J Mol Biol, 1988 Apr 5, 200(3), 439 - 47 Evidence for gene conversion between the phosphoglycerate kinase genes of Trypanosoma brucei; Le Blancq SM et al.; Trypanosoma brucei contains a tandem array of three genes for phosphoglycerate kinase (PGKase), genes A, B and C, each coding for a different protein . We have compared allelic variants of this gene array and find evidence for gene conversion between the three genes . Near the 3' end, the different alleles and gene B contain a variable sequence that is similar to the corresponding sequence in either gene A or gene C . This sequence is flanked by glycine triplets that are conserved in all PGKases from bacteria to mammals . The triplets are encoded by (GGT)n, resulting in sequences that resemble the recombination-promoting chi-sites of Escherichia coli . Upstream of the variable sequence, there is an area of 800 base-pairs in which genes A, B and C are highly homologous; in all three genes this region ends with a sharp boundary at which gene B again shows segmental homology with both genes A and C . These results suggest that repeated gene conversion events partially erase the differences between genes A, B and C that arise in evolution and suggest that chi-like sequences may act as recombinational hotspots in protozoa such as T . brucei. Biochemistry, 1988 Apr 5, 27(7), 2277 - 81 Diethyl pyrocarbonate reaction with the lactose repressor protein affects both inducer and DNA binding; Sams CF et al.; Modification of the lactose repressor protein of Escherichia coli with diethyl pyrocarbonate (DPC) results in decreased inducer binding as well as operator and nonspecific DNA binding . Spectrophotometric measurements indicated a maximum of three histidines per subunit was modified, and quantitation of lysine residues with trinitrobenzenesulfonate revealed the modification of one lysine residue . The loss of DNA binding, both operator and nonspecific, was correlated with histidine modification; removal of the carbethoxy groups from the histidines by hydroxylamine was accompanied by significant recovery of DNA binding function . The presence of inducing sugars during the DPC reaction had no effect on histidine modification or the loss of DNA binding activity . In contrast, inducer binding was not recovered upon reversal of the histidine modification . However, the presence of inducer during reaction protected lysine from reaction and also prevented the decrease in inducer binding; these results indicate that reaction of the lysine residue(s) may correlate to the loss of sugar binding activity . Since no difference in incorporation of radiolabeled carbethoxy was observed following reaction with diethyl pyrocarbonate in the presence or absence of inducer, the reagent appears to function as a catalyst in the modification of the lysine . The formation of an amide bond between the affected lysine and a nearby carboxylic acid moiety provides a possible mechanism for the activity loss . Reaction of the isolated NH2-terminal domain resulted in loss of DNA binding with modification of the single histidine at position 29 . Results from the modification of core domain paralleled observations with intact repressor. J Biol Chem, 1988 Apr 5, 263(10), 4740 - 4 Directed mutations of the strongly conserved lysine 155 in the catalytic nucleotide-binding domain of beta-subunit of F1-ATPase from Escherichia coli; Parsonage D et al.; The amino acid sequence -Gly-X-X-X-X-Gly-Lys- occurs in many, diverse, nucleotide-binding proteins, and there is evidence that it forms a flexible loop which interacts with one or other of the phosphate groups of bound nucleotide . This sequence occurs as -Gly-Gly-Ala-Gly-Val-Gly-Lys- in the beta-subunit of the enzyme F1-ATPase, where it is thought to form part of the catalytic nucleotide-binding domain . Mutants of Escherichia coli were generated in which residue beta-lysine 155, at the end of the above sequence, was replaced by glutamine or glutamate . Properties of the soluble purified F1-ATPase from each mutant were studied . The results showed: 1) replacement of lysine 155 by Gln or Glu decreased the steady-state rate of ATP hydrolysis by 80 and 66%, respectively . 2) Characteristics of ATP hydrolysis at a single site were not markedly changed in the mutant enzymes, implying that lysine 155 is not directly involved in bond cleavage during ATP hydrolysis or bond formation during ATP synthesis . 3) The binding affinity for MgATP was weakened considerably in the mutants (Lys much much greater than Gln greater than Glu), whereas the binding affinity for MgADP was affected only mildly (Lys = Gln greater than Glu), suggesting that lysine 155 interacts with the gamma-phosphate of ATP bound at a single high affinity catalytic site . 4) The major determinant of inhibition of steady-state ATPase turnover rate in the mutant enzymes was an attenuation of positive catalytic cooperativity . 5) The data are consistent with the idea that during multisite catalysis residue 155 of beta-subunit undergoes conformational movement which changes substrate and product binding affinities. J Biol Chem, 1988 Apr 5, 263(10), 4619 - 23 A mutation in the alpha-subunit of F1-ATPase from Escherichia coli affects the binding of F1 to the membrane; Maggio MB et al.; The mutation Gly-29----Asp in the alpha-subunit of the F1-ATPase from Escherichia coli was characterized and shown to cause the following effects . 1) Oxidative phosphorylation was markedly impaired in vivo 2) Membrane ATPase and ATP-driven proton-pumping activities were decreased markedly . 3) Membranes were proton-permeable, and membrane-bound ATPase was dicyclohexylcarbodiimide-insensitive . Therefore, it appeared that integration between F1 and F0 was abnormal . This was confirmed directly by the demonstration that the mutant F1 bound poorly to stripped membranes from a normal strain . Purified, soluble mutant F1 had normal ATPase activity . These results suggest that residue Gly-29, which is strongly conserved in alpha-subunits of F1-ATPases, lies in a region of the alpha-subunit important for membrane binding . Thus, three regions of the F1-alpha-subunit have now been recognized, specialized for membrane binding, nucleotide binding, and alpha/beta intersubunit signal transmission, respectively . The approximate locations of the three regions are described. Eur J Biochem, 1988 Apr 5, 173(1), 227 - 31 Electrostatic potential of macromolecules measured by pKa shift of a fluorophore . 1 . The 3' terminus of 16S RNA; Friedrich K et al.; We have investigated the use of the pH-sensitive fluorescein label as a probe for electrostatic potential in macromolecules . The practicality of this technique is demonstrated by its application to the 16S RNA molecule . The dependence of the electrostatic potential upon ionic conditions and upon the presence of ribosomal proteins and the state of the RNA was studied . The combination of electrostatic and anisotropy data emphasizes the role of the 30S ribosomal proteins, rather than of the renaturation of the 16S RNA or the presence of the 50S subunit, in shaping the environment of the 3' terminus of the 16S RNA in the active ribosome. J Biol Chem, 1988 Apr 5, 263(10), 4984 - 90 The effect of H2O2 upon thioredoxin-enriched lens epithelial cells; Spector A et al.; Thioredoxin, a dithiol polypeptide, has been examined as a potential contributor to the recovery of lens epithelial cells from oxidative insult . It is reported that Escherichia coli thioredoxin can (a) effectively reduce lens-soluble protein disulfide bonds generated by H2O2, (b) restore to its initial activity H2O2-inactivated glyceraldehyde-3-phosphate dehydrogenase, (c) act as an effective source of reducing potential for lens methionine sulfoxide peptide reductase, and (d) act as a free radical quencher based on studies with a stable free radical system generated by ascorbic acid and 2,6-dimethoxy-p-benzoquinone . Thioredoxin is much more effective than dithiothreitol in restoring glyceraldehyde-3-phosphate dehydrogenase activity and as a cofactor for methionine sulfoxide peptide reductase . Upon incubation with epithelial cells, thioredoxin can be observed in the cell using rocket immunoelectrophoresis . These cells recover from H2O2 insult more rapidly than control cell preparations based upon 1) analyses of plasma membrane-related activities: leucine and 86Rb uptake and 2) analyses of parameters primarily related to the internal cell metabolism: ATP concentration and glyceraldehyde-3-phosphate dehydrogenase activity . Analysis of thioredoxin in cell preparations indicates that only about 9% is in the reduced state implying a low effective concentration of the polypeptide . The experiments suggest that low levels of thioredoxin may significantly increase the ability of lens epithelial cells to recover from exposure to H2O2. J Biol Chem, 1988 Apr 5, 263(10), 4641 - 6 Derived amino acid sequence and identification of active site residues of Escherichia coli beta-hydroxydecanoyl thioester dehydrase; Cronan JE Jr et al.; The nucleotide sequence of the fabA gene encoding beta-hydroxydecanoyl thioester dehydrase, a key enzyme of the unsaturated fatty acid synthesis pathway of Escherichia coli, has been determined by the dideoxynucleotide sequencing technique . Most of the sequence was obtained by sequencing intragenic insertions of the transposon, Tn1000, isolated in vivo . A synthetic primer complementary to a portion of the inverted repeat sequences at the ends of the transposon was used to prime DNA synthesis into the flanking fabA sequences . The gene is composed of 516 nucleotides (171 amino acid residues) encoding a protein with a molecular weight of 18,800 . Approximately half of the derived amino acid sequence was confirmed by automated Edman sequencing of peptides obtained by cyanogen bromide cleavage . The active site histidine residue (His-70) has been identified by analysis of the peptides labeled by reaction with 14C-labeled 3-decynoyl-N-acetylcysteamine, a specific mechanism-activated inhibitor . A cysteine residue (Cys-69) adjacent to the active site histidine may play the role in catalysis previously assigned to a tyrosine residue . We also report a simplified purification process for the dehydrase beginning with extracts of a brain which greatly overproduces the enzyme. Biochemistry, 1988 Apr 5, 27(7), 2629 - 34 Yeast redoxyendonuclease, a DNA repair enzyme similar to