA polymerase I activity in Escherichia coli is influenced by spot 42 RNA; Polayes DA et al.; We have shown that the level of DNA polymerase I (Pol I) activity in Escherichia coli is influenced by the level of a 109-nucleotide RNA, spot 42 RNA . Deletion of the gene for spot 42 RNA results in a 20 to 25% decrease in Pol I activity, as assayed by nucleotide incorporation in cell extracts and a decrease in the ability of cells to grow in the presence of the DNA-alkylating agent methyl methanesulfonate . Also, a physiological reduction of the level of spot 42 RNA, by growth in media containing poor carbon sources, results in a corresponding decrease in Pol I activity . Conversely, overproduction of spot 42 RNA results in a 10 to 15% increase in Pol I activity in vitro . Thus, changes in the amount of spot 42 RNA result in relatively small but significant changes in Pol I activity.

J Virol, 1988 May, 62(5), 1513 - 9
Recognition of adenovirus E1A gene products on immortalized cell surfaces by cytotoxic T lymphocytes; Bellgrau D et al.; The experiments described in this report were designed to examine whether target cells transfected with the adenovirus E1A gene and exhibiting increased susceptibility to lysis by natural killer cells and activated macrophages (J . L . Cook, T . A . Walker, A . M . Lewis, Jr., H . E . Ruley, F . L . Graham, and S . H . Pilder, Proc . Natl . Acad . Sci . USA 83:6965-6969, 1986) also express E1A proteins on their surfaces . MT1A, 12S, and 13S are strain Fischer baby rat kidney (BRK) cell lines immortalized by transfection with plasmids containing only the E1A gene of nononcogenic adenovirus . All of these cell lines were effective in stimulating the generation of cytotoxic T lymphocytes (CTL) in vitro, provided that the cultures were supplemented with an exogenous source of lymphokine and that the responding lymphocytes were from syngeneic Fischer rats previously immunized with a cell line containing the intact E1A gene . HrA2, a Fischer BRK cell line immortalized by transfection with a plasmid containing only exon 1 of the E1A gene, did not generate, nor was it lysed by, E1A-specific CTL . The cytolytic activity of E1A-specific CTL was blocked by antiserum from Fischer rats immunized with purified E1A proteins synthesized in Escherichia coli, supporting the conclusion that an epitope on E1A proteins encoded by the intact E1A gene constitutes part of the CTL target structure on adenovirus-transformed cells . These data suggest that in addition to their functions within host cells, E1A gene products are important immunogenic determinants on the surfaces of adenovirus-transformed cells.

Gene, 1988 Apr 29, 64(2), 313 - 9
A rapid and efficient method for targeted random mutagenesis; Shiraishi H et al.; We describe a new rapid method for random introduction of single-nucleotide (nt) substitutions into a small segment of cloned DNA . A DNA fragment containing a sequence to be mutagenized is inserted into a multiple cloning site sequence of a vector plasmid . The plasmid is linearized with two adjacent cuts (generating 5' and 3' protruding ends) and then synchronously and unidirectionally digested with exonuclease III (Exo III) so that the 3' termini generated are localized within the target region . A non-complementary alpha-thiophosphate nucleotide is misincorporated into the 3' terminus generated by Exo III . Since the nucleotide analogue is resistant to the 3'-5' exonuclease activity of DNA polymerase I, its misincorporation into the 3' termini is irreversible . Then, the single-stranded region is filled-in with four canonical nucleotides, and the plasmid is recircularized . This procedure was used to mutagenize a specific region of the rnpB gene of E . coli . By sequencing 72 randomly selected clones, we found that 27 clones (37.5%) had nucleotide substitutions distributed within the desired region of a 55-nt-long segment of the gene . The procedure is simple and is applicable to any DNA molecule.

Biochem Biophys Res Commun, 1988 Apr 29, 152(2), 674 - 9
Globotriosyl ceramide is specifically recognized by the Escherichia coli verocytotoxin 2; Waddell T et al.; Two Escherichia coli cytotoxins (verotoxins 1 and 2) have been previously implicated in the cytopathology of the Hemolytic Uremic Syndrome . We have examined the glycolipid binding specificity of verotoxin (VT)2 . This toxin specifically binds to globotriosyl ceramide (galactose alpha 1-4 galactose beta 1-4 glucosyl ceramide) . Removal, or substitution of the terminal a galactose residue with N-acetyl galactosamine in beta 1-3 linkage, deletes binding activity . The toxin does not recognize similar terminal a galactose residues on a glycoglycerolipid . Thus the binding specificity of VT2 is the same as previously reported for VT1 . Liposomes containing globotriosyl ceramide are able to specifically remove VT1 and VT2 cytotoxicity and cell lines selected in vitro for resistance to VT1 are cross resistant to VT2.

Biochem Biophys Res Commun, 1988 Apr 29, 152(2), 879 - 85
Interleukin-2 self-association; Fleischmann JD et al.; The self-association of human recombinant interleukin-2 (IL-2) from E . coli was explored . Self-association, with an apparent Kd of 0.6 micromolar, has pronounced effects on (1) the surface exposure of Trp-121, deduced from quenching studies employing potassium iodide and acrylamide, (2) the apparent quantum yield of Trp-121, the fluorescence of Trp-121 in IL-2 aggregates is 4-fold lower than in IL-2 "monomers", and (3) IL-2-mediated phospholipid vesicle fusion/aggregation.

Gene, 1988 Apr 29, 64(2), 305 - 11
High-level expression of the cloned ada gene of Escherichia coli by deletion of its regulatory sequence; Tano K et al.; The Ada protein, a methyltransferase for repair of several alkyl adducts in DNA, was expressed in its native form at a high level in Escherichia coli from a pUC9 recombinant plasmid carrying ada gene from which the sequence controlling the Ada induction was deleted . The regulatory sequence appears to act as a terminator of transcription initiated from the lac promoter of the vector . However, deletion of the regulatory sequence resulted in elimination of ada induction by alkylating agents, providing confirmation of its role in activation of ada expression.

Gene, 1988 Apr 29, 64(2), 265 - 75
Hybrid enterotoxin LTA::STa proteins and their protection from degradation by in vivo association with B-subunits of Escherichia coli heat-labile enterotoxin; Sanchez J et al.; Chimeric proteins exhibiting antigenic determinants of the heat-labile enterotoxin (LT) and heat-stable (STa) enterotoxins on the same molecule may provide a means to obtain immunoprophylactic and diagnostic reagents for Escherichia coli-caused diarrhea . We recently showed that fusion of two different lengths of the STa gene to the C end of the A-subunit of LT (LTA) results in LTA::STa fusion proteins as monitored by GM1-ELISA {Sanchez et al.: FEBS Lett . 208 (1986) 194-198} . Here we determine the approximate molecular size of the LTA::STa fusion proteins and provide further evidence of their hybrid nature by immunoblot analysis . Using this technique we also demonstrate that to obtain detectable amounts of these recombinant proteins it is essential to coexpress them with the respective B-subunit of LT (LTB) . We propose that this dependence on coexpression reflects the association between the LTA::STa hybrids and LTB subunits . The resulting LTA::STa/LTB complexes were found in the E . coli periplasm . This indicated that the exported hybrids, once associated with LTB, were stabilized and formed molecules that behaved essentially as native LT . The protective effect exerted by the B-subunit might conceivably be extended to other LTA-derived hybrid proteins, thus allowing the fusion of other foreign peptides to LTA and their subsequent recovery in the same fashion.

Gene, 1988 Apr 29, 64(2), 207 - 15
Promoter-detection vectors for Escherichia coli with multiple useful features; Malo MS et al.; Two promoter-detection vectors have been constructed which enable the cloning and characterization of promoters recognized by the RNA polymerase of Escherichia coli K-12 . The intergenic region of phage M13 DNA, present in opposite orientations in the two vectors, permits the preparation of single-stranded DNA of either strand of the insert thus facilitating oligodeoxyribonucleotide heteroduplex mutagenesis and sequencing of both strands by the dideoxy method of chain termination . After mutagenesis, isolates can be screened for changed function by replica-plating colonies to plates containing XGal . Selected isolates can be characterized by nucleotide sequence analysis, to determine the change in structure, and by beta-galactosidase assays, thus measuring the effect of mutagenesis on promoter function . The vectors could also be used like other protein-fusion vectors.

Gene, 1988 Apr 29, 64(2), 199 - 205
Nucleotide sequence of a mutation in the proB gene of Escherichia coli that confers proline overproduction and enhanced tolerance to osmotic stress; Csonka LN et al.; We determined the nucleotide (nt) sequence of a mutation that confers proline overproduction and enhanced tolerance of osmotic stress on bacteria . The mutation, designated as proB74, is an allele of the Escherichia coli proB gene which results in a loss of allosteric regulation of the protein product, gamma-glutamyl kinase . Our sequencing indicated that the proB74 mutation is a substitution of an A for a G at nt position 319 of the coding strand of the gene, resulting in a change of an aspartate to an asparagine at amino acid (aa) residue 107 of the predicted protein product . Rushlow et al . {Gene 39 (1984) 109-112} determined that another proB mutation (designated as DHPR), that resulted in a loss of allosteric inhibition by proline of the E . coli gamma-glutamyl kinase, was due to a substitution of an alanine for a glutamate at aa residue 143 . Therefore, even though both the DHPR and the proB74 mutations caused a loss of allosteric inhibition of gamma-glutamyl kinase, they are due to different amino acid substitutions.

Biochim Biophys Acta, 1988 Apr 28, 954(1), 114 - 25
Interaction of alkylputrescines with ornithine decarboxylase from rat liver and Escherichia coli: an in vitro and in vivo study; Ruiz O et al.; The inhibitory effect of a series of 2-alkylputrescines on rat liver and Escherichia coli ornithine decarboxylase (L-ornithine carboxy-lyase, EC 4.1.1.17) was examined . At 2.5 mM concentrations, 2-methyl-, 2-propyl-, 2-butyl-, 2-pentyl- and 2-hexylputrescines were stronger inhibitors of the mammalian enzyme than putrescine . Only the higher homologues (from 2-propyl- to 2-hexylputrescine) were inhibitors of the E . coli enzyme . An analysis of the effect of increasing concentrations of the 2-alkylputrescines showed that the main difference in the behaviour of the mammalian and E . coli decarboxylases toward 2-alkylputrescines was that the former was strongly inhibited by 2-methylputrescine whereas the latter was not . 2-Alkylputrescines were found to be competitive inhibitors of both the bacterial and mammalian enzyme . The smallest Ki values (0.1 and 0.5 mM) were found for the 2-hexyl- and 2-pentylputresciens . N-Methyl-, N-ethyl-, N-propyl- and N-butylputrescines (50 mumol per 100 g body weight) were assayed as inhibitors of thioacetamide-induced rat liver ornithine decarboxylase . N-Propylputrescine was found to be the most inhibitory (66% inhibition) and although the N-alkylputrescines were taken up by the liver, they did not inhibit the liver polyamine pools . Both putrescine and N-methylputrescine were found to stabilize the thioacetamide-induced ornithine decarboxylase at the onset of the enzyme's degradation, while 2-alkylputrescines were inhibitory under similar conditions . N-Methylputrescine induced antizyme in thioacetamide-treated rats . In thioacetamide- or dexamethasone-treated rats, 2-methylputrescine was found to be the strongest in vivo inhibitor of the liver decarboxylase . Although 2-alkylputrescines were efficiently taken up by the liver, they did not noticeably inhibit its polyamine pools . 2-methylputrescine decreased the putrescine concentration of the liver, but not its spermidine and spermine content . No induction of ornithine decarboxylase antizyme by 2-methylputrescine could be detected . The intrahepatic concentration of the latter decreased with time, very likely due to its degradation by a diamine oxidase, since the decrease was inhibited by aminoguanidine.

