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, 19