Escherichia coli endonuclease III; Gossett J et al.; A DNA repair endonuclease (redoxyendonuclease) was isolated from bakers' yeast (Saccharomyces cerevisiae) . The enzyme has been purified by a series of column chromatography steps and cleaves OsO4-damaged, double-stranded DNA at sites of thymine glycol and heavily UV-irradiated DNA at sites of cytosine, thymine, and guanine photoproducts . The base specificity and mechanism of phosphodiester bond cleavage for the yeast redoxyendonuclease appear to be identical with those of Escherichia coli endonuclease III when thymine glycol containing, end-labeled DNA fragments of defined sequence are employed as substrates . Yeast redoxyendonuclease has an apparent molecular size of 38,000-42,000 daltons and is active in the absence of divalent metal cations . The identification of such an enzyme in yeast may be of value in the elucidation of the biochemical basis for radiation sensitivity in certain yeast mutants. Genomics, 1988 Apr, 2(3), 215 - 9 The complete nucleotide sequence of murine beta-glucuronidase mRNA and its deduced polypeptide; Gallagher PM et al.; The complete nucleotide sequence of murine beta-glucuronidase (GUS) mRNA has been compiled from three overlapping cloned cDNAs and a single GUS-specific genomic clone . The sequence is composed of 2455 nucleotides, exclusive of the poly(A) tail . The 5' and 3' untranslated regions contain 12 and 499 bases, respectively, with the open reading frame encoding a polypeptide of 648 amino acids (74.2 kDa), including a 22 amino acid signal sequence . The nucleotide and deduced amino acid sequences of murine GUS are compared to those published for rat and human GUS and the results are presented . Murine GUS also shares amino acid sequence identity with Escherichia coli GUS and beta-galactosidase . The complete sequences of murine GUS mRNA and its deduced polypeptide provide a basis from which to study the mechanisms responsible for the well-characterized variation in GUS expression among inbred mouse strains. J Clin Microbiol, 1988 Apr, 26(4), 648 - 53 Immunoblot analysis of immunoglobulin G response to the Lyme disease agent (Borrelia burgdorferi) in experimentally and naturally exposed dogs; Greene RT et al.; Immunoblots were used to study the immunoglobulin G response to Borrelia burgdorferi in experimentally and naturally exposed dogs . Adsorption studies confirmed that the antibodies were specific for B . burgdorferi . Experimentally exposed dogs were asymptomatic . Naturally exposed dogs included both asymptomatic animals and animals showing signs compatible with Lyme disease . Naturally exposed dogs were from four geographic regions of the country . No differences were detected between immunoblot patterns of naturally exposed symptomatic or asymptomatic dogs from different areas of the country . The immunoblot patterns obtained with sera from experimentally exposed dogs were different from those obtained with sera from naturally exposed dogs and were characterized by reactivity to fewer and different protein bands . Immunoblot analysis using an OspA-protein-producing Escherichia coli recombinant showed that experimentally exposed dogs produced antibodies to OspA, whereas naturally exposed dogs did not . Modifications of the immune response over time, different routes of antigen presentation, and strain variation are factors postulated to account for the observed differences. Proc Natl Acad Sci U S A, 1988 Apr, 85(8), 2568 - 72 Evidence that glutamic acid 167 is an active-site residue of Shiga-like toxin I; Hovde CJ et al.; Escherichia coli Shiga-like toxin I, a close relative of Shiga toxin and a distant relative of the ricin family of plant toxins, inhibits eukaryotic protein synthesis by catalyzing the depurination of adenosine 4324 in 28S rRNA . By comparing the crystallographic structure of ricin with amino acids conserved between the Shiga and ricin toxin families, we identified seven potential active-site residues of Shiga-like toxin I . The structural gene encoding Shiga-like toxin I A chain (Slt-IA), the enzymatically active subunit, was engineered for high expression in E . coli . Oligonucleotide-directed mutagenesis of the gene for Slt-IA was used to change glutamic acid 167 to aspartic acid . As measured by an in vitro assay for inhibition of protein synthesis, the specific activity of mutant Slt-IA was decreased by a factor of 1000 compared to wild-type Slt-IA . Immunoblots showed that mutant and wild-type Slt-IA were synthesized as full-length proteins and were processed correctly by signal peptidase . Both proteins were equally susceptible to trypsin digestion, suggesting that the amino acid substitution did not produce a major alteration in Slt-IA conformation . We conclude that glutamic acid 167 is critical for activity of the Shiga-like toxin I A chain and may be located at the active site. Arch Biochem Biophys, 1988 Apr, 262(1), 337 - 44 6-Nitro-L-tryptophan: a novel spectroscopic probe of trp aporepressor and human serum albumin; Phillips RS et al.; The binding of 6-nitro-L-tryptophan to trp aporepressor and human serum albumin has been examined by visible difference spectroscopy and circular dichroism . 6-Nitro-L-tryptophan, prepared by nitration of L-tryptophan with nitric acid in glacial acetic acid, exhibits a visible and near-uv absorption spectrum with lambda max at about 330 nm (epsilon = 7 X 10(3) M-1 cm-1) and a shoulder near 380 nm in H2O . In the presence of trp aporepressor, the visible absorption intensity is sharply diminished . Visible difference spectral titration data give KD = 1.27 X 10(-4) M and n = 0.95 per subunit at 25 degrees C . While 6-nitro-L-tryptophan exhibits no significant circular dichroism between 300 and 500 nm, the complex with trp aporepressor exhibits strong circular dichroism signals, with a negative maximum at 386 nm (delta epsilon = -7.5 M-1 cm-1) and a positive maximum at 310 nm (delta epsilon = +6 M-1 cm-1) . Circular dichroism titration data give KD = 1.69 X 10(-4) M and n = 0.90 per subunit at 25 degrees C . The KD values determined spectroscopically are in excellent agreement with that determined by equilibrium dialysis, KD = 1.5 X 10(-4) M at 25 degrees C . In the presence of human serum albumin, the spectrum of 6-nitro-L-tryptophan exhibits a blue shift and an increase in absorption intensity; similar changes are observed in solvents of low dielectric contrast such as 80% aqueous dioxane . Visible difference spectral titration data give KD = 8.0 X 10(-5) M and n = 0.95 for human serum albumin . The complex of 6-nitro-L-tryptophan with human serum albumin exhibits a strong positive circular dichroism maximum at 380 nm (delta epsilon = +9.8 M-1 cm-1) with a shoulder at 310-320 nm . Circular dichroism titration data give KD = 6.4 X 10(-5) M and n = 0.83, in good agreement with the visible difference spectral results . Taken together, our results demonstrate the utility of 6-nitro-L-tryptophan as a spectroscopic probe for tryptophan-binding proteins. Virology, 1988 Apr, 163(2), 268 - 75 Formation of transmembraneous hepatitis B e-antigen by cotranslational in vitro processing of the viral precore protein; Bruss V et al.; The gene encoding the major core protein P22c of hepatitis B virus is preceded by a precore sequence . Expression of the core gene with the precore in Escherichia coli results in a membrane protein of HBe antigenicity . Expression in mammalian cells generates secreted HBeAg . To study the biosynthetic pathway of HBeAg and the function of precore in this process, we translated mRNAs for core proteins with and without precore using reticulocyte lysates and microsomal vesicles . The precore sequence was cleaved cotranslationally as a signal peptide, probably at alanine 19 . The processed product P23e was partially translocated to the lumen of the microsomes . The arginine-rich carboxy-terminal domain of P23e was however not translocated and susceptible to trypsin . Clusters of positive-charged amino acids seem to act as a novel type of translocation stop signal . Trypsin generated a P16e which no longer had a transmembraneous configuration . The findings may explain the biosynthesis and potential function of HBeAg in hepatitis B virus-infected hepatocytes. Genomics, 1988 Apr, 2(3), 231 - 9 Genomic mapping by fingerprinting random clones: a mathematical analysis; Lander ES et al.; Results from physical mapping projects have recently been reported for the genomes of Escherichia coli, Saccharomyces cerevisiae, and Caenorhabditis elegans, and similar projects are currently being planned for other organisms . In such projects, the physical map is assembled by first "fingerprinting" a large number of clones chosen at random from a recombinant library and then inferring overlaps between clones with sufficiently similar fingerprints . Although the basic approach is the same, there are many possible choices for the fingerprint used to characterize the clones and the rules for declaring overlap . In this paper, we derive simple formulas showing how the progress of a physical mapping project is affected by the nature of the fingerprinting scheme . Using these formulas, we discuss the analytic considerations involved in selecting an appropriate fingerprinting scheme for a particular project. Genetika, 1988 Apr, 24(4), 622 - 7 {Suppression of dnaZ mutation during integration of factor F' into the chromosome of a mutant strain}; Oskolkova OB et al.; The phenomenon of suppression of dnaZ mutation has been revealed in the course of F' factor integration into the chromosome of the mutant strain . We have shown that under non-permissive conditions (t = 43 degrees C), chromosome replication in dnaZts strains proceeds under control of the factor F' replicon stably integrated into the chromosome . Possible mechanism of suppression effect, based on the formation of a bireplicon replication system, is discussed. Br J Pharmacol, 1988 Apr, 93(4), 955 - 63 Modification by steroids of pulmonary oedema and prostaglandin E2 pharmacokinetics induced by endotoxin in rats; Izumi T et al.; 1 . A single i.p . injection of bacterial endotoxin in rats (3.5 mg kg-1) caused lung injury assessed as changes in lung dry:wet weight ratio and leukopaenia over the subsequent 28 h . 2 . This treatment also slowed the efflux of 14C from {14C}-prostaglandin E2 (PGE2), i.e., increased t1/2 and increased the survival of PGE2 in isolated perfused lungs over the same period . 