Biochim Biophys Acta, 1988 Apr 28, 954(1), 1 - 13
The inducible trimethylamine-N-oxide reductase of Escherichia coli K12: biochemical and immunological studies; Silvestro A et al.; The inducible trimethylamine-N-oxide reductase which migrates on non-denaturing polyacrylamide gels with an RF of 0.22, has been purified from the soluble fraction of wild-type E . coli K12 . The molecular weight of the purified enzyme estimated by molecular-sieve chromatography is about 230,000 . It is composed of two subunits of molecular weight 110,000 . Antiserum specific for the enzyme has been produced . Gel filtration on Sephadex G-200 of the soluble fraction gave two peaks of trimethylamine-N-oxide reductase, one with an Mr of 230,000 and an RF of 0.22, and another with an Mr of 120,000 and an RF of 0.36 . Since the anti-trimethylamine-N-oxide reductase serum recognises the two forms and shows a single subunit with an Mr of 110,000, we conclude that in E . coli there is a single inducible trimethylamine-N-oxide reductase which can exist as a dimer or a monomer . Other immunological studies with anti-trimethylamine-N-oxide reductase serum on crude extracts prepared from cells grown in the absence of inducer showed that the constitutive trimethylamine-N-oxide reductase was not recognised by the antiserum . The same analyses carried out on a tor mutant (defective in the structural gene of the inducible enzyme) confirmed without ambiguity that the constitutive enzyme is immunologically distinct from the inducible enzyme . In the same way, using the anti-trimethylamine-N-oxide reductase serum, rocket immunoelectrophoresis analyses were able to show that the inducible apoenzyme is not regulated by the fnr gene product and that molybdate does not seem necessary for the synthesis or stabilisation of this enzyme.

J Biol Chem, 1988 Apr 25, 263(12), 5815 - 9
The structure of crystalline Escherichia coli-derived rat intestinal fatty acid-binding protein at 2.5-A resolution; Sacchettini JC et al.; Rat intestinal fatty acid-binding protein (I-FABP) is an abundant cytoplasmic protein which is synthesized in the small intestinal lining cell where it is thought to participate in the absorption and intracellular metabolism of fatty acids . Each mole of this 132-residue polypeptide binds 1 mol of long chain fatty acid in a noncovalent fashion . Because of its small size and single ligand-binding site, I-FABP represents an attractive model for defining the molecular details of long chain fatty acid-protein interactions . The structure of Escherichia coli-derived rat I-FABP has now been solved to 2.5 A resolution using three isomorphous heavy atom derivatives . The protein consists of 10 anti-parallel beta-strands present as two orthogonal beta-sheets . Together a "clam shell-like" structure is formed with an opening located between two beta-strands and an interior that is lined with the side chains of nonpolar amino acids . The bound fatty acid ligand is located in the interior of the protein and has a bent conformation, possibly reflecting the presence of several gauche bonds in the hydrocarbon tail . Our present interpretation of the electron density map suggests that the fatty acid is oriented with its carboxylate group facing the guanidinium group of Arg127, whereas the end of its hydrocarbon tail is in close proximity to Val106 . The indole side chain of Trp83 forms the molecular framework around which the principal bend of the hydrocarbon chain occurs.

J Biol Chem, 1988 Apr 25, 263(12), 5674 - 80
Reversible dissociation of active octamer of cyanase to inactive dimer promoted by alteration of the sulfhydryl group; Anderson PM et al.; Cyanase is an inducible enzyme in Escherichia coli that catalyzes the reaction of cyanate with bicarbonate resulting in the decomposition of cyanate to ammonia and bicarbonate . In this study, the role of the single sulfhydryl group in each of the eight identical subunits of cyanase was investigated . Tetranitromethane, methyl methanethiosulfonate, N-ethylmaleimide, and Hg2+ all reacted with the sulfhydryl group to give derivatives which had reduced activities and which dissociated reversibly to inactive dimer . Association of inactive dimer to active octamer was facilitated by the presence of azide (cyanate analog) and bicarbonate, increased temperature and enzyme concentration, and presence of phosphate . Nitration of tyrosine residues by tetranitromethane occurred only in the absence of azide and bicarbonate, suggesting that at least some of the tyrosine residues become exposed when octamer dissociates to dimer . Site-directed mutagenesis was used to prepare a mutant enzyme in which serine was substituted for cysteine . The mutant enzyme was catalytically active and had properties very similar to native enzyme, except that it was less stable to treatment with urea and to high temperatures . These results establish that in native cyanase the sulfhydryl group per se is not required for catalytic activity, but it may play a role in stabilizing octameric structure, and that octameric structure is required for catalytic activity.

J Biol Chem, 1988 Apr 25, 263(12), 5640 - 5
Pyridoxal 5'-diphospho-5'-adenosine binds at a single site on isolated alpha-subunit from Escherichia coli F1-ATPase and specifically reacts with lysine 201; Rao R et al.; Pyridoxal 5'-diphospho-5'-adenosine (PLP-AMP), an adenine nucleotide affinity analog, was found to bind in a saturable fashion to isolated alpha-subunit from Escherichia coli F1-ATPase with a stoichiometry of one mol/mol and a Kd approximately 150 microM . The binding was shown to be specific by the following criteria: 1) ATP reduced the binding of PLP-AMP by 80%, and 2) PLP-AMP, like ATP, induced a conformational change which increased the mobility of alpha-subunit in nondenaturing polyacrylamide gel electrophoresis and rendered alpha-subunit resistant to mild trypsin proteolysis . A stable adduct was formed between isolated alpha-subunit and {3H} PLP-AMP after reduction with NaBH4 . alpha-Subunit labeled to the extent of 0.4-0.7 mol/mol was digested with trypsin and subjected to high pressure liquid chromatography purification, yielding a single labeled peptide . Automated amino acid sequencing showed that residue alpha-Lys-201 was specifically labeled . The results suggest that Lys-201 occupies a position proximate to the phosphate groups of bound ATP in the alpha.ATP complex . PLP-AMP did not support repolymerization of isolated alpha-, beta-, and gamma-subunits, consistent with previous reports that subunit repolymerization in vitro is dependent upon the presence of nucleoside triphosphate . Further, PLP-AMP-labeled alpha-subunit could not be reconstituted with isolated beta- and gamma-subunits in the presence of ATP, showing that occupation of the alpha-subunit nucleotide site by PLP-AMP impairs normal subunit-subunit interaction.

J Biol Chem, 1988 Apr 25, 263(12), 5569 - 73
Trinitrophenyl-ATP and -ADP bind to a single nucleotide site on isolated beta-subunit of Escherichia coli F1-ATPase . In vitro assembly of F1-subunits requires occupancy of the nucleotide-binding site on beta-subunit by nucleoside triphosphate; Rao R et al.; The stoichiometry of nucleotide binding to the isolated alpha- and beta-subunits of Escherichia coli F1-ATPase was investigated using two experimental techniques: (a) titration with fluorescent trinitrophenyl (TNP) derivatives of AMP, ADP, and ATP and (b) the centrifuge column procedure using the particular conditions of Khananshvili and Gromet-Elhanan (Khananshvili, D., and Gromet-Elhanan, Z . (1985) FEBS Lett . 178, 10-14) . Both procedures showed that alpha-subunit contains one nucleotide-binding site, confirming previous work . TNP-ADP and TNP-ATP bound to a maximal level of 1 mol/mol beta-subunit, consistent with previous equilibrium dialysis studies which showed isolated beta-subunit bound 1 mol of ADP or ATP per mol (Issartel, J . P., and Vignais, P . V . (1984) Biochemistry 23, 6591-6595) . However, binding of only approximately 0.1 mol of ATP or ADP per mol of beta-subunit was detected using centrifuge columns . Our results are consistent with the conclusion that each of the alpha- and beta-subunits contains one nucleotide-binding domain . Because the subunit stoichiometry is alpha 3 beta 3 gamma delta epsilon, this can account for the location of the six known nucleotide-binding sites in E . coli F1-ATPase . Studies of in vitro assembly of isolated alpha-, beta-, and gamma- subunits into an active ATPase showed that ATP, GTP, and ITP all supported assembly, with half-maximal reconstitution of ATPase occurring at concentrations of 100-200 microM, whereas ADP, GDP, and IDP did not . Also TNP-ATP supported assembly and TNP-ADP did not . The results demonstrate that (a) the nucleotide-binding site on beta-subunit has to be filled for enzyme assembly to proceed, whereas occupancy of the alpha-subunit nucleotide-binding site is not required, and (b) that enzyme assembly requires nucleoside triphosphate.

Biochim Biophys Acta, 1988 Apr 25, 969(2), 185 - 93
31P-NMR saturation transfer studies of aerobic Escherichia coli cells; Mitsumori F et al.; 31P-NMR measurements of saturation transfer have been used to measure the flux between Pi and ATP in Escherichia coli cells respiring on an endogenous carbon source . Measurements were made in the wild type and in cells genetically modified to give a 5-fold higher concentration of the F1F0-ATP synthase . The flux in the two cell types was not significantly different . This, together with studies using inhibitors specific for the glycolytic enzyme, glyceraldehyde-3-phosphate dehydrogenase and the ATP synthase, suggests that the observed flux arises predominantly from glycolytic rather than ATP synthase activity . Although this conclusion is in disagreement with previous experiments on E . coli, it is in agreement with recent experiments on yeast.

FEBS Lett, 1988 Apr 25, 231(2), 389 - 92
Bovine seminalplasmin {corrected} is a DNA unwinding protein; Gopal V et al.; The duplex DNA unwinding ability of seminalplasmin {corrected} from bovine semen was examined by treatment of plasmid-protein complexes with calf thymus topoisomerase I and resolution of the topoisomer distributions by agarose gel electrophoresis . Binding of seminalplasmin {corrected} results in a moderate degree of unwinding of supercoiled plasmid . The elongation of the RNA chain by E . coli RNA polymerase over promoter containing template is not inhibited by seminalplasmin {corrected} . However, the reinitiation of transcription is blocked in such cases indicating that seminalplasmin {corrected} inhibits transcription by binding to the initiation site of RNA polymerase.

FEBS Lett, 1988 Apr 25, 231(2), 361 - 5
Photoaffinity labelling shows that Escherichia coli isocitrate dehydrogenase kinase/phosphatase contains a single ATP-binding site; Varela I et al.; Ultraviolet irradiation of E.coli isocitrate dehydrogenase kinase/phosphatase in the presence of 8-azidoATP resulted in parallel losses of its kinase and phosphatase activities, and in covalent attachment of the reagent to the protein at a single site . ATP and ADP protected the two activities to similar extents . The data suggest that the activation of the phosphatase by adenine nucleotides results from binding of the nucleotides to the active site of the kinase.

J Biol Chem, 1988 Apr 25, 263(12), 5512 - 8
The primosomal protein n' of Escherichia coli is a DNA helicase; Lasken RS et al.; Protein n' of Escherichia coli functions in assembly and translocation of the primosome, a mobile multiprotein complex involved in priming DNA replication (Kornberg, A . (1982) Supplement to DNA Replication, Freeman Publications, San Francisco) . By itself, protein n' translocates on single-stranded DNA and destabilizes duplex regions by acting as a DNA helicase, using the energy of ATP or dATP hydrolysis . Single-stranded DNA binding protein was required for melting of duplex regions longer than 40 base pairs . Initial binding of protein n' to a specific site on DNA (Shlomai, J., and Kornberg, A . (1980) Proc . Natl . Acad . Sci . U.S.A . 77, 799-803) is essential for its helicase function . The polarity of protein n' translocation on DNA, in the 3' to 5' direction of the chain, suggests a mechanism for how the primosome may contribute to concurrent replication of both strands at a replication fork.

J Biol Chem, 1988 Apr 25, 263(12), 5560 - 8
Synthesis, purification, and characterization of the cytoplasmic domain of the human insulin receptor using a baculovirus expression system; Herrera R et al.; The cytoplasmic domain of the beta subunit of the human insulin receptor has been overexpressed in insect cells using the baculovirus expression system . A recombinant baculovirus (BIR-2) was constructed by inserting the human insulin proreceptor cDNA fragment that encodes the cytoplasmic domain of the receptor into the genome of Autographa californica nuclear polyhedrosis virus adjacent to the strong polyhedrin promoter . Synthesis of the protein (baculovirus insulin receptor kinase (BIRK), Mr 48,000) in BIR-2-infected Spodoptera frugiperda (Sf9) cells was detected 24 h after infection and maximal accumulation (2-3% of the cytosolic protein) was achieved 48-72 h post-infection . The expressed protein is active as a soluble protein tyrosine kinase, both in Sf9 cells and in vitro . Rapid purification to near homogeneity was accomplished by sequential chromatography on Fast-Q-Sepharose and phenyl-Superose with an overall yield of 35% and a specific activity with histone as substrate of 20 nmol/min/mg protein . Autophosphorylation activated the intrinsic kinase activity of BIRK and decreased its mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis . Using a combination of tryptic digestion and immunoprecipitation with specific antipeptide antisera, it was ascertained that 30-40% of the 32P incorporated into BIRK by autophosphorylation is in the carboxyl-terminal domain (that includes tyrosyl residues 1316 and 1322 of the human proreceptor) . Of the remaining radioactivity, 75% is in the amino-terminal domain (that includes tyrosyl residues 953, 960, 972, 999, and 1075) and 25% is in the conserved autophosphorylation domain (including tyrosyl residues 1146, 1150, and 1151) . Limited digestion of BIRK with trypsin yielded a fragment, Mr 38,000, that lacks the carboxyl-terminal domain . This fragment exhibits protein tyrosine kinase activity that is stimulated by autophosphorylation . The properties of the soluble, monomeric BIRK are similar to those of the intact, activated, oligomeric insulin receptor kinase with respect to specificity, immunoreactivity, divalent cation requirements, and specific activity . These observations coupled with the ease of producing 0.4 mg of purified enzyme from 100 ml of suspension culture suggest that BIRK will be useful for biochemical and biophysical analysis of the insulin receptor protein tyrosine kinase.

Cell, 1988 Apr 22, 53(2), 267 - 72
Novel reactions of RNAase P with a tRNA-like structure in turnip yellow mosaic virus RNA; Guerrier-Takada C et al.; A quasi-continuous double hellix, containing a pseudoknot and ending in a single-stranded region which contains CCA, can be formed at the 3' terminus of the genomic RNAs of certain plant viruses . M1 RNA (the catalytic subunit) alone and the RNAase P holoenzyme from E . coli cleave the tRNA-like structure of TYMV RNA in vitro at the 5' side of the quasi-helical structure to generate 5' phosphate and 3' hydroxyl groups in the cleavage products . The intact pseudoknot structure in the substrate is not required for the reaction catalyzed by M1 RNA alone, but its presence markedly improves the efficiency of the reaction . In addition to its essential role in the biosynthesis of tRNA, RNAase P may have another function in vivo, namely, in the physiology of viral infections.

Science, 1988 Apr 22, 240(4851), 521 - 3
Evidence from cassette mutagenesis for a structure-function motif in a protein of unknown structure; Clarke ND et al.; The three-dimensional structure of most enzymes is unknown; however, many enzymes may have structural motifs similar to those in the known structures of functionally related enzymes . Evidence is presented that an enzyme of unknown structure {Ile-transfer RNA (tRNA) synthetase} may share a functionally important structural motif with an enzyme of related function (Tyr-tRNA synthetase) . This approach involves (i) identifying segments of Ile-tRNA synthetase that have been unusually conserved during evolution, (ii) predicting the function of one such segment by assuming a structural relation between Ile-tRNA synthetase and Tyr-tRNA synthetase, and (iii) testing the predicted function by mutagenesis and subsequent biochemical analysis . Random mutations were introduced by cassette mutagenesis into a ten-amino-acid segment of Ile-tRNA synthetase that was predicted to be involved in the formation of the binding site for isoleucine . Few amino acid substitutions appear to be tolerated in this region . However, one substitution (independently isolated twice) increased the Michaelis constant Km for isoleucine in the adenylate synthesis reaction by greater than 6000-fold, but had little effect on the Km for adenosine triphosphate, the apparent Km for tRNA, or the rate constant kcat.

Cell, 1988 Apr 22, 53(2), 257 - 66
Target immunity of Mu transposition reflects a differential distribution of Mu B protein; Adzuma K et al.; A DNA molecule carrying Mu end DNA sequence(s) is a poor target in the Mu DNA strand-transfer reaction, a phenomenon which is referred to as "target immunity." We find that Mu B protein stimulates intermolecular strand-transfer by binding to the target DNA . Our results show that a differential distribution of Mu B protein between "immune" and "non-immune" DNA molecules is responsible for target immunity; in the presence of Mu A protein and ATP, Mu B protein dissociates preferentially from immune DNA molecules . Hydrolysis of ATP is implicated in establishing the differential distribution of Mu B protein between immune and non-immune DNA molecules in the presence of Mu A protein; nonhydrolyzable ATP gamma S can support an efficient strand-transfer reaction even with a target DNA that is immune in a reaction with ATP.

Biochim Biophys Acta, 1988 Apr 22, 933(2), 241 - 8
The proton pore in the Escherichia coli F0F1-ATPase: substitution of glutamate by glutamine at position 219 of the alpha-subunit prevents F0-mediated proton permeability; Lightowlers RN et al.; Three mutations in the uncB gene encoding the a-subunit of the F0 portion of the F0F1-ATPase of Escherichia coli were produced by site-directed mutagenesis . These mutations directed the substitution of Glu-219 by Gln, or of Lys-203 by Ile, or of Glu-196 by Ala . Strains carrying either the Lys-203 or Glu-196 substitutions showed growth characteristics indistinguishable from the coupled control strain . Properties of membrane preparations from these strains were also similar to those from the coupled control strain . The substitution of Glu-219 by Gln resulted in a strain which was unable to utilise succinate as sole carbon source and had a growth-yield characteristic of an uncoupled strain . Membrane preparations of the Glu-219 mutant were proton impermeable and the F1-ATPase activity was inhibited by about 50% when membrane-bound . The results are discussed with reference to a previously proposed intramembranous proton pore involving subunits a and c.

J Chromatogr, 1988 Apr 22, 438(2), 347 - 57
Analytical strategy for determination of active site sequences in aminoacyl-tRNA synthetases; Beauvallet C et al.; Affinity labelling with radioactive, periodate-oxidized tRNA has been used to investigate the structures of tRNA-binding sites in Escherichia coli aminoacyl-tRNA synthetases . Labelled peptides were isolated by means of a combination of techniques involving chymotryptic digestion of the enzyme, gel filtration, ribonuclease digestion of tRNA, chromatography on a TSK 2000 column and reversed-phase chromatography . An isocratic phenylthiohydantoin identification system has been interfaced to a sequencer, allowing the characterization of modified lysine residues by means of both chromatographic retention and liquid scintillation counting.

J Mol Biol, 1988 Apr 20, 200(4), 753 - 4
Preliminary X-ray crystallographic study of adenylosuccinate synthetase from Escherichia coli; Serra MA et al.; Adenylosuccinate synthetase, a dimeric enzyme of 96,000 Mr, catalyzes the first committed step toward the de novo biosynthesis of AMP . Large, single crystals of adenylosuccinate synthetase from Escherichia coli grow from solutions of polyethylene glycol and ammonium sulfate . Crystals from ammonium sulfate belong to the orthorhombic space group P212121 with unit cell parameters a = 79.0 A, b = 70.2 A and c = 152.6 A . Crystals from polyethylene glycol belong to the space group P21, having unit cell parameters of a = 71.16 A, b = 71.99 A, c = 82.95 A, and beta = 71.52 degrees . The asymmetric units of both crystal forms probably contain the entire dimeric enzyme.

J Mol Biol, 1988 Apr 20, 200(4), 751 - 2
Crystallization of O6-methylguanine-DNA methyltransferase from Escherichia coli; Moody PC et al.; The 19,000 Mr C-terminal domain of the Escherichia coli ada gene product that contains O6-methylguanine-DNA methyltransferase DNA repair activity has been crystallized in a low-salt environment . The crystals, which diffract to 2.3 A (1 A = 0.1 nm), are suitable for detailed structural studies . The space group is P21 with unit cell dimensions a = 46.3 A, b = 45.8 A, c = 46.9 A and beta = 113.3 degrees.

J Mol Biol, 1988 Apr 20, 200(4), 709 - 23
Selection of DNA binding sites by regulatory proteins . II . The binding specificity of cyclic AMP receptor protein to recognition sites; Berg OG et al.; The statistics of base-pair usage within known recognition sites for a particular DNA-binding protein can be used to estimate the relative protein binding affinities to these sites, as well as to sites containing any other combinations of base-pairs . As has been described elsewhere, the connection between base-pair statistics and binding free energy is made by an equal probability selection assumption; i.e . that all base-pair sequences that provide appropriate binding strength are equally likely to have been chosen as recognition sites in the course of evolution . This is analogous to a statistical-mechanical system where all configurations with the same energy are equally likely to occur . In this communication, we apply the statistical-mechanical selection theory to analyze the base-pair statistics of the known recognition sequences for the cyclic AMP receptor protein (CRP) . The theoretical predictions are found to be in reasonable agreement with binding data for those sequences for which experimental binding information is available, thus lending support to the basic assumptions of the selection theory . On the basis of this agreement, we can predict the affinity for CRP binding to any base-pair sequence, albeit with a large statistical uncertainty . When the known recognition sites for CRP are ranked according to predicted binding affinities, we find that the ranking is consistent with the hypothesis that the level of function of these sites parallels their fractional saturation with CRP-cAMP under in-vivo conditions . When applied to the entire genome, the theory predicts the existence of a large number of randomly occurring "pseudosites" with strong binding affinity for CRP . It appears that most CRP molecules are engaged in non-productive binding at non-specific or pseudospecific sites under in-vivo conditions . In this sense, the specificity of the CRP binding site is very low . Relative specificity requirements for polymerases, repressors and activators are compared in light of the results of this and the first paper in this series.

J Mol Biol, 1988 Apr 20, 200(4), 639 - 54
Spinach chloroplast rpoBC genes encode three subunits of the chloroplast RNA polymerase; Hudson GS et al.; Sequence analysis of a 12,400 base-pair region of the spinach chloroplast genome indicates the presence of three genes encoding subunits of the chloroplast RNA polymerase . These genes are analogous to the rpoBC operon of Escherichia coli, with some significant differences . The first gene, termed rpoB, encodes a 121,000 Mr homologue of the bacterial beta subunit . The second and third genes, termed rpoC1 and rpoC2, encode 78,000 and 154,000 Mr proteins homologous to the N and C-terminal portions, respectively, of the bacterial beta' subunit . RNA mapping analysis indicates that the three genes are cotranscribed, and that a single intron occurs in the rpoC1 gene . No splicing occurs within the rpoC2 gene or between rpoC1 and rpoC2 . Furthermore, the data indicate the possibility of an alternative splice acceptor site for the rpoC1 intron that would give rise to a 71,000 Mr gene product . Thus, with the inclusion of the alpha subunit encoded by rpoA at a separate locus, the chloroplast genome is predicted to encode four subunits (respectively called alpha, beta, beta', beta") equivalent to the three subunits of the core enzyme of the E . coli RNA polymerase.

J Mol Biol, 1988 Apr 20, 200(4), 745 - 8
Proton-nuclear magnetic resonance studies of the aromatic spin systems of Escherichia coli adenylate kinase; Bock I et al.; Escherichia coli adenylate kinase has a very well resolved proton nuclear magnetic resonance spectrum in the region containing signals from aromatic amino acid side-chains . We found that the protein is structurally stable over a wide pH range and renatures spontaneously after acidic as well as basic denaturation . Only one out of the three histidyl imidazole rings titrates on changing the pH and has a pka value of 7.6 . Two-dimensional nuclear magnetic resonance spectroscopy studies allowed use to identify most of the enzyme's aromatic spin systems, and by investigation of a mutant protein we were able to assign the aromatic part of the spin system of Tyr24 unambiguously.

Biochemistry, 1988 Apr 19, 27(8), 3011 - 8
Sequence specificity in photoreaction of various psoralen derivatives with DNA: role in biological activity; Boyer V et al.; The sequence specificity in the photoreaction of various psoralen derivatives with DNA is investigated by using DNA sequencing methodology . The 3'-5' exonuclease activity associated with T4 DNA polymerase serves as a probe to map the psoralens' photoaddition (monoadducts plus biadducts) on DNA fragments of defined sequence . This approach has already allowed us to demonstrate a strong sequence context effect on the 8-methoxypsoralen photobinding to DNA {Sage, E., & Moustacchi, E . (1987) Biochemistry 26, 3307-3314} . The psoralens studied include bifunctional derivatives {8-methoxypsoralen, 5-methoxypsoralen, and 4'-(hydroxymethyl)-4,5',8-trimethylpsoralen} and monofunctional derivatives (angelicin, 3-carbethoxypsoralen, and three pyridopsoralens) . Maps of photochemical binding on two DNA fragments of the lacI gene of Escherichia coli are established for all the derivatives . These maps demonstrate the following general qualitative rules in the photoreaction of the furocoumarins with DNA: thymine residues in a GC environment are cold, adjacent thymines are better targets, 5'-TpA sites are strongly preferred versus 5'-ApT, and alternating (AT)n sequences are hot spots for photoaddition . Depending on the chemical structure of the derivatives and on their affinity for DNA, some minor differences in the binding spectrum are detected . A most interesting example is 3-carbethoxypsoralen, which specifically reacts with (AT)n sites . Our observations lead us to define two types of target sites: the "strong sites", which are preferential targets for all psoralen derivatives, and the "weak sites", which are targets only for derivatives having a high affinity for DNA . The frequency of DNA lesions is much higher in the former sites.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1988 Apr 19, 27(8), 2983 - 90
Expression of active rat DNA polymerase beta in Escherichia coli; Date T et al.; A recombinant plasmid for expression of rat DNA polymerase beta was constructed in a plasmid/phage chimeric vector, pUC118, by an oligonucleotide-directed mutagenesis technique . The insert contained a 1005 bp coding sequence for the whole rat DNA polymerase beta . The recombinant plasmid was designed to use the regulatory sequence of Escherichia coli lac operon and the initiation ATG codon for beta-galactosidase as those for DNA polymerase beta . The recombinant clone, JMp beta 5, obtained by transfection of E . coli JM109 with the plasmid, produced high levels of DNA polymerase activity and a 40-kDa polypeptide that were not detected in JM109 cell extract . Inducing this recombinant E . coli with isopropyl beta-thiogalactopyranoside (IPTG) yielded amounts of 40-kDa polypeptide as high as 19.3% of total protein . Another recombinant clone, JMp beta 2-1, which was constructed by an oligonucleotide-directed mutagenesis to use the second ATG codon for the initiation codon, thus deleting the first 17 amino acid residues from the amino terminus, produced neither high DNA polymerase activity nor the 40-kDa polypeptide . The evidence suggests that this amino-terminal structure is important for stability of this enzyme in E . coli . The DNA polymerase was purified to homogeneity from the IPTG-induced JMp beta 5 cells by fewer steps than the procedure for purification of DNA polymerase beta from animal cells . The properties of this enzyme in activity, chromatographic behavior, size, antigenicity, and also lack of associated nuclease activity were indistinguishable from those of DNA polymerase beta purified from rat cells, indicating the identity of the overproduced DNA polymerase in the JMp beta 5 and the rat DNA polymerase beta.

Biochemistry, 1988 Apr 19, 27(8), 3038 - 45
Photoaffinity labeling of Escherichia coli RNA polymerase/poly{d(A-T)} transcription complexes by nascent RNA; Stackhouse TM et al.; To elucidate the molecular interactions during transcription by Escherichia coli RNA polymerase, we have performed a quantitative analysis of the photoaffinity labeling produced by an aryl azide positioned at the leading (5') end of the nascent RNA . Macromolecular contacts on the path of RNA across the transcription complex containing the template poly{d(A-T)} are observed as a function of the length of the transcript . Quantitative analysis provides the percent yield of photoaffinity labeling in the transcription complex by each length of RNA . Significant yields are observed for DNA, the beta/beta' subunits (analyzed together), and the sigma subunit . The alpha subunit is not labeled under these experimental conditions . The DNA template is labeled by the leading ends of RNA molecules 5-18 bases long, with yields ranging from 1% to 6% . Photoaffinity labeling of poly{d(A-T)} is also observed for many transcript lengths longer than 18 nucleotides, but the yields are too low to quantitate . Labeling of the beta/beta' subunits occurs with approximately 50% yields for transcripts of lengths greater than or equal to 12 nucleotides; low but significant labeling yields of 1-8% by shorter RNAs (3-10 nucleotides) are observed . Labeling of the sigma subunit is detectable for transcripts from 7 to more than 19 nucleotides long; quantitative measurements were possible up to the 19-mer . The RNAs most likely to be photoattached to the sigma subunit are 9-12 nucleotides long, with a maximum photoaffinity labeling yield of 15% by the decanucleotide.(ABSTRACT TRUNCATED AT 250 WORDS)

Gene, 1988 Apr 15, 64(1), 147 - 53
Suppression of the btuB451 mutation by mutations in the tonB gene suggests a direct interaction between TonB and TonB-dependent receptor proteins in the outer membrane of Escherichia coli; Heller KJ et al.; In cells of Escherichia coli, the function of the tonB gene is needed for energy-dependent transport processes mediated by the outer-membrane receptors for iron siderophore complexes and vitamin B12 . The btuB451 mutation has the same effect on vitamin B12 transport as does a tonB mutation . When a btuB451 strain carried a plasmid with the intact tonB gene, partial revertant strains were isolated which had acquired the ability to grow on 5 nM vitamin B12 . This suppression activity was associated with the plasmid, suggesting that a mutation within the tonB gene on the plasmid allowed the mutant BtuB receptor to function in the transport of the vitamin . The nucleotide sequence of the entire tonB gene of ten independently isolated suppressing plasmids was determined . Only a single nucleotide change had occurred in each of the cases . The same codon was always affected resulting in the conversion of glutamine-165 to a leucine in seven of the ten isolates and to a lysine in the other three . The phenotype of strains carrying both types of altered tonB genes showed the retention of their function for other TonB-dependent processes in addition to their suppressor properties with respect to the btuB451 mutation . The fact that mutations suppressing the btuB451 mutation occurred in the tonB gene suggests that there is a direct interaction between TonB and TonB-dependent receptors in the outer membrane of E . coli.

J Biol Chem, 1988 Apr 15, 263(11), 5368 - 72
Efficient in vitro translocation into Escherichia coli membrane vesicles of a protein carrying an uncleavable signal peptide . Characterization of the translocation process; Yamane K et al.; The translocation into Escherichia coli cytoplasmic membrane vesicles of a protein containing an uncleavable signal peptide was studied . The signal peptide cleavage site of the ompF-lpp chimeric protein, a model secretory protein, was changed from Ala-Ala to Phe-Pro through oligonucleotide-directed site-specific mutagenesis of the ompF-lpp gene on a plasmid . The mutant protein was no longer processed by the signal peptidase . When proteinase K treatment was adopted as a probe for protein translocation into inverted membrane vesicles, the mutant protein exhibited rapid and almost complete translocation, most likely due to the lack of premature cleavage of the signal peptide before the translocation . This result also indicates that cleavage of the signal peptide is not required for translocation of the mature domain of the protein . The establishment of an efficient system made it possible to perform precise and quantitative analysis of the translocation process . The translocation was time-dependent, vesicle-dependent, and required ATP and NADH . Translocation into membrane vesicles was also observed with the uncleavable precursor protein purified by means of immunoaffinity chromatography, although the efficiency was appreciably low . The translocation required only ATP and NADH . Addition of the cytosolic fraction did not enhance the translocation.

J Biol Chem, 1988 Apr 15, 263(11), 5235 - 40
The active form of the cytochrome d terminal oxidase complex of Escherichia coli is a heterodimer containing one copy of each of the two subunits; Miller MJ et al.; The cytochrome d complex is a component of the aerobic respiratory system of Escherichia coli . The enzyme functions as a terminal oxidase, oxidizing ubiquinol-8 within the cytoplasmic membrane and reducing oxygen to water . The enzyme is of particular interest because it is a coupling site in the electron transfer chain . The electron transfer reaction catalyzed by this enzyme is coupled to the translocations of protons across the membrane (H+/e-approximately equal to 1) . The oxidase contains two subunits by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, with molecular weights of 58,000 and 43,000 . In this paper, the question of the quaternary structure is addressed . Quantitative N-terminal analysis of the isolated enzyme and relative mass quantitation following sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicate the subunits are present in equimolar amounts . Sedimentation velocity and sedimentation equilibrium studies were used to characterize the hydrodynamic properties of the purified enzyme solubilized in Triton X-100, under conditions where the enzyme is active . It is concluded that the active enzyme in Triton X-100 is a heterodimer, containing one copy of each subunit . This is likely the structure of the enzyme in the E . coli membrane.

Eur J Biochem, 1988 Apr 15, 173(2), 389 - 94
Increase in nucleolytic activity of ribonuclease T1 by substitution of tryptophan 45 for tyrosine 45; Nishikawa S et al.; The preparation and analysis of a mutant ribonuclease (RNase) T1 which possesses higher nucleolytic activity than the wild-type enzyme are described . The gene for the mutant RNase T1 (Tyr45----Trp45), in which a single amino acid at the binding site of the guanine base has been changed, was constructed by the cassette mutangenesis method using a chemically synthesized gene {Ikehara, M . et al . (1986) Proc . Natl Acad . Sci . USA 83, 4695-4699} . In order to reduce the nucleolytic activity of the enzyme in vivo, this gene was expressed in Escherichia coli as a fused protein connected through methionine residues to other proteins at both the N- and C-termini . After liberation from the fused protein by cleavage with cyanogen bromide at the methionine junctions, the mutant RNase T1 was purified by column chromatography . The nucleolytic activity toward pGpC increased to 120% of that of wild-type RNase T1 . The kinetic parameters of the mutant enzyme demonstrate that this higher nucleolytic activity is due to a higher affinity for the substrate, probably because of an increased stacking effect in the binding pocket for the guanine base . This mutant enzyme also possessed a higher nucleolytic activity against pApC than wild-type RNase T1.

J Biol Chem, 1988 Apr 15, 263(11), 5098 - 103
Induction of kappa light chain synthesis in 70Z/3 B lymphoma cells by chemically defined lipid A precursors; Sibley CH et al.; The murine B cell lymphoma 70Z/3 is a tumor line that resembles an early B cell . It responds to lipopolysaccharide by synthesizing kappa light chains, resulting in the appearance of surface IgM . We now demonstrate that this effect is triggered by the lipid A domain of lipopolysaccharide since a defined tetraacyl disaccharide 1,4'-bisphosphate precursor of mature lipid A, designated lipid IVA (Raetz, C . R . H., Purcell, S., Meyer, M . V., Qureshi, N., and Takayama, K . (1985) J . Biol . Chem . 260, 16080-16088), is fully active in stimulating kappa chain mRNA synthesis . In contrast, the monosaccharide lipid A precursor, lipid X, shows only slight activity; but a large excess of lipid X appears to complete with lipid IVA, partially blocking its effects . Mutants of the 70Z/3 line that are unresponsive to lipopolysaccharide also fail to respond to lipid IVA . The somatic cell system described here, coupled with the use of chemically defined agonists and antagonists, offers a new approach to understanding the early events in lipopolysaccharide/animal cell interactions.

Biochem J, 1988 Apr 15, 251(2), 313 - 22
The overexpression, purification and complete amino acid sequence of chorismate synthase from Escherichia coli K12 and its comparison with the enzyme from Neurospora crassa; White PJ et al.; The enzyme chorismate synthase was purified in milligram quantities from an overproducing strain of Escherichia coli . The amino acid sequence was deduced from the nucleotide sequence of the aroC gene and confirmed by determining the N-terminal amino acid sequence of the purified enzyme . The complete polypeptide chain consists of 357 amino acid residues and has a calculated subunit Mr of 38,183 . Cross-linking and gel-filtration experiments show that the enzyme is tetrameric . An improved purification of chorismate synthase from Neurospora crassa is also described . Cross-linking and gel-filtration experiments on the N . crassa enzyme show that it is also tetrameric with a subunit Mr of 50,000 . It is proposed that the subunits of the N . crassa enzyme are larger because they contain a diaphorase domain that is absent from the E . coli enzyme.

J Biol Chem, 1988 Apr 15, 263(11), 5254 - 9
The structure of the amino terminus of tropomyosin is critical for binding to actin in the absence and presence of troponin; Heald RW et al.; Analysis of two recombinant variants of chicken striated muscle alpha-tropomyosin has shown that the structure of the amino terminus is crucial for most aspects of tropomyosin function: affinity to actin, promotion of binding to actin by troponin, and regulation of the actomyosin MgATPase . Initial characterization of variants expressed and isolated from Escherichia coli has been published (Hitchcock-DeGregori, S . E., and Heald, R . W . (1987) J . Biol . Chem . 262, 9730-9735) . Fusion tropomyosin contains 80 amino acids of a nonstructural influenza virus protein (NS1) on the amino terminus . Nonfusion tropomyosin is a variant because the amino-terminal methionine is not acetylated (unacetylated tropomyosin) . The affinity of tropomyosin labeled at Cys190 with N-{14C}ethylmaleimide for actin was measured by cosedimentation in a Beckman Airfuge . Fusion tropomyosin binds to actin with an affinity slightly greater than that of chicken striated muscle alpha-tropomyosin (Kapp = 1-2 X 10(7) versus 0.5-1 X 10(7) M-1) and more strongly than unacetylated tropomyosin (Kapp = 3 X 10(5) M-1) . Both variants bind cooperatively to actin . Troponin increases the affinity of unacetylated tropomyosin for actin (+Ca2+, Kapp = 6 X 10(6) M-1; +EGTA, Kapp = 2 X 10(7) M-1), but the affinity is still lower than that of muscle tropomyosin for actin in the presence of troponin (Kapp much greater than 10(8) M-1) . Troponin has no effect on the affinity of fusion tropomyosin for actin indicating that binding of troponin T to the over-lap region of the adjacent tropomyosin, presumably sterically prevented by the fusion peptide in fusion tropomyosin, is required for troponin to promote the binding of tropomyosin to actin . The role of troponin T in regulation and the mechanisms of cooperative binding of tropomyosin to actin have been discussed in relation to this work.

Gene, 1988 Apr 15, 64(1), 21 - 31
An engrailed class homeo box gene in sea urchins; Dolecki GJ et al.; The homeo box, a conserved DNA element first recognized in Drosophila development-controlling genes, is present in the genomes of many higher metazoan species and provides a valuable probe for the isolation of regulatory genes from diverse phylogenetic groups . We have employed these probes to isolate and study the homeo-box genes in sea urchins . As in other species, the sea urchin homeo boxes fall into at least two classes defined by nucleotide sequence similarity to the homeo boxes of the Drosophila Antennapedia (Antp) and engrailed (en) genes . In this study, we characterize the only detectable sea urchin en class homeo box . Its nucleotide sequence similarity and lack of an intron indicate that it is more closely related to the two mouse en class homeo boxes than to the two Drosophila en class homeo boxes . These relationships are most parsimoniously explained if the single sea urchin en class homeo-box gene represents the primitive condition and the two mouse and the two Drosophila en class homeo-box genes represent independent duplications which occurred in the evolutionary lines leading to the vertebrates and arthropods, respectively . The most abundant en class gene transcripts detected by gel transfer analysis of RNA extracted from sea urchin tissues were found in Aristotle's lantern . Rare transcripts were present in ovary, testis and coelomocytes.

J Biol Chem, 1988 Apr 15, 263(11), 5402 - 7
Effect of an uncE ribosome-binding site mutation on the synthesis and assembly of the Escherichia coli proton-translocating ATPase; Solomon KA et al.; Plasmid pRPG54, which carries the genes for the eight subunits of the proton-translocating ATPase of Escherichia coli, has been found to carry a single base change of a G to an A in the ribosome-binding site for uncE, the gene which codes for the N,N'-dicyclohexylcarbodiimide-binding subunit c of the Fo . This noncoding region mutation both lowers expression of uncE by a factor of 2-3 and affects the function of the ATPase, specifically of the Fo sector . The presence of the mutation results in a decrease in the proton permeability of the Fo or of the entire F1Fo-ATPase complex when either is synthesized from genes on a multicopy plasmid . Expression of uncE from an F1Fo plasmid carrying the wild type ribosome binding site results in increased membrane proton permeability and decreased ability of the resultant ATPase to couple a transmembrane proton gradient to ATP synthesis both in vitro and in vivo . Also, although an Fo plasmid carrying the correct ribosome-binding site causes harmful, F1-dependent proton permeability in unc+ cells (Brusilow, W . S . S . (1987) J . Bacteriol . 169, 4984-4990), an identical plasmid carrying the mutation does not, even though it still codes for a functional reconstitutable Fo . The results show a relationship between the relative level of expression of uncE from a multicopy plasmid and the assembly pathway, proton permeability, and energy-coupling characteristics of the ATPase.

J Biol Chem, 1988 Apr 15, 263(11), 5362 - 7
Isolation and characterization of a receptor for type 1 fimbriae of Escherichia coli from guinea pig erythrocytes; Giampapa CS et al.; The adhesion of Escherichia coli to eukaryotic cells is mediated by proteinaceous surface appendages called fimbriae and complementary receptors on host cells . Although type 1 fimbriae, which contain a D-mannose-reactive lectin, have been well studied little is known about the binding mechanism of isolated fimbriae to individual cell receptors . This report describes the isolation and purification of a guinea pig erythrocyte receptor for type 1 fimbriae . Erythrocyte membranes were dissolved in 0.5% Triton X-100 and the receptor isolated and purified by affinity chromatography using type 1 fimbriae immobilized on Sepharose . The 65-kDa receptor, which inhibits the agglutination of guinea pig erythrocytes by type 1 fimbriated E . coli, has a pI of 8.5-8.7, and binds concanavalin A and type 1 fimbriae in a dose-dependent and saturable manner . The fimbrial binding activity of the receptor was reduced when treated with sodium metaperiodate, endoglycosidase H, trypsin, and V8 protease, suggesting the isolated receptor is a glycoprotein with N-linked carbohydrate units . Isolated type 1 fimbriae inhibited the binding of fimbriated E . coli to purified receptor in a dose- and time-related fashion . The calculated binding affinity was 6 X 10(6) M-1, a value consistent with the low binding affinity expected from previous studies of the agglutination of guinea pig erythrocytes by isolated type 1 fimbriae.

Gene, 1988 Apr 15, 64(1), 135 - 45
Bidirectional chain-termination nucleotide sequencing: transposon Tn5seq1 as a mobile source of primer sites; Nag DK et al.; The sequencing of large DNA fragments by the chain-termination method {Sanger et al., Proc . Natl . Acad . Sci . USA 74 (1977) 5463-5467} has generally required extensive manipulations to bring all parts of the fragment near a specific primer-binding site, or the repeated synthesis of new oligodeoxynucleotide primers . Here we develop a more efficient approach, the use of a transposable element to insert primer binding sites at random in the DNA of interest . We constructed a Tn5 derivative called Tn5seq1 with unique DNA segments near each end so that oligodeoxynucleotides matching them could serve as primers for sequencing in each direction from any Tn5seq1 insertion site . Our experiments demonstrate the use of Tn5seq1 for sequencing in pBR322 plasmids and also in uncloned DNAs of the Escherichia coli chromosome . The unique segments near the left and right ends of Tn5seq1 are promoters from phages T7 and SP6, respectively, to permit the efficient transcription of adjacent DNAs in vivo or in vitro.

J Biol Chem, 1988 Apr 15, 263(11), 5271 - 6
Trypsin proteolysis of the cytochrome d complex of Escherichia coli selectively inhibits ubiquinol oxidase activity while not affecting N,N,N',N'-tetramethyl-p-phenylenediamine oxidase activity; Lorence RM et al.; The cytochrome d complex is one of two membrane-bound terminal oxidases of the Escherichia coli aerobic respiratory chain . Previous studies have shown that this enzyme reconstituted into proteoliposomes rapidly oxidizes ubiquinol-8 as well as the soluble homologue, ubiquinol-1, and that quinol oxidase activity is accompanied by the formation of a transmembrane H+ electrochemical gradient . The enzyme also oxidizes the artificial reductant, N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) with the generation of a H+ electrochemical gradient . In this work, it is established that trypsin digestion of the purified cytochrome d complex cleaves subunit I while subunit II is unaffected . Proteolysis of subunit I is correlated with loss of ubiquinol-8 and ubiquinol-1 oxidase activities . Trypsin digestion has no effect on TMPD oxidase activity . The cytochrome d complex is concluded to possess three distinct active sites for 1) ubiquinol oxidation, 2) TMPD oxidation, and 3) oxygen binding and reduction . Data also suggest that both sites of ubiquinol and TMPD oxidations are located on the periplasmic side of the E . coli membrane while the site of oxygen reduction is on the opposite side.

Gene, 1988 Apr 15, 64(1), 107 - 19
Characterization of immunoreactive epitopes of the HIV-1 p41 envelope protein using fusion proteins synthesized in Escherichia coli; Windheuser MG et al.; We have identified several immunoreactive epitopes on the human immunodeficiency virus (HIV) type 1 transmembrane envelope protein by synthesizing various regions of the protein as fusions to the trpE gene in Escherichia coli . Ten fusion clones which expressed overlapping peptides were found to contain epitopes reactive with antibodies in sera of North American (NAm) and West African (WAf) patients with acquired immune deficiency syndrome (AIDS) . An immunodominant epitope which reacted with all HIV-infected patients' sera was mapped to a 51-amino acid sequence in the N terminus of p41 . A novel epitope was also identified in the C terminus of p41 which was reactive with 41% and 48% of the sera tested from NAm and WAf, respectively . In addition, several minor epitopes were identified . We observed that sera from WAf reacted more strongly to minor HIV-1 p41 epitopes than did sera from similarly infected individuals in NAm . We also report on the detection of antibodies from patients with HIV-2 infection in WAf which cross react with HIV-1 p41 recombinant envelope antigens.

Gene, 1988 Apr 15, 64(1), 1 - 8
Cloning and expression of the MspI restriction and modification genes; Nwankwo DO et al.; The genes for the MspI restriction (R) and modification enzymes (recognition sequence CCGG) have been cloned into Escherichia coli using the vector pBR322 . Clones carrying both genes have been isolated from libraries prepared with EcoRI, HindIII and BamHI . The smallest fragment that encodes both activities is a 3.6-kb HindIII fragment . Plasmids purified from the clones are fully resistant to digestion by MspI, indicating that the modification gene is functional in E . coli . The clones remain sensitive to phage infection, however, indicating that the endonuclease is dysfunctional . When the R gene is brought under the control of the inducible leftward promoter from phage lambda, the level of endonuclease increases and the level of methylase decreases, suggesting that the genes are transcribed in opposite directions.

Nature, 1988 Apr 14, 332(6165), 649 - 50
The JUN oncoprotein, a vertebrate transcription factor, activates transcription in yeast; Struhl K; Transcriptional activation of RNA polymerase II in eukaryotic organisms ranging from yeasts to mammals has many common features such as enhancer elements, TATA elements, and activator proteins that bind specifically to promoter DNA . The JUN oncoprotein, which causes sarcomas in chickens, shows significant homology to the DNA-binding domain of GCN4, a yeast protein that stimulates transcription of the amino acid biosynthetic genes . The GCN4 and JUN proteins bind the same DNA sequences, consensus ATGA(C/G)TCAT, even though the DNA-binding domains are only 45% identical in amino acid sequence . The JUN protein almost certainly represents the oncogenic version of the normal AP-1 transcription factor, suggesting an evolutionary relationship between yeast and vertebrate activator proteins . Here, I demonstrate that JUN efficiently activates transcription in yeast either through its own or a heterologous DNA-binding domain . As is the case for yeast activator proteins, transcriptional stimulation by JUN requires an acidic activation region distinct from the DNA-binding domain . The functional interchangeability between yeast and vertebrate transcription factors strongly suggests a basic similarity in the molecular mechanism of eukaryotic transcriptional activation.

FEBS Lett, 1988 Apr 11, 231(1), 99 - 101
Processing by inverted plasma membrane vesicles of in vitro synthesized major lipoprotein from Escherichia coli; Krishnabhakdi SS et al.; Synthesis, lipid modification and proteolytic processing of the major lipoprotein from Escherichia coli is shown to occur in a homologous in vitro transcription-translation system containing inverted plasma membrane vesicles . The primary translation product (cross-reacting with anti-lipoprotein antiserum) is a precursor which is converted into a lower molecular mass species of the size of mature lipoprotein by the addition of inverted membrane vesicles from E . coli . Conversion is prevented by globomycin, a specific inhibitor of the unique lipoprotein-signal peptidase II, which is active only on lipid-containing precursors . The inverted plasma membrane vesicles used here must therefore contain active lipid-modifying enzymes and signal peptidase II.

FEBS Lett, 1988 Apr 11, 231(1), 243 - 6
Formation of the 680 nm-absorbing form of the cytochrome bd oxidase complex of Escherichia coli by reaction of hydrogen peroxide with the ferric form; Poole RK et al.; Reduced minus aerated difference spectra of membranes from Escherichia coli (grown under oxygen-limited conditions) show, in addition to the 650 nm trough attributed to the oxygenated form of cytochrome d, a smaller trough centred at about 680 nm of unknown origin . When the reference spectrum is that of a sample oxidized with ferricyanide and to which hydrogen peroxide was added, the trough proportions changed, the 680 nm species being more dominant . Similarly, when 8.8 mM hydrogen peroxide is added to a persulphate-oxidized sample, a peak at 680 nm is immediately formed . No such compound is observed when peroxide is added to persulphate-oxidized membranes from a cytochrome d-deficient mutant . It is concluded that the 680 nm species represents a peroxy form of haem d, which is stable at room temperature and is probably an intermediate in the reaction mechanism of this oxidase.

FEBS Lett, 1988 Apr 11, 231(1), 187 - 91
Human ubiquitin genes: one member of the UbB gene subfamily is a tetrameric non-processed pseudogene; Cowland JB et al.; The human ubiquitin gene family consists of three subfamilies . One of these, the UbB subfamily, includes a functional gene coding for a polyubiquitin protein that contains three ubiquitin copies tandemly repeated, as well as three pseudogenes of the processed type . We have now isolated a fifth human UbB type gene, different from any of the previously identified ones . This newly isolated gene is a tetrameric pseudogene which has presumably arisen by unequal crossing-over of two ancestral trimeric alleles . Southern blotting data indicate that all members of the human UbB gene subfamily are now accounted for.

Nucleic Acids Res, 1988 Apr 11, 16(7), 2931 - 42
Dependence of M1 RNA substrate specificity on magnesium ion concentration; Nichols L et al.; We have constructed a plasmid expressing E . coli M1 RNA, the catalytic RNA subunit of ribonuclease P, under the control of a phage T7 promoter . The active M1 RNA species synthesized in vitro by T7 RNA polymerase from this vector was reacted with the tRNA(Gln) - tRNA(Leu) precursor RNA (Band K) encoded by phage T4 . Only the tRNA(Leu) moiety of this dimeric precursor RNA contains the 3' terminal C-C-A sequence common to all tRNAs . We observed that protein-free M1 RNA was capable of processing the precursor RNA at the 5' ends of both tRNA tRNA sequences . The rate of cleavage of the tRNA(Gln) sequence was more strongly dependent on {Mg2+} than that of tRNA(Leu), increasing severalfold between 100 and 500 mM Mg2+, conditions under which the rate of cleavage at the tRNA(Leu) sequence was constant.

Science, 1988 Apr 8, 240(4849), 199 - 201
Genetic and crystallographic studies of the 3',5'-exonucleolytic site of DNA polymerase I; Derbyshire V et al.; Site-directed mutagenesis of the large fragment of DNA polymerase I (Klenow fragment) yielded two mutant proteins lacking 3',5'-exonuclease activity but having normal polymerase activity . Crystallographic analysis of the mutant proteins showed that neither had any alteration in protein structure other than the expected changes at the mutation sites . These results confirmed the presumed location of the exonuclease active site on the small domain of Klenow fragment and its physical separation from the polymerase active site . An anomalous scattering difference Fourier of a complex of the wild-type enzyme with divalent manganese ion and deoxythymidine monophosphate showed that the exonuclease active site has binding sites for two divalent metal ions . The properties of the mutant proteins suggest that one metal ion plays a role in substrate binding while the other is involved in catalysis of the exonuclease reaction.

Biochim Biophys Acta, 1988 Apr 7, 939(2), 282 - 8
Solubilization and reconstitution of proline carrier in Escherichia coli; quantitative analysis and optimal conditions; Hanada K et al.; Proline carrier of Escherichia coli was extracted from the carrier-overproducing membranes with dodecylmaltoside in the presence of phospholipid . The solubilized carrier showed the same proline binding activity as that in normal membranes . As judged from determinations of the binding activity in the micellar state as a marker of active carrier and the radioactivity of N-{ethyl-2-3H}ethylmaleimide-labeled carrier as a marker of carrier polypeptide, 80% of the carrier molecules in the membranes were extracted . Optimal conditions for reconstitution of the solubilized carrier were established . By a combination of freeze-thawing, sonication and dilution procedures, 70% of the solubilized carrier molecules were incorporated into proteoliposomes and the restored active transport of proline showed an apparent Kt of 1 microM and turnover number of 0.6 s-1 . The transport of proline was driven by a membrane potential in a Na+ (or Li+)-dependent manner.

Biochim Biophys Acta, 1988 Apr 7, 939(2), 289 - 94
Use of a fluorescein derivative of phosphatidylethanolamine as a pH probe at water/lipid interfaces; Soucaille P et al.; A fluorescent indicator for the determination of pH in the vicinity of water/lipid interfaces was produced by the covalent linkage of fluoresceinisothiocyanate to Escherichia coli phosphatidylethanolamine . When embedded in monolayers spread on an air/water interface, its apparent pK was shown to be only slightly affected by the nature of the polar headgroups or the packing density of the host phospholipids . Its properties were not affected by the ion content of the subphase . For small unilamellar vesicles, its properties were only affected when localized in the inner layer . This probe could therefore be of value in the study of proton fluxes along biological membranes.

Biochemistry, 1988 Apr 5, 27(7), 2603 - 8
Affinity labeling of the P site of Drosophila ribosomes: a comparison of results from (bromoacetyl)phenylalanyl-tRNA and mercurated fragment affinity reactions; North D et al.; The binding site of the peptidyl group of peptidyl-tRNA in the P site of Drosophila ribosomes was probed with (bromoacetyl)phenylalanyl-tRNA (BrAcPhe-tRNA) . This affinity label binds specifically to the P site by virtue of its ability to participate in peptide bond formation with puromycin following its attachment to ribosomes . As many as nine ribosomal proteins may be labeled under these conditions; however, the majority of the labeling is associated with three large-subunit proteins and two small-subunit proteins . Two of the large-subunit proteins, L4 and L27, are electrophoretically very similar to the proteins labeled by the same reagent in Escherichia coli ribosomes L2 and L27 . Reexamination by a different two-dimensional gel system of the ribosomal components labeled by a second P site reagent, the 3' pentanucleotide fragment of N-acetylleucyl-tRNA which is derivatized to contain mercury atoms at the C-5 position of all three cytosine residues, shows two major and three minor labeled proteins . These proteins, L10/L11, L26, S1/S4, S13, and S20, are likely present in the binding site of the 3' end of peptidyl-tRNA, a site that appears to span both subunits . These results have allowed us to construct a model for the protein positions in and near the peptidyl-tRNA binding site of Drosophila ribosomes.

J Biol Chem, 1988 Apr 5, 263(10), 4939 - 44
Wheat germ cytoplasmic ribosomes . Localization of 7-methylguanosine and 6-methyladenosine by electron microscopy of immune complexes; Montesano L et al.; Nucleoside analysis of the RNA from the small subunit of wheat germ cytoplasmic ribosomes shows 1 mol each of N7-methylguanosine and N6-methyladenosine/mol of RNA . Antibodies directed against each methylated nucleoside were used to localize these residues within the subunit by electron microscopy of immune complexes . Antibodies to 7-methylguanosine bound 40 S subunits at a single site, at or slightly above the division between the upper and lower segments of the particle and on the surface furthest from the platform (or large lobe) of the subunit . This site is essentially equivalent to that previously seen with Escherichia coli and chloroplast 30 S subunits (Trempe, M . R., Ohgi, K., and Glitz, D . G . (1982) J . Biol . Chem . 257, 9822-9829) . Antibodies to N6-monomethyladenosine were induced in rabbits with a nucleoside-albumin conjugate and shown to be specific for the modified nucleoside . Electron microscopy of antibody-subunit complexes placed the methyladenosine residue in a position that is essentially indistinguishable from that of 7-methylguanosine.

J Mol Biol, 1988 Apr 5, 200(3), 501 - 11
Genetic analysis of the membrane insertion and topology of MalF, a cytoplasmic membrane protein of Escherichia coli; Froshauer S et al.; MalF is an essential cytoplasmic membrane protein of the maltose transport system of Escherichia coli . We have developed a general approach for analysis of the mechanism of integration of membrane proteins and their membrane topology by characterizing a series of fusions of beta-galactosidase to MalF . The properties of the fusion proteins indicate the following . (1) The first two presumed transmembrane segments of MalF are sufficient to anchor beta-galactosidase firmly to the inner membrane . (2) Hybrid proteins with beta-galactosidase fused to a presumed cytoplasmic domain of MalF have high beta-galactosidase specific activity; fusions to periplasmic domains have low activity . We propose therefore, that periplasmic and cytoplasmic domains of integral membrane proteins can be distinguished by the enzymatic properties of such hybrid proteins . In general, it appears that cleaved or non-cleaved signal sequences when attached to beta-galactosidase cause it to become embedded in the membrane, and this results in the inability of the hybrid proteins to assemble into active enzyme . Additional properties of these fusion proteins contribute to our understanding of the regulation of MalF synthesis . The MalF protein, synthesized as part of the malEFG operon of E . coli, is approximately 30-fold less abundant in the cell than MalE protein (the maltose-binding protein) . Differential amounts of the fusion proteins indicate that a regulatory signal occurs within the malF gene that is responsible for the step-down in expression from the malE gene to the malF gene.

J Mol Biol, 1988 Apr 5, 200(3), 427 - 38
Generation of a detailed physical and genetic map of the ilv-metE-udp region of the Escherichia coli chromosome; Aldea M et al.; The entire ilv-metE-udp region of the Escherichia coli chromosome has been cloned in two steps using the lambda replacement vector EMBL4 . A detailed restriction map for approximately 70 X 10(3) bases of DNA has been generated . The gpp and udp structural genes have been identified, the cya and metE genes have been physically located, and the direction of recQ gene transcription has been determined . By examining a variety of plasmid subclones, 44 polypeptides have been detected using maxicell and minicell analysis, accounting for 70% of the maximum coding capacity of the entire region . On the basis of the observed gene density in the ilv-metE-udp region, a total number of 3000 genes is predicted for the entire E . coli chromosome . In addition, anomalies in cotransduction frequencies that have been observed in this region have been interpreted by employing a new formula that incorporates the effects of different transducing fragment representations and recombination probabilities.

Biochemistry, 1988 Apr 5, 27(7), 2635 - 40
ATP-stimulated hydrolysis of GTP by RecA protein: kinetic consequences of cooperative RecA protein-ATP interactions; Menge KL et al.; The cooperativity of the single-stranded DNA dependent nucleoside triphosphatase activity of the recA protein was investigated by examining the influence of a good substrate (ATP) on the hydrolysis of a poor substrate (GTP) . At pH 7.5 and 37 degrees C, both ATP and GTP are hydrolyzed with a turnover number of 17.5 min-1 . The S0.5 for GTP (750 microM), however, is nearly 20-fold higher than the S0.5 for ATP (45 microM) . Low concentrations of ATP activate the GTPase activity of the recA protein by lowering the S0.5 for GTP; in the presence of 50 microM ATP, the S0.5 for GTP is reduced from 750 microM to 200 microM . Concentrations of ATP greater than 50 microM result in competitive inhibition of the ATP-activated GTPase activity . Although GTP is a substrate for hydrolysis, it will not substitute for ATP as a high-energy cofactor in the standard recA protein promoted three-strand exchange reaction . To account for these results, a minimal kinetic model is presented in which ATP binding induces specific conformational changes in the recA protein that do not occur with GTP binding.

Biochemistry, 1988 Apr 5, 27(7), 2597 - 603
Cytoplasmic membrane is the target organelle for transition metal mediated damage induced by paraquat in Escherichia coli; Kohen R et al.; Bacterial survival indicates that copper or iron is an essential mediator in paraquat toxicity in Escherichia coli {Kohen, R., & Chevion, M . (1985) Free Radical Res . Commun . 1, 79-88; Korbashi, P., Kohen, R., Katzhendler, J., & Chevion, M . (1986) J . Biol . Chem . 261, 12472-12476} . In this study we have identified the cytoplasmic membrane as a target organelle in metal-mediated paraquat toxicity and have demonstrated the complete correlation of the membrane damage with the levels of adventitious copper (or iron) . The extent of membrane damage was related by use of four parameters: (a) the level of cellular ATP, (b) the level of cellular potassium, (c) the cellular capacity to accumulate and retain radiolabeled leucine, and (d) the cellular integrity as reflected by transmission electron microscopy (TEM) . Exposure of bacterial cells to a combination of paraquat and copper caused a marked decline in parameters a, b, and c . This decline was found to occur in parallel with, or even to precede, the sharp loss of survival of E . coli under the same conditions . Likewise, TEM micrographs clearly indicated alterations in cellular structure that possibly reflect sites of detachment of the cytoplasmic membrane from the bacterial capsule . In contradistinction, copper alone or paraquat alone could not bring about similar changes in cellular structure . These findings are in accord with the suggested site-specific metal-mediated Haber-Weiss mechanism for paraquat toxicity and support our notion that specific chelators of transition metals could reduce or prevent the biological deleterious effects of this herbicide.

Biochemistry, 1988 Apr 5, 27(7), 2260 - 5
Negative cooperativity within individual tetramers of Escherichia coli single strand binding protein is responsible for the transition between the (SSB)35 and (SSB)56 DNA binding modes; Lohman TM et al.; We have examined the binding of the oligonucleotide dT (pT)34 to the Escherichia coli SSB protein as a function of NaCl and MgCl2 concentration (25 degrees C, pH 8.1) by monitoring the quenching of the intrinsic protein fluorescence . We find two binding sites for dT(pT)34 per single strand binding (SSB) protein tetramer, with each site possessing widely different affinities depending on the salt concentration . At 200 mM NaCl, we observe nearly stoichiometric binding of dT(pT)34 to both binding sites within the SSB tetramer, although a difference in the affinities is still apparent . However, when the NaCl concentration is lowered, the overall affinity of dT(pT)34 for the second site on the SSB tetramer decreases dramatically . At 1.5 mM NaCl, only a single molecule of dT(pT)34 can bind per SSB tetramer, even with a 10-fold molar excess of dT(pT)34 . MgCl2 is effective at 100-fold lower concentrations than NaCl in promoting the binding of the second molecule of dT(pT)34 . This binding behavior reflects an intrinsic property of the SSb tetramer, since it is also observed upon binding of smaller oligonucleotides, and the simplest explanation is that a salt-dependent negative cooperativity exists between DNA binding sites within the SSB tetramer . This phenomenon is also responsible for the transition between the two SSB-single strand (ss) polynucleotide binding modes that cover 35 and 56 nucleotides per tetramer {Bujalowski, W., & Lohman, T . M . (1986) Biochemistry 25, 7799-7802} . Extreme negative cooperativity stabilizes the (SSB)35 binding mode, in which the SSB tetramer binds tightly to ss DNA with only two of its subunits while the other two subunits remain unligated.(ABSTRACT TRUNCATED AT 250 WORDS)

Eur J Biochem, 1988 Apr 5, 173(1), 65 - 72
The conformation and stability of recombinant-derived granulocyte-macrophage colony stimulating factors; Wingfield P et al.; The conformation and stability of recombinant-derived human and murine granulocyte-macrophage colony stimulating factors produced in Escherichia coli have been investigated by analytical ultracentrifugation, urea-gradient polyacrylamide gel electrophoresis and several spectroscopic methods . The proteins were demonstrated to be physically homogeneous monomeric proteins with compact globular shapes and shown to have similar secondary structures containing both alpha-helix and beta-sheet structure . The intramolecular disulphide linkages of both proteins were shown to be essential for maintaining native conformation as reduction with dithiothreitol resulted in protein unfolding . Comparison of the human E . coli-derived (non-glycosylated) and mammalian cell culture-derived (glycosylated) proteins by urea-gradient electrophoresis indicated that glycosylation had no major effect on the conformational stability and kinetics of urea induced unfolding and refolding.

Eur J Biochem, 1988 Apr 5, 173(1), 233 - 9
Electrostatic potential of macromolecules measured by pKa shift of a fluorophore . 2 . Transfer RNA; Friedrich K et al.; The procedures developed earlier (Friedrich and Woolley, preceding paper in this journal) for probing electrostatic potential with the fluorescein label were applied to transfer RNA . By using tRNA species that contain chemically reactive bases we were able to label these bases with fluorescein derivatives and thus to 'map' the electrostatic potential around the molecule . Both the electrostatic potential and the fluorescence emission anisotropy data that were obtained at the same time could be understood in terms of the well-known, paradigmatic crystal structure of tRNA(Phe) . However, within the distribution of the various tRNA species, tRNA(Met)f appeared to occupy an extreme position, which suggests a relation between the conformation in solution and the initiation function of this molecule . Comparison with theoretical predictions by others of the electrostatic potential map of tRNA showed agreement in respect of trends, but the values of the potentials measured were orders of magnitude lower than predicted . This we attribute primarily to solvation.

Eur J Biochem, 1988 Apr 5, 173(1), 109 - 14
Expression, renaturation and purification of recombinant human interleukin 4 from Escherichia coli; van Kimmenade A et al.; The lymphokine human interleukin 4 (IL-4) has been expressed from a plasmid in the cytoplasm of Escherichia coli . Advantage has been taken of insolubility of the human IL-4 in E . coli for rapid purification of this protein in only a few steps . We describe extraction and renaturation procedures which solubilize human IL-4 yielding biologically active protein . The protein was purified to homogeneity by one passage over a gel-filtration column . The refolded human IL-4 was characterized by N-terminal sequence analysis, amino acid analysis and bioassays . The refolded E . coli-derived human IL-4 has biological activity on T and B cells and binds to the human IL-4 receptor, comparable to mammalian expressed human IL-4, indicating that the protein is folded correctly.

J Biol Chem, 1988 Apr 5, 263(10), 4837 - 43
Expression of soluble and fully functional ricin A chain in Escherichia coli is temperature-sensitive; Piatak M et al.; Linkage of ricin A chain (RA) to a cell surface binding antibody or other ligand can result in a potent cytotoxic agent . We expressed the primary sequence for RA in Escherichia coli to facilitate production and to obtain protein free of naturally occurring contaminants, i.e . ricin B chain . Differences in the level of expression and in the characteristics of the expressed protein were noted when several different host/vector systems were tested . Recombinant RA (rRA) was expressed directly under control of the phage lambda major leftward promoter (PL) and the E . coli trp promoter . It was also expressed fused to E . coli alkaline phosphatase sequences, both in the same reading frame for secretion and out-of-reading frame for expression in a cistron-like arrangement . Expression in the PL promoter system, which is temperature-regulated, was achieved at 37 degrees C as well as at 42 degrees C . The protein expressed at these different temperatures had grossly different properties . Whereas rRA expressed at 37 degrees C was soluble and fully active, that produced at 42 degrees C was aggregated, insoluble, and reduced in activity . Soluble rRA could be converted to the insoluble form by incubation at 42 degrees C in vivo, but not in vitro . Hence, this difference in properties does not simply reflect an inherent thermal instability of the protein . Conditions present in vivo, including the possible association with other proteins, are apparently required for this effect on rRA.

J Biol Chem, 1988 Apr 5, 263(10), 4801 - 6
Immune electron microscopic localization of dinitrophenyl-modified ribosomal protein S19 in reconstituted Escherichia coli 30 S subunits using antibodies to dinitrophenol; Olson HM et al.; Escherichia coli small ribosomal subunits have been reconstituted from RNA and high performance liquid chromatography-purified proteins including protein S19 that had been modified at its amino-terminal proline residue with 1-fluoro-2,4-dinitrobenzene . As detailed in the accompanying paper (Olah, T . V., Olson, H . M., Glitz, D . G., and Cooperman, B . S . (1988) J . Biol . Chem . 263, 4795-4800), dinitrophenyl (DNP)-S19 was efficiently incorporated into the site ordinarily occupied by S19 . Antibodies to DNP bound effectively to the reconstituted subunits and did not cause dissociation of the modified protein from the subunit . Electron microscopy of the immune complexes was used to localize the modified protein on the subunit surface . More than 95% of the antibody binding sites seen were consistent with a single location of protein S19 on the upper portion or head of the subunit, on the surface that faces the 50 S particle in a 70 S ribosome, and in an area relatively distant from the subunit platform . The S19 site is close to the region in which 30 S subunits are photoaffinity labeled with puromycin . Protein S19 is thus near protein S14 in the small subunit and in proximity to the peptidyl transferase center of the 70 S ribosome.

J Biol Chem, 1988 Apr 5, 263(10), 4629 - 40
Binding mode transitions of Escherichia coli single strand binding protein-single-stranded DNA complexes . Cation, anion, pH, and binding density effects; Bujalowski W et al.; We have extended our investigations of the multiple binding modes that form between the Escherichia coli single strand binding (SSB) protein and single-stranded DNA (Lohman, T . M . & Overman, L . B . (1985) J . Biol . Chem . 260, 3594-3603; Bujalowski, W . & Lohman, T . M . (1986) Biochemistry 25, 7799-7802) by examining the effects of anions, pH, BaCl2, and protein binding density on the transitions among these binding modes . "Reverse" titrations that monitor the quenching of the intrinsic tryptophan fluorescence of the SSB protein upon addition of poly(dT) have been used to measure the apparent site size of the complex at 25 degrees C in pH 8.1 and 6.9 as a function of NaF, NaCl, NaBr, and MgCl2 concentrations . Under all conditions in which "reverse" titrations were performed, we observe three distinct binding modes with site sizes of 35 +/- 2, 56 +/- 3, and 65 +/- 3 nucleotides/SSB tetramer; however, the transitions among the three binding modes are strongly dependent upon both the cation and anion valence, type, and concentration as well as the pH . A net uptake of both cations and anions accompanies the transitions from the (SSB)35 to the (SSB)56 binding mode at pH 6.9, whereas at pH 8.1 this transition is anion-independent, and only a net uptake of cations occurs . The transition from the (SSB)56 to the (SSB)65 binding mode is dependent upon both cations and anions at both pH 6.9 and 8.1 (25 degrees C), and a net uptake of both cations and anions accompanies this transition . We have also examined the transitions by monitoring the change in the sedimentation coefficient of the SSB protein-poly(dT) complex as a function of MgCl2 concentration (20 degrees C, pH 8.1) and observe an increase in s20,w, which coincides with the increase in apparent site size of the complex, as measured by fluorescence titrations . The frictional coefficient of the complex decreases by a factor of two in progressing from the (SSB)35 to the (SSB)65 binding mode, indicating a progressive compaction of the complex throughout the transition . The transition between the (SSB)35 and the (SSB)56 complex is dependent on the protein binding density, with the lower site size (SSB)35 complex favored at higher binding density . These results indicate that the transitions among the various SSB protein-single-stranded DNA binding modes are complex processes that depend on a number of solution variables that are thermodynamically linked.(ABSTRACT TRUNCATED AT 400 WORDS)

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