3 . These effects of endotoxin were reversed by methylprednisolone (30 mg kg-1), given 30 min after the endotoxin . 4 . Another synthetic corticosteroid, budesonide (1.2 mg kg-1) given 1 h before endotoxin partially prevented the lung injury and leukopaenia but did not affect the increased t1/2 for PGE2 nor its survival . 5 . The reversal by methylprednisolone of both the physical signs of lung injury and the changes in PGE2 pharmacokinetics caused by endotoxin suggests that changes in PGE2 pharmacokinetics could serve as an index of acute lung injury following sepsis. Mol Biochem Parasitol, 1988 Apr, 28(3), 235 - 47 Characterization of a surface antigen of Eimeria tenella sporozoites and synthesis from a cloned cDNA in Escherichia coli; Brothers VM et al.; An antigenic surface protein of Eimeria tenella sporozoites has been identified that is the target of two neutralizing monoclonal antibodies Ptn 7.2A4/4 and Ptn 9.9D12 . The antigen as isolated from the parasite is composed of a 17 kDa polypeptide and a 8 kDa polypeptide linked by a disulfide bridge . De novo synthesis of the antigen does not begin until approximately 16-20 h after the initiation of oocyst sporulation . A cDNA library was constructed using mRNA from sporulated oocysts and a clone encoding the antigen was isolated . The Ta4 gene encodes a single polypeptide of 25 kDa which contains the 17 and 8 kDa polypeptides . The protein has been synthesized in Escherichia coli either directly or as part of a beta-galactosidase fusion protein . The products synthesized in E . coli are single polypeptides and are not cleaved to two polypeptides as is seen in the parasite . The products accumulate in bacteria in an insoluble form which can be solubilized and renatured to an immunoreactive form. Biotechnol Appl Biochem, 1988 Apr, 10(2), 143 - 53 Characteristics and applications of adsorbents for pyrogen removal; Minobe S et al.; Characteristics and applications of immobilized histidine and immobilized histamine for pyrogen removal were investigated . Immobilized histidine showed a high affinity for pyrogen at low ionic strength and over a wide pH range . The adsorption capacity was 0.53 mg of lipopolysaccharide per milliliter of the adsorbent . The apparent dissociation constant was 1.57 X 10(-9) M . The adsorption of pyrogen to immobilized histidine decreased with increasing ionic strength, but pyrogen could be adsorbed even at ionic strengths of gamma/2 = 0.05-0.1, at which other substances were little adsorbed; that is, specific adsorption of pyrogen was observed . The adsorption of pyrogen could be increased at ionic strengths of gamma/2 = 0.05-0.1 by using a lower flow rate or a longer column length . Immobilized histidine and immobilized histamine could be used for the removal of natural pyrogens contaminating various useful low-molecular-weight compounds as well as high-molecular-weight compounds such as proteins. Biochem Med Metab Biol, 1988 Apr, 39(2), 176 - 81 Effect of enterotoxigenic Escherichia coli heat stable toxin on intestinal lysosomal enzymes in the suckling rat; Aye-Kyaw et al.; A preliminary study on 9 suckling Wistar rats, which received E . coli stable toxin, and on 12 sham-operated controls showed that acid phosphatase, the marker enzyme for lysosome, was significantly increased in the infected group whereas alkaline phosphatase, glucose 6-phosphatase, succinic dehydrogenase, and proteinase, the marker enzymes for brush border, microsome, mitochondria, and the soluble fraction, respectively, remained unaffected . The results suggest that lysosome, the subcellular organelle responsible for intracellular digestion could be modified by E . coli stable toxin . In another set of experiments, where 7 infected suckling rats and 7 sham-operated controls were used, the maximal activities of lysosomal enzymes (released by Triton X-100) were found to be increased in the infected group confirming the results obtained in the preliminary experiment . The values of the ratio between maximal and basal activity (an expression of the degree of retention of enzymes to lysosome) of acid phosphatase and cathepsin D were also significantly increased, indicating that lysosomal membrane may also be stabilized during the infection . The increased activities of lysosomal enzymes and the increased lysosomal membrane stability suggest that intracellular digestion by lysosome could be increased during E . coli stable toxin infection. Mol Gen Genet, 1988 Apr, 212(1), 76 - 84 Characterization of a sequence (hlyR) which enhances synthesis and secretion of hemolysin in Escherichia coli; Vogel M et al.; A sequence (hlyR) of about 600 bp which enhances the expression of hemolysin (HlyA) more than 50-fold was identified in the plasmid pHly152-specific hemolysin (hly) determinant . Deletion of this entire hlyR sequence led to the same low level of hemolysin synthesis and excretion as that expressed by the recombinant plasmid pANN202-312 . HlyR was active in cis but its activity was orientation-dependent . The enhancing sequence, hlyR, is separated from the promoter phlyI transcribing hlyC, hlyA and possibly hlyB by more than 1.5 kb including an IS2 element . Stepwise removal of the hlyR sequence from its 5' end by exonuclease III (ExoIII) digestion yielded several types of deletion mutants which expressed decreasing amounts of hemolysin . A similar observation was made when hlyR was shortened by ExoIII from its 3' end, which suggests that more than one functional region may be present in the hlyR sequence . A deletion of 717 bp within the adjacent IS2 element reduced the activity of hlyR only slightly, indicating that IS2 is not directly involved in the enhancement mechanism but that it may support an optimal positioning in hlyR relative to the hly promoter . The nucleotide sequence of hlyR is rich in A + T and does not contain an extended open reading frame, but exhibits several sequence motives that may represent sites for protein binding and DNA bending. Mol Gen Genet, 1988 Apr, 212(1), 177 - 81 The phenotypic suppression of a mutation in the gene rplX for ribosomal protein L24 by mutations affecting the lon gene product for protease LA in Escherichia coli K12; Nishi K et al.; A suppressor mutation of a temperature-sensitive mutant of ribosomal protein L24 (rplX19) was mapped close to the lon gene by genetic analysis and was shown to affect protease LA . The degradation and the synthesis rates of individual ribosomal proteins were determined . Proteins L24, L14, L15 and L27 were found to be degraded faster in the original rplX19 mutant than in the rplX19 mutant containing the suppressor mutation . Other ribosomal proteins were either weakly or not at all degraded in both mutants . Temperature-sensitive growth was also suppressed by the overproduction of mutant protein L24 from a plasmid . Our results suggest that (1) either free ribosomal proteins or proteins bound to abortive assembly precursors are highly susceptible to the lon gene product and (2) the mutationally altered protein L24 can still function at the nonpermissive growth temperature of the mutant, if it is present in sufficient amounts. DNA, 1988 Apr, 7(3), 211 - 7 Site-specific oligonucleotide-directed mutagenesis using T4 DNA polymerase; Chang GJ et al.; A simple and efficient mutagenesis procedure is described which uses both the 3'----5' exonuclease and 5'----3' polymerase activities of T4 DNA polymerase . Different types of mutation-deletion, insertion, and substitution-can be introduced into the DNA in a single reaction . The technique uses recombinant M13 single-stranded DNA and two complementary DNA oligonucleotides to target and control the extent of deletions catalyzed by T4 DNA polymerase . The second oligonucleotide not only directs ligation, but also serves as a template for insertion or substitution of nucleotides by T4 polymerase . Mutant phages in a genetically pure form can be obtained at high efficiency, allowing their characterization directly by nucleotide sequencing without prior enrichment, plaque purification, and screening . We tested the versatility of this method by manipulating five regions of cDNA encoding the structural proteins of eastern equine encephalitis virus. Can J Vet Res, 1988 Apr, 52(2), 280 - 2 Natural infection with an attaching and effacing Escherichia coli in a diarrheic puppy; Broes A et al.; Enteric infection with an attaching and effacing Escherichia coli was diagnosed in a puppy with protracted diarrhea . Extensive colonization of the small intestinal mucosa was observed by light and scanning electron microscopy and characteristic lesions of bacterial attachment of the brush border of the enterocytes were demonstrated by transmission electron microscopy . The E . coli strain isolated from the small intestine belonged to serotype O49:H10, did not produce any known E . coli enterotoxin or cytotoxin, was not invasive, and was negative for the known fimbrial colonization factors produced by animal and human enterotoxigenic E . coli . A positive immunoperoxidase reaction was obtained on the bacteria attached to the enterocytes with an anti-E . coli O49 antiserum. J Clin Microbiol, 1988 Apr, 26(4), 784 - 6 Evaluation of a nonisotopically labeled oligonucleotide probe to detect the heat-stable enterotoxin gene of Escherichia coli by the DNA colony hybridization test; Nishibuchi M et al.; A commercially available, alkaline-phosphatase-conjugated oligonucleotide probe for detecting the heat-stable enterotoxin gene of Escherichia coli was compared with cloned gene probes by examining E . coli isolates from traveler's diarrhea by DNA colony hybridization tests . The oligonucleotide probe was useful in specifically identifying the so-called STh gene . No deproteinization of sample was necessary to prepare the colony blots. Genetics, 1988 Apr, 118(4), 593 - 600 Repair of single base-pair transversion mismatches of Escherichia coli in vitro: correction of certain A/G mismatches is independent of dam methylation and host mutHLS gene functions; Lu AL et al.; Six different base-pair transversion mismatches are repaired with different efficiencies in an in vitro mismatch repair system . In particular, the T/T and C/C mismatches appear to be less efficiently repaired than the A/A and G/G mismatches . Four A/G and four C/T mismatches at different positions are repaired to different extents . One of the A/G mismatches is repaired equally efficiently when DNA heteroduplexes are fully methylated or hemi-methylated at the d(GATC) sequences . This type of mismatch repair appears to be unidirectional with A to C conversion by acting at A/G mispairs to restore the C/G pairs . This methylation-independent correction is not controlled by the mutH, mutL, mutS, uvrE, uvrB, phr, recA, recF, and recJ gene products . The independence of the transversion mismatch repair of these genes and methylation distinguishes this from the known mismatch repair pathways. Genetics, 1988 Apr, 118(4), 571 - 9 A mutation affecting the regulation of a secA-lacZ fusion defines a new sec gene; Riggs PD et al.; It was shown previously that the secA gene of Escherichia coli is derepressed in cells that have a defect in protein export . Here it is demonstrated that the beta-galactosidase produced by a secA-lacZ gene fusion strain is regulated in the same way . Studies on the fusion strain reveal that the promoter or a site involved in regulation of the secA gene is located considerably upstream from the structural gene . The properties of the fusion strain provide a new selection for mutants that are defective in protein export . Selection for increased lac expression of a secA-lacZ fusion strain yields mutations in three of the known sec genes, secA, secD and prlA/secY . In addition, mutations in several genes not previously known to affect secA expression were obtained . A mutation in one of these genes causes a pleiotropic defect in protein export and a cold-sensitive growth defect; this gene, which maps at approximately 90 min on the bacterial chromosome, has been named secE. J Surg Res, 1988 Apr, 44(4), 417 - 24 Neutrophil phagocytosis during endotoxin-induced lung injury; Griswold J et al.; Depressed neutrophil (PMN) phagocytosis in patients with ARDS may contribute to the known increased incidence of pulmonary sepsis . To evaluate changes in phagocytosis, circulating PMNs from normal rats were compared to circulating and alveolar PMNs (obtained by bronchoalveolar lavage, BAL) from rats after 72 hr of endotoxin infusion (LPS-Rx)-induced acute lung injury . Since phagocytosis correlates with adherence, PMN adherence to coverslips and to a standard nylon wool column was also measured . PMN adherence to nylon wool was 65% for control, 77% for circulating LPS-Rx, and 20% for BAL PMNs . As a measure of phagocytosis the PMNs were incubated for 30 min with opsonized fluorescent (FITC) tagged yeast . Total PMN with yeast were 95.4 +/- 2.1% for control; 96.4 +/- 1.8% for circulating LPS-Rx; and 78.7 +/- 7.8% (P less than 0.05 compared to control) for BAL PMNs . Total numbers of yeast particles per 100 PMN are 270 +/- 64 for control, 300 +/- 42 for circulating LPS-Rx, and 170 +/- 45 (P less than 0.05 compared to control) for BAL PMN . Conclusions: (1) Intraalveolar (BAL) PMNs have decreased adherence; (2) nonadherent PMNs have decreased uptake of yeast; (3) BAL PMNs, overall, have a significantly decreased uptake of yeast; (4) this depression in BAL PMN phagocytosis may partially explain the known decreased rate of bacterial clearance in injured lungs and the increased risk of pulmonary sepsis with adult respiratory distress syndrome. J Surg Res, 1988 Apr, 44(4), 397 - 403 The role of intestinal flora on the interactions between nonparenchymal cells and hepatocytes in coculture; Billiar TR et al.; Kupffer cells are exposed directly to a number of factors in the portal circulation that can modify or regulate their responses to septic stimuli . The gut represents a potential source of a number of these factors including endotoxin, lymphokines, and prostaglandins . We examined Kupffer cells from germfree rats and germfree rats exposed to endotoxin or bacteria via their GI tracts to determine the importance of the intestinal flora in maintaining or modulating Kupffer cell responses . Kupffer cells from germfree animals were reduced in numbers and failed to respond to LPS in Kupffer cell: hepatocyte coculture . When germfree rats were exposed to bacterial endotoxin or bacteria via the gastrointestinal tract their Kupffer cells increased in numbers to normal and the cells responded to LPS in culture . Intestinal overgrowth with Escherichia coli for 2 days activated the Kupffer cells and significantly increased Kupffer cell sensitivity to LPS . These data suggest that the environment of the gastrointestinal tract is important for normal Kupffer cell responses and that intestinal bacterial overgrowth can modify Kupffer cell responses to septic stimuli. Proc Natl Acad Sci U S A, 1988 Apr, 85(8), 2449 - 53 An 11-kDa form of human immunodeficiency virus protease expressed in Escherichia coli is sufficient for enzymatic activity; Graves MC et al.; In order to define the protease domain of human immunodeficiency virus 1, various regions of the pol open reading frame were cloned and expressed in Escherichia coli . Antiserum directed against the conserved retroviral protease active site was used to identify pol precursor and processed species containing the presumed protease domain . The smallest product that accumulates is about 11 kDa as measured by NaDodSO4/PAGE . This size agrees with that predicted from the presence in this region of two Phe-Pro sequences, which is one of the cleavage sites recognized by HIV protease . DNA encoding only the predicted 11-kDa protein was cloned, bypassing the need for autoprocessing, and the protein was expressed to a high level in E . coli . This form is active as demonstrated by its ability to specifically cleave protease-deficient pol protein in vivo in E . coli . Extracts of E . coli containing the 11-kDa protease also process human immunodeficiency virus gag substrates in vitro . These results demonstrate that the 11-kDa protease is sufficient for enzymatic activity and are consistent with a major role for this form in virus maturation. Epidemiol Infect, 1988 Apr, 100(2), 213 - 20 Managemental influences on the selective proliferation of two strains of haemolytic Escherichia coli in weaned pigs; Hampson DJ et al.; In an experimental study on a piggery it was found that haemolytic Escherichia coli of O-serotypes 138 or 139 proliferated in the intestinal tracts of pigs following weaning, with E . coli of the O-138 type also being occasionally recovered from unweaned pigs, and once from a sow . Organisms of the O-138 type produced heat labile enterotoxin and their presence in weaned pigs was associated with the development of severe post-weaning diarrhoea . E . coli of O-139 type produced a vero cell cytotoxin and were associated with a milder diarrhoea in weaned pigs . Under various managemental circumstances the O-138 type E . coli almost invariably proliferated after weaning . The O-139 strain of E . coli did however proliferate rather than the O-138 strain following the movement of weaned pigs to new accommodation, after weaned pigs were returned to their sow and then weaning again 5 days later, and very occasionally in pigs weaned at 5 weeks of age . In all these cases earlier proliferation of the O-138 E . coli had been detected, suggesting that this may be a prerequisite for proliferation of the O-139 strain. Am Rev Respir Dis, 1988 Apr, 137(4), 783 - 9 Pulmonary edema after Escherichia coli peritonitis correlates with thiobarbituric-acid-reactive materials in bronchoalveolar lavage fluid; Ishizaka A et al.; We developed a new model of acute lung injury caused by live Escherichia coli peritonitis in guinea pigs . Arterial blood gas determinations, arterial blood pressure, and white blood cell counts were monitored serially for 12 h after the injection of either 2 x 10(9) E . coli J96 or saline . Lung water, albumin concentration in bronchoalveolar lavage fluid (BALF) and in lung tissue, WBC counts in BALF, and thiobarbituric-acid-reactive materials (TBARM) in plasma, lung tissue, and BALF were examined . Increased TBARM might be associated with pulmonary injury and are produced either by the generation of lipoperoxides secondary to oxygen-free radicals or as metabolic byproducts of prostanoid metabolism . Lung tissue sections were studied by light microscopy . E . coli peritonitis, as compared with control animals, caused significant peripheral neutropenia, histopathologic evidence of lung inflammation, acidosis, and hypotension . The wet-to-dry lung ratio was increased in the peritonitis group when compared with that in the control group (p less than 0.01) . Pulmonary edema in the peritonitis group was associated with significantly increased albumin concentrations in BALF and lung tissue . We report the new finding of increased TBARM concentrations in BALF after E . coli peritonitis (p less than 0.01 and p less than 0.05, respectively) . In contrast, plasma TBARM concentrations were unchanged . The levels of TBARM in the BALF correlated significantly with both lung water (p less than 0.01) and lung tissue albumin concentration (p less than 0.01) . The measurement of elevated TBARM in BALF may allow acute lung injury to be detected . We conclude that this model may be useful for further studies of acute lung injury caused by E . coli peritonitis. Proc Soc Exp Biol Med, 1988 Apr, 187(4), 408 - 15 Enhanced production of monokines by canine alveolar macrophages in response to endotoxin-induced shock; Tabor DR et al.; The enhanced production of soluble mediators by alveolar macrophages may be responsible for promoting lung injury in canines administered endotoxin . One of the most prominent monokines, interleukin 1 (IL-1), has the potential to significantly influence the responses of host tissues . In this study we analyzed alveolar macrophages from canines that were experimentally administered endotoxin (AMEC) for their ability to produce IL-1 . When concentrated AMEC supernatants from in vitro cultures were incubated with fresh C3H/HEJ thymocytes, a threefold greater incorporation of {3H}thymidine resulted as compared to the response produced by controls . Heat treatment of the experimental preparations ablated this difference . Conversely, the activity of AMEC intracellular lysates did not significantly differ from the controls . Silver-staining the preparations separated by SDS-PAGE revealed a low-molecular-weight species (17 kD) in the AMEC supernatant lane while a similar molecular distribution was absent in all of the control preparations examined . Moreover, using the L929 cell line in a cytolytic bioassay we found that these same AMEC supernatants also contained significantly elevated levels of tumor necrosis factor . Collectively, this study suggests that during endotoxin-induced canine lung injury, the alveolar macrophages generate soluble species that can substantially regulate the hosts cellular response . This activity in the canine lung may play a critical role in the development and/or maintenance of the pathology associated with exposure to endotoxin. Mutat Res, 1988 Apr, 203(2), 81 - 94 Evaluation of the SOS chromotest; von der Hude W et al.; In the present investigation, the SOS chromotest with E . coli PQ37 was evaluated . The potential to identify different kinds of bacterial mutagens was examined . 124 chemicals of different chemical classes were tested . Their responses in the SOS chromotests were compared to reported test results obtained with the Ames test. Mutat Res, 1988 Apr, 198(2), 343 - 50 Mutagenic DNA repair in Escherichia coli . XVI . Mutagenesis by ultraviolet light plus delayed photoreversal in recA strains; Bridges BA; Mutagenesis was demonstrable after delayed photoreversal of UV-irradiated strains carrying a recA deletion indicating that RecA protein is not essential for the misincorporation process that is revealed by delayed photoreversal . Moreover, the data suggest that RecA protein actually depresses misincorporation to varying extents depending on the recA allele . No delayed photoreversal was demonstrable in reA1 or recA56 bacteria unless the lexA102(ind-) allele was also present . It is suggested that the level of these RecA proteins may be lower in the lexA102(ind-) strains thus minimising their depressive effect . Delayed photoreversal mutagenesis in strains carrying the recA441 allele was not affected by either adenine or guanosine plus cytidine, substances which affect the proteolytic activity of RecA441 protein. J Bacteriol, 1988 Apr, 170(4), 1973 - 4 A progenitor of the outer membrane LamB trimer; Stader J et al.; During its localization to the outer membrane, LamB possesses distinctive biochemical properties as it passes through the cytoplasmic membrane . Because LamB entered this dynamic state with an attached signal sequence and leaves after cleavage, we call this export-related form of LamB the early-translocation form (et-LamB). J Bacteriol, 1988 Apr, 170(4), 1582 - 8 Abnormal induction of heat shock proteins in an Escherichia coli mutant deficient in adenosylmethionine synthetase activity; Matthews RG et al.; Most prototrophic strains of Escherichia coli become restricted for methionine at 44 degrees C . A mutant strain (RG62 metK) in which the level of S-adenosylmethionine synthetase activity is only 10 to 20% of normal shows constitutive expression of one of the heat shock proteins, the lysU gene product, lysyl-tRNA synthetase form II, at 37 degrees C . These findings suggested a possible linkage between methionine metabolism and heat shock . We examined the induction of heat shock polypeptides in strain RG62 (metK) and in its parent, RG (metK+), from which it was derived by spontaneous mutation . Exponential-phase cultures of the two strains were pulse-labeled with {3H}leucine shortly after a shift from 37 to 44 degrees C, and the total cellular polypeptides were examined by two-dimensional electrophoresis . The results confirmed the constitutive production of the lysU gene product previously reported for strain RG62, but also revealed that the induction of 2 of the 17 heat shock polypeptides, C14.7 and G13.5, was markedly depressed . Otherwise the heat shock induction pattern was similar in timing and magnitude in the two strains . Transformation of the mutant strain with a plasmid, pK8, containing the metK coding sequence and promoter region as a 1.8-kilobase insert into pBR322 restored normal induction of C14.7 and G13.5, but did not prevent constitutive expression of the lysU gene product in the medium required for growth of this strain . The three heat shock polypeptides abnormally controlled in strain RG62 are the three polypeptides which are not induced when rapid synthesis of the htpR gene product is induced by isopropyl-beta-D-thiogalactopyranoside at 28 degree C (R . A . VanBogelen, M . A . Acton, and F . C . Neidhardt, Genes Dev . 1:525-531, 1987) . We postulate that induction of these three polypeptides involves metabolic signals in addition to the synthesis of the htpR gene product and that strain RG62 (metK) fails to produce the signals involved in induction of C14.7 and G13.5 on a shift-up in temperature and produces the signal related to lysU induction even at 37 degree C. J Bacteriol, 1988 Apr, 170(4), 1505 - 10 Purification and properties of Escherichia coli dimethyl sulfoxide reductase, an iron-sulfur molybdoenzyme with broad substrate specificity; Weiner JH et al.; Dimethyl sulfoxide reductase, a terminal electron transfer enzyme, was purified from anaerobically grown Escherichia coli harboring a plasmid which codes for dimethyl sulfoxide reductase . The enzyme was purified to greater than 90% homogeneity from cell envelopes by a three-step purification procedure involving extraction with the detergent Triton X-100, chromatofocusing, and DEAE ion-exchange chromatography . The purified enzyme was composed of three subunits with molecular weights of 82,600, 23,600, and 22,700 as identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis . The native molecular weight was determined by gel electrophoresis to be 155,000 . The purified enzyme contained 7.5 atoms of iron and 0.34 atom of molybdenum per mol of enzyme . The presence of molybdopterin cofactor in dimethyl sulfoxide reductase was identified by reconstitution of cofactor-deficient NADPH nitrate reductase activity from Neurospora crassa nit-I mutant and by UV absorption and fluorescence emission spectra . The enzyme displayed a very broad substrate specificity, reducing various N-oxide and sulfoxide compounds as well as chlorate and hydroxylamine. Infect Immun, 1988 Apr, 56(4), 930 - 5 Genetic control in the susceptibility of germfree inbred mice to infection by Escherichia coli O115a,c:K(B); Itoh K et al.; We studied the susceptibility of five germfree inbred strains of mice to oral infection by murine pathogenic Escherichia coli O115a,c:K(B) (MPEC), the causative agent of mouse megaenteron . Although MPEC colonized all strains of mice at 10(9)/g of feces, the mouse strains could be divided into three groups according to their intestinal lesions . In CF1 and C3H/He mice, intestinal lesions were produced in the cecum and colon with hyperplasia of epithelial cells accompanied by severe inflammatory reactions and erosion . The lesions in NC and C57BL/6 mice were restricted to the tip of the cecum, and hyperplasia of epithelial cells was more severe in these mice than in CF1 or C3H/He mice . BALB/c mice had no lesions . Analysis of F1 hybrids of CF1, NC, and BALB/c mice and offsprings from backcrosses of F1 mice to parental strains showed that susceptibility to MPEC seemed to be controlled genetically by a single locus which may be related to the receptors on epithelial cells for MPEC adherence . However, the differences in lesions between CF1 and NC mice suggest that a combination of this locus and another locus to which it may be related regulates the hyperplasia of intestinal epithelial cells. EMBO J, 1988 Apr, 7(4), 1061 - 9 Identification of three critical regions within mouse interleukin 2 by fine structural deletion analysis; Zurawski SM et al.; We have analyzed structure--function relationships of the protein hormone murine interleukin 2 by fine structural deletion mapping . A total of 130 deletion mutant proteins, together with some substitution and insertion mutant proteins, was expressed in Escherichia coli and analyzed for their ability to sustain the proliferation of a cloned murine T cell line . This analysis has permitted a functional map of the protein to be drawn and classifies five segments of the protein, which together contain 48% of the sequence, as unessential to the biological activity of the protein . A further 26% of the protein is classified as important, but not crucial, for the activity . Three regions, consisting of amino acids 32-35, 66-77 and 119-141 contain the remaining 26% of the protein and are critical to the biological activity of the protein . The functional map is discussed in the context of the possible role of the identified critical regions in the structure of the hormone and its binding to the interleukin 2 receptor complex. J Clin Microbiol, 1988 Apr, 26(4), 641 - 7 Antibodies reactive with human immunodeficiency virus gag-coded antigens (gag reactive only) are a major cause of enzyme-linked immunosorbent assay reactivity in a blood donor population; Tribe DE et al.; Normal blood donors were examined for human immunodeficiency virus (HIV)-reactive antibodies with both virus- and Escherichia coli-expressed env- and gag-coded antigens . The frequency of samples from normal (low-risk) donors that were repeatedly reactive with an HIV enzyme-linked immunosorbent assay blood screening test (Du Pont Co.) was 0.6% . Two classes of HIV serological reactivity were identified: a minor env-reactive class (0.03 to 0.06% of donors) and the predominant env-nonreactive gag-reactive class (gag reactive only {GRO}) (0.4 to 0.5% of donors) . Assignment of env reactivity was made by a synthetic (recombinant) env enzyme-linked immunosorbent assay and virus immunoblot . Most GRO sera reacted with p15/p17 bands on HIV immunoblot . Antibody specificity in GRO sera was confirmed by competition-binding studies with viral gag and E . coli-expressed p55gag . This study provides independent verification that gag-specific antibodies are present in many env-nonreactive sera . More serological and virological studies of individuals with this antibody pattern should be pursued to determine the origin of these gag-reactive antibodies. J Appl Physiol, 1988 Apr, 64(4), 1700 - 8 Effect of verapamil on pulmonary and eicosanoid responses to endotoxin in awake sheep; Ahmed T et al.; Leukotrienes have been suggested to play a role in the endotoxin-induced changes of the pulmonary hemodynamics and airway mechanics . Since Ca2+ is necessary for contraction of airway and vascular smooth muscle as well as for activation of phospholipase A2 and 5-lipoxygenase enzymes, we wondered whether the calcium antagonist verapamil would modify the endotoxin-mediated pulmonary effects as well as the generation of circulating eicosanoids . In twelve conscious sheep, measurements of pulmonary vascular resistance (PVR), systemic vascular resistance (SVR), lung resistance (RL), arterial PO2 (PaO2), leukocyte (WBC) count, arterial thromboxane B2 (TxB2), prostaglandin (PG) F2 alpha, and 6-ketoprostaglandin F1 alpha (6-keto-PGF1 alpha) concentrations were obtained before and at predetermined intervals after a 10-min infusion of Escherichia coli endotoxin (0.3 microgram/kg) . On separate occasions, the sheep received a bolus injection of verapamil (150 micrograms/kg) before endotoxin, followed by a continuous infusion of verapamil {10 micrograms.kg-1.min-1 (n = 5) or 20 micrograms.kg-1.min-1 (n = 7)} for up to 4 h post-endotoxin . Endotoxin caused a biphasic response with an increase in mean PVR and RL to 326 and 276% of base line during phase I (0-1 h) and lesser increases to 177 and 157% of base line during phase II (1.5-4 h), respectively (P less than 0.05) . SVR also showed biphasic increases of 44 and 42% during phase I and II, respectively . Mean PaO2 decreased by 16 Torr and WBC count decreased from 6.4 +/- 1.5 to 3.3 +/- 1.1 thousand/mm3, associated with marked increases in plasma TxB2, PGF2 alpha, and 6-keto-PGF1 alpha.(ABSTRACT TRUNCATED AT 250 WORDS) Protein Eng, 1988 Apr, 2(1), 63 - 8 A new method for random mutagenesis of complete genes: enzymatic generation of mutant libraries in vitro; Lehtovaara PM et al.; A new efficient in vitro mutagenesis method for the generation of complete random mutant libraries, containing all possible single base substitution mutations in a cloned gene is described . The method is based on controlled use of polymerases . Four populations of DNA molecules are first generated by primer elongation so that they terminate randomly, but always just before a known type of base (before A, C, G or T respectively) . Each of the four populations is then mutagenized in a separate misincorporation reaction, where the correct base can now be omitted . The regeneration of wild-type sequences can thus be efficiently avoided . Also, the misincorporating nucleotide concentrations can be optimized to give the three possible single mutations in close to equal ratio . The mutagenesis can be precisely localized within a predetermined target region of any size, and vector sequences remain intact . We have mutagenized the DNA coding for the alpha-fragment of Escherichia coli beta-galactosidase, and identified 176 different base substitution mutations by sequencing . The present method gives mutant yields of 40-60%, when the mutants contain about one amino acid change per protein molecule . All types of base substitution mutations can be generated and deletions are rare . The efficiency of this method permits the use of relatively elaborate screening systems to isolate mutants of either structural genes or regulatory regions. Mol Gen Mikrobiol Virusol, 1988 Apr, (4), 37 - 41 {Expression in Escherichia coli of the human leukocyte interferon alpha2 gene fused with N-terminal fragment of beta-galactosidase}; Petrenko VA et al.; Genes for leucocyte interferon and alpha-donor of galactosidase were fused by deletion mutagenesis or by site-directed mutagenesis . In both cases the fused protein was expressed . The protein having an antiviral activity of leucocyte interferon was easily detected in bacteria and solutions by the reaction of beta-galactosidase alpha-complementation and retained the antigenic determinants of interferon and beta-galactosidase . The use of fused proteins for optimization of gene expression and for the analysis of interferon structure-function relationship is discussed. Nippon Geka Gakkai Zasshi, 1988 Apr, 89(4), 482 - 93 {Postoperative debasement of the host-defense system and wound healing in elderly patients, and nutritional support as a therapeutic implication}; Kuroiwa K; Plasma opsonic activity and several kinds of plasma protein levels in each pre- and post-operative days were compared between older 25 cases (70 years or older) and 31 younger cases (59 years or younger) which both underwent elective abdominal surgery . The activity to opsonize E . coli 075 as a host-defense capacity was reduced on postoperative days 1,3 and returned gradually in either young or elderly . But opsonic activity levels in elderly patients in each postoperative days were significantly lower than in younger group . Plasma albumin level and rapid turnover protein levels in elderly patients tended to be lower than those in younger group through the postoperative period but statistically not significant . Plasma levels of C3 and fibronectin overshot the each preoperative value on days 5-7, after sharp reduction on day 1 in younger patients, but those in elderly didn't . Opsonic activity levels were closely correlated with plasma protein levels . Based on these clinical results, the experiments were designed to evaluate the effect of nutritional support in the early postoperative period on opsonic activity and protein synthesis in aged rats . These studies elucidated that early nutritional support brought beneficial effects on opsonic activity and plasma protein levels on postoperative days 7, but not on days 3 in aged rats . This may be because in aged animals, protein synthetic function has not fully recovered in the early postoperative period. Eur J Clin Microbiol Infect Dis, 1988 Apr, 7(2), 167 - 71 Identification of enterotoxigenic Escherichia coli using alkaline phosphatase-labeled synthetic oligodeoxyribonucleotide probes; Olive DM et al.; Alkaline phosphatase-conjugated synthetic oligodeoxyribonucleic acid probes were used to detect enterotoxigenic Escherichia coli strains containing either the heat stable or heat labile toxin genes . Both of the synthetic probes detected as little as 5 ng of purified plasmid DNA bearing the appropriate toxin gene . In addition, both probes could detect 5 X 10(6) toxigenic bacteria by colony hybridisation . No cross reactivity was observed between probes . When 197 clinical isolates of Escherichia coli were examined for toxigenicity using bioassays, 13 heat stable and 17 heat labile toxin strains were identified . Of the 13 heat stable toxin strains, 12 were positive using the heat stable toxin synthetic probe (sensitivity, 92%; specificity, 98%) while 16 of 17 bioassay heat labile toxin positive samples were identified using the heat labile toxin synthetic probe (sensitivity, 94%; specificity, 97%) . Alkaline phosphatase-conjugated synthetic probes with high sensitivity and specificity should provide a rapid means of identifying toxigenic Escherichia coli. J Interferon Res, 1988 Apr, 8(2), 169 - 78 Effect of recombinant interferon-gamma on protein content, phagocytic, and cytotoxic activity of mouse peritoneal macrophages; Rollag H et al.; We have studied the effect of recombinant murine interferon-gamma (rMuIFN-gamma) on the protein content, phagocytic activity, and cytotoxicity of mouse peritoneal macrophages (MPM) . The aim of this study was to see whether rMuIFN-gamma alone could influence these parameters of MPM activity or if an additional stimulus, like elicitation or cultivation with lipopolysaccharide (LPS), was required . The MPM cultures were treated with rMuIFN-gamma for 24, 48, or 72 h . Generally, rMuIFN-gamma treatment of the cultures increased the protein content of the MPM . MPM were generally cytotoxic and they phagocytized IgG-opsonized Escherichia coli under the experimental conditions of this study . The effects of rMuIFN-gamma on phagocytic and cytotoxic activities were complex and highly dependent on the dose and length of treatment . Low or high concentrations may exert opposite effects on the same functions . Addition of a low dose of LPS to the cultures did not generally amplify the rMuIFN-gamma-induced alterations of MPM activities . However, the combination of LPS, high doses of rMuIFN-gamma, and long incubation time reduced the protein content, suppressed the phagocytic activity, and negatively influenced the viability of the MPM . Thioglycolate-elicited MPM had a higher baseline activity than resident MPM, but the rMuIFN-gamma effect on elicited MPM was parallel to the effect on resident MPM. J Antimicrob Chemother, 1988 Apr, 21(4), 439 - 43 Standardization of inoculum size for disc susceptibility testing: a preliminary report of a spectrophotometric method; Moosdeen F et al.; Many methods of disc susceptibility testing aim at an inoculum to yield a semi-confluent growth . We have improved the means of standardizing inoculum by measuring the turbidity with a spectrophotometer . Absorbance measurements were made for various bacterial suspensions and dilutions thereof, from 1 in 2 to 1 in 1000 . Volumes of 0.1 ml of each dilution were spread on to Diagnostic Sensitivity Test agar . Viable counts of bacteria were made for each absorbance reading and for what constituted semi-confluent growth . Suspensions of bacteria having similar absorbance readings contained variable numbers of viable bacteria depending on the species . The number of bacterial cells that yielded semi-confluent growth also varied with different bacterial species . A chart was prepared to indicate the appropriate dilutions of bacterial suspensions with different absorbance readings to produce semi-confluent growth in sensitivity testing of each species. Br J Exp Pathol, 1988 Apr, 69(2), 169 - 76 Observations on the delay in onset of the acute phase protein response; Myers MA et al.; The early time course of the acute phase protein response (APPR) and mediators involved in its control were investigated in the rat and mouse . After turpentine-induced inflammation in the rat C-reactive protein and fibrinogen increased in concentration peaking at 48 h and 18-24 h, respectively . A 9 h delay prior to elevation of these protein was observed . After injection of endotoxin into mice, a 4-6 h delay was observed prior to any increase in the concentration of the acute phase protein serum amyloid P-component . This delay was shortened to 2 h after injection of leucocytic endogenous mediators (LEM) produced from rabbit peritoneal exudate cells . It is concluded that the delay between the initiating stimulus and the increases in the acute phase proteins is due to some obligatory intermediate steps which lead to the production of the final mediators of the APPR, and that these mediators are present in LEM. Arthritis Rheum, 1988 Apr, 31(4), 506 - 14 Quantitative immunoassay of anti-La antibodies using purified recombinant La antigen; St Clair EW et al.; A purified recombinant La fusion protein was tested in an enzyme-linked immunosorbent assay to quantitate anti-La responses . This protein contained the immunodominant region of the La molecule fused to beta-galactosidase . In solid-phase assays, recombinant La protein was solubilized in urea and bound to polystyrene wells without loss of immunoreactivity . The recombinant-based enzyme-linked immunosorbent assay proved to be a sensitive method for the detection of anti-La binding, and it accurately distinguished anti-La precipitin positive sera from normal sera. Proc Natl Acad Sci U S A, 1988 Apr, 85(8), 2709 - 13 The mutY gene: a mutator locus in Escherichia coli that generates G.C----T.A transversions; Nghiem Y et al.; We have used a strain with an altered lacZ gene, which reverts to wild type via only certain transversions, to detect transversion-specific mutators in Escherichia coli . Detection relied on a papillation technique that uses a combination of beta-galactosides to reveal blue Lac+ papillae . One class of mutators is specific for the G.C----T.A transversion as determined by the reversion pattern of a set of lacZ mutations and by the distribution of forward nonsense mutations in the lacI gene . The locus responsible for the mutator phenotype is designated mutY and maps near 64 min on the genetic map of E . coli . The mutY locus may act in a similar but reciprocal fashion to the previously characterized mutT locus, which results in A.T----C.G transversions. Proc Natl Acad Sci U S A, 1988 Apr, 85(8), 2603 - 7 Fluorescence-activated cell analysis and sorting of viable mammalian cells based on beta-D-galactosidase activity after transduction of Escherichia coli lacZ; Nolan GP et al.; We demonstrate that individual cells infected with and expressing a recombinant retrovirus carrying the Escherichia coli beta-galactosidase gene (lacZ) can be viably stained, analyzed, sorted, and cloned by fluorescence-activated cell sorting based on the levels of lacZ expressed . To accomplish this we have devised a method to enzymatically generate and maintain fluorescence in live mammalian cells . Accumulation of fluorescent products in cells is linear with time, with a direct correlation of fluorescence to enzymatic activity . This technology for beta-galactosidase detection is more sensitive than other available cytochemical or biochemical methods . We have used this procedure to show that the expression of psi-2-MMuLVSVnlsLacZ in the T-cell lymphoma BW5147 and the B-cell hybridoma SP2/0 is not completely stable and that subclones selected by the fluorescence-activated cell sorter for low lacZ activity demonstrate distinctly lower average expression of LacZ . These findings indicate the utility of beta-galactosidase as a reporter molecule at the single-cell level for studies of gene regulation, including studies of promoter efficacy, enhancer activity, trans-acting factors, and other regulatory elements. Proc Natl Acad Sci U S A, 1988 Apr, 85(8), 2474 - 8 Phosphorylation of the RAS2 gene product by protein kinase A inhibits the activation of yeast adenylyl cyclase; Resnick RJ et al.; The RAS2 gene product of Saccharomyces cerevisiae expressed in Escherichia coli was phosphorylated by protein kinase A in vitro to approximately 0.5-0.7 mol of phosphate per mol of protein . Neither protein kinase C nor protein kinase P phosphorylated the RAS2 protein significantly . The RAS2 protein is known to activate, in the presence of either Mg2+ and GTP or Mn2+, a yeast membrane preparation with an overexpressed adenylyl cyclase and a deficiency in endogenous RAS1 and RAS2 proteins . When the RAS2 protein was phosphorylated by protein kinase A prior to exposure to the yeast membranes, its capacity to activate the adenylyl cyclase was diminished by 40-60%, while activation by Mn2+ remained unaffected . The phosphorylated protein retained, however, its ability to bind GTP . Incubation of protein kinase A with a specific protein kinase A inhibitor prior to phosphorylation prevented the inhibition . Furthermore, the hydrolysis of GTP was not required for the observed inhibition . These data suggest that phosphorylation of the RAS2 gene product by protein kinase A may function as one mechanism by which the intracellular level of cAMP in yeast is regulated. J Gen Virol, 1988 Apr, 69 ( Pt 4), 765 - 76 Functional studies on the p10 gene of Autographa californica nuclear polyhedrosis virus using a recombinant expressing a p10-beta-galactosidase fusion gene; Vlak JM et al.; The beta-galactosidase gene (lacZ) of Escherichia coli was inserted in phase with the coding sequence of the Autographa californica nuclear polyhedrosis virus (AcMNPV) late-expressed Mr 10,000 (p10) gene . The fusion gene was inserted into the AcMNPV genome by cotransfection of a recombinant plasmid pAcR159Z, consisting of the EcoRI P fragment-containing pBR325-derived plasmid pAcR159 and the lacZ insert in the p10 gene, and wild-type AcMNPVDNA . Infection of Spodoptera frugiperda cells by the resulting recombinant AcMNPV/p10Z-2 showed high level expression of a p10-lacZ fusion protein, but no synthesis of p10 . Therefore, the p10 gene is dispensable for virus replication and the p10 promoter is effective in driving the expression of foreign genes . Cells infected with AcMNPV/p10Z recombinants resembled those infected with wild-type AcMNPV in the amounts of polyhedrin synthesized and polyhedra formed, although p10 was absent . The nucleus and cytoplasm of AcMNPV/p10Z-2-infected cells lacked the fibrous structures that are associated with p10 in wild-type AcMNPV-infected cells . Instead, large granular structures were observed that were found by immunogold labelling to contain the lacZ gene product . The electron-dense 'spacers', thought to be precursors of the polyhedron membrane, were absent from cells infected by the recombinant virus and the polyhedra did not have a membrane . The recombinant AcMNPV/p10Z-2 was at least twice as virulent for second instar S . exigua larvae than was wild-type AcMNPV . The increased virulence of the recombinant is an important property for the control of insects. J Bacteriol, 1988 Apr, 170(4), 1975 - 7 The Escherichia coli K-12 lexA2 gene encodes a hypocleavable repressor; Peterson KR et al.; LexA2 repressor was partially inactivated after mitomycin C or UV light treatment in a recA+ or recA85(Prtc) (protease constitutive) host background . LexA2 protein was cleaved, but the reaction was slower than that observed for LexA+ repressor . lexA2 had a C-to-T transition at nucleotide 461 (Thr-154 to Ile). Biotechniques, 1988 Apr, 6(4), 332 - 9 An enrichment selection for mutants resulting from oligonucleotide-directed mutagenesis of double-stranded DNA; McCracken AA et al.; We report here a simple and rapid procedure for enrichment and selection of mutants from oligonucleotide-directed mutagenesis on double-stranded plasmid DNA . Mutagenic oligonucleotides were designed to insert or delete a unique restriction site with silent codon changes . After mutagenesis, plasmid DNA from all resulting colonies was pooled, restricted with the appropriate endonuclease, and the resulting unique form of DNA (linear or circular) was isolated and used for transformation of competent E . coli . These procedures provided an enrichment of mutant plasmid from the 4% obtained by more conventional techniques to greater than 65%. Pathology, 1988 Apr, 20(2), 167 - 72 Enterotoxigenic Escherichia coli in Central Australia: diagnosis using cloned and synthetic nucleic acid probes; Higgins GD et al.; Feces from 169 children admitted with diarrhea to Alice Springs Hospital, were screened for enterotoxigenic E . coli (ETEC) using specific DNA probes . E . coli which hybridized with probes for ST-P, ST-H and LT were confirmed by bioassay for toxin production . Fifty children were shown to excrete ETEC . The probes for LT correlated well with bioassay; however, probes for ST-H and ST-P hybridized with more E . coli than were shown to produce toxin by bioassay . When probing for ST was repeated using a synthetic oligonucleotide probe, only those specimens which were bioassay-positive hybridized with the probe. Bioorg Khim, 1988 Apr, 14(4), 472 - 7 {Affinity modification of Escherichia coli ribosomes with the non-hydrolyzed GTP analog, gamma-{4-N-(2-chloroethyl)-N-methylaminobenzyl}amide of GTP}; Babkina GT et al.; Substituted gamma-amides of GTP viz . GTP gamma-{4-N-(2-chloro- and gamma-{4-N-(2-hydroxyethyl)-N-methylaminobenzyl}amide (CIRCH2NHpppG and OHRCH2NHpppG, resp.) were shown to be unhydrolisable GTP analogues in the EF-Tu-dependent GTP-ase reaction of ribosomes . The reactive analogue, CIRCH2NHpppG, was used for affinity labelling within the 70S ribosome.poly(U).tRNAPhe(P-site).Phe-tRNAPhe.EF-Tu.CIR{14C}CH2.NHpppG complex . Both 50S and 30S subunits were thus labelled but 50S subunit was modified considerably more than 30C subunit . Labelled were proteins L17, L21, S16, S21, and rRNA of both subunits, 23C rRNA within 50C subunit being labelled preferentially as compared with 50C proteins . No labelling of EF-Tu within the complex was detected. Vet Q, 1988 Apr, 10(2), 117 - 25 Pathophysiological effects of endotoxins in ruminants . 2 . Metabolic aspects; Lohuis JA et al.; Metabolic disturbances following intravenous and intramammary administration of endotoxins in ruminants are described . In contrast to the similarity in response of blood biochemical parameters after intravenous and intramammary administrations of endotoxins, responses in plasma concentrations of enzyme activities, the thyroid hormones, cortisol, and somatotropin differ markedly . Biochemical changes in blood after endotoxin administration are predominantly dose-dependent; thus some of the biochemical parameters - especially plasma concentrations of Fe and Zn - serve also to evaluate the effects of certain drugs in endotoxin models . Changes in milk composition have been documented only after intramammary infusion of endotoxins and can partly be explained by the increased permeability of the blood/milk barrier . Appearance and production of milk returns to normal within a week after intramammary endotoxin treatment, indicating that the mammary gland is only temporarily damaged by endotoxin-induced mastitis. Vet Q, 1988 Apr, 10(2), 109 - 16 Pathophysiological effects of endotoxins in ruminants . 1 . Changes in body temperature and reticulo-rumen motility, and the effect of repeated administration; Lohuis JA et al.; Data from the literature on the clinical effects of bacterial endotoxins in ruminants are reviewed . Special attention is paid to the effects on body temperature and reticulo-rumen motility . Furthermore, the effects of repeated intravenous injection of endotoxin are summarised . Pathophysiological disturbances after intramammary infusion of endotoxins proved to be identical to those found after intravenous injection of non-lethal doses . Strikingly, however, no marked inhibitory effect on rumen motility nor abortion was observed after intramammary infusion of endotoxins . Moreover, in cows that were made tolerant to endotoxin by daily intravenous injections, intramammary infusion of one-fifth of this daily dose produced a maximum effect on body temperature and plasma Zn concentrations . This suggests that inflammatory endogenous mediators were released in the udder and then absorbed into the blood circulation, rather than the absorption of endotoxin. Zhonghua Liu Xing Bing Xue Za Zhi, 1988 Apr, 9(2), 84 - 7 {Serotypes of enteroinvasive Escherichia coli in China}; Yang ZS; results of identification and serotyping of enteroinvasive escherichia in china from 13 provinces in China in 1984-85 were reproted. Behring Inst Mitt, 1988 Apr, (82), 349 - 59 A histidin alanine rich recombinant antigen protects Aotus monkeys from P . falciparum infection; Knapp B et al.; We have isolated a cDNA clone coding for 165 amino acids of a histidine alanine rich protein . An extended repeat region of this clone codes for the tripeptide Ala-His-His, which is extremely conserved, and for the tripeptide Ala-Ala-Asp, which shows only slight variability among the repeat units . This antigen exhibits high homology to the HRPII antigen described by Wellems and Howard (1986) . The coding region was expressed in E . coli as a MS2-polymerase fusion protein, which was purified and used for immunization of Aotus monkeys . The animals immunized with this fusion protein showed only low parasitemias (less than 2%) after infection with P . falciparum, while animals from the control group or animals immunized with a MS2-fusion protein carrying other malaria specific sequences were not protected . The result suggests that this antigen is a good candidate for a malaria vaccine. Behring Inst Mitt, 1988 Apr, (82), 338 - 48 Immunoreactivity of human immunodeficiency virus (HIV-1) envelope polypeptides expressed in Escherichia coli; Broker M et al.; Defined cDNA sequences encoding segments of envelope glycoproteins of the human immunodeficiency virus were expressed in E . coli . The recombinant env fusion proteins were tested for immunoreactivity with patient sera by radio immunoprecipitation procedures . The regions of the env proteins, carrying relevant antigenic determinants in respect to diagnostic purposes, could be identified. Behring Inst Mitt, 1988 Apr, (82), 26 - 34 Efficient expression system for human antithrombin III in baby hamster kidney cells; Zettlmeissl G et al.; A DNA fragment carrying the enhancer of an immediate early gene of human cytomegalovirus (hCMV) was tested as a transcriptional control element by fusion downstream of a transcription unit for human antithrombin III (AT III) driven by the Simian virus (SV40) enhancer and promoter . Measurement of transient AT III expression in baby hamster kidney cells (BHK) shows that by the presence of the hCMV enhancer the synthesis of AT III is increased considerably (three to fourfold) . The AT III expression vectors carrying the hCMV enhancer were used to establish stable BHK cell-lines by a new and fast G418/methotrexate selection protocol, which express up to 12 micrograms AT III/10(6) cells/24h after 40-50 days . The given system might be generally useful for the fast expression of recombinant proteins in mammalian cells, e.g . the screening of altered AT III molecules obtained by site directed mutagenesis. Behring Inst Mitt, 1988 Apr, (82), 59 - 67 Isolation and expression of cDNA coding for a new member of the phospholipase A2 inhibitor family; Grundmann U et al.; The placental protein PP41,2 was shown to have thromboplastin-inhibitor activity . We used partial amino acid sequence information from PP4 cyanogen bromide fragments to design oligonucleotide probes for the screening of a human placental cDNA library . In addition to the PP4 cDNA we isolated a cDNA coding for a protein with considerable homology which we subsequently termed PP4-X . PP4 and PP4-X belong to the phospholipase A2 inhibitor family, as judged by their homology to lipocortin I and calpactin I3 . The full-length PP4-X cDNA encodes a protein of 321 amino acid residues including a fourfold repeat structure . Northern blot analysis using the PP4-X cDNA reveals two hybridizing RNA species of approximately 1400 nucleotides and 2500 nucleotides, respectively . The shorter one could well represent the PP4-X transcript which is in good agreement with the isolated cDNA insert of 1326 nucleotides . Expression of the PP4-X coding sequence in E . coli resulted in the appearance of a protein which crossreacts with antibodies raised against PP4. Genetics, 1988 Apr, 118(4), 551 - 60 Changes in DNA base sequence induced by gamma-ray mutagenesis of lambda phage and prophage; Tindall KR et al.; Mutations in the cI (repressor) gene were induced by gamma-ray irradiation of lambda phage and of prophage, and 121 mutations were sequenced . Two-thirds of the mutations in irradiated phage assayed in recA host cells (no induction of the SOS response) were G:C to A:T transitions; it is hypothesized that these may arise during DNA replication from adenine mispairing with a cytosine product deaminated by irradiation . For irradiated phage assayed in host cells in which the SOS response had been induced, 85% of the mutations were base substitutions, and in 40 of the 41 base changes, a preexisting base pair had been replaced by an A:T pair; these might come from damaged bases acting as AP (apurinic or apyrimidinic) sites . The remaining mutations were 1 and 2 base deletions . In irradiated prophage, base change mutations involved the substitution of both A:T and of G:C pairs for the preexisting pairs; the substitution of G:C pairs shows that some base substitution mechanism acts on the cell genome but not on the phage . In the irradiated prophage, frameshifts and a significant number of gross rearrangements were also found. Am J Physiol, 1988 Apr, 254(4 Pt 2), R567 - 71 Immunoreactive atrial natriuretic factor is increased in ovine model of endotoxemia; Lubbesmeyer HJ et al.; A bolus of Escherichia coli endotoxin (1.5 micrograms/kg) was administered to chronically instrumented sheep . Immunoreactive atrial natriuretic factor (IR-ANF) was measured in extracted plasma by radioimmunoassay . There was a thirteenfold increase in IR-ANF 2 h after endotoxin administration, and IR-ANF levels remained significantly elevated during the first 6 h . A marked diuresis and natriuresis occurred between 4 and 6 h . ANF not only affects renal function but is also associated with decreased cardiac output, increased peripheral resistance (in sheep), and decreased capillary absorption (in rats) . These renal and hemodynamic changes are also characteristic of the early (first 6 h) response to endotoxin . Therefore ANF should be considered as a potential mediator of renal and hemodynamic changes induced by sepsis . It is difficult to determine if ANF elevation is an epiphenomenon or a causative factor, because no antagonist of ANF is currently available. Zh Mikrobiol Epidemiol Immunobiol, 1988 Apr, (4), 90 - 3 {Detection of the gene sequences of the leukocytosis (lymphocytosis)-stimulating factor of Bordetella pertussis in Bordetella parapertussis and Bordetella bronchiseptica by using molecular hybridization}; Rozinov MN et al.; The 4.7 Kb EcoRI-fragment of phase I B . pertussis 475 (serovar 1.2.3) chromosome DNA carrying the pertussis toxin (PT) operon was cloned on vector plasmid pUC19 in Escherichia coli . Three fragments (1.14 Kb KpnI-PstI, 1.27 Kb PstI-PstI, and 0.96 Kb PstI-PstI) were obtained from the resulting hybrid plasmid, coded pRH119, by electrophoretic techniques and used as a combined molecular probe for analysis of the EcoRI-digested and PstI-digested chromosomal DNA of B . pertussis strain 475 in phase I, B . pertussis in phase IV, B . parapertussis strains 504 and 17903, B . bronchiseptica strain 214, and B . parapertussis strain 17903 (a convertant obtained by means of B . pertussis phage 134), as well as B . pertussis phage 134 . Southern blot hybridization under the conditions of 100% DNA-DNA homology showed the presence of DNA sequences characteristic of the PT operon in all cases except the DNA of phage 134; moreover, the use of the above-mentioned probe made it possible to hybridize all EcoRI-fragments of chromosomal DNA, having the same molecular size (4.7 Kb) . Consequently, the PT genes in the above Bordetella species were mapped in identical loci.(ABSTRACT TRUNCATED AT 250 WORDS) Zh Mikrobiol Epidemiol Immunobiol, 1988 Apr, (4), 86 - 90 {Cloning and gene expression of the leukocytosis (lymphocytosis) stimulating factor of Bordatella pertussis in Escherichia coli by bringing the PT genes close to the lactose promoter of plasmid pUC19}; Rozinov MN et al.; The 4.7 Kb EcoRI-fragment of phase I B . pertussis 475 (serovar 1.2.3.) chromosome carrying all five genes of the pertussis toxin (PT) operon was cloned on plasmid pUC19 in E . coli . The resulting hybrid plasmid pRH119 contained the PT operon in the same orientation of transcription as the lac promoter of plasmid pUC19 . Nevertheless, the expression of the PT operon was not observed even after induction with isopropyl thio-beta-D-galactopyranoside (IPTG), which suggested either the inability of the PT operon to work in E . coli, or the presence of a transcription terminator between the lac promoter and the PT operon in plasmid pRH119 . The expression was determined by the incubation of the clones harboring plasmid pRH119 with antiserum to PT and their subsequent in situ treatment with 125I-labeled protein A . Three deletion variants of plasmid pRH119 were constructed with the aim of approaching the PT genes to the lac promoter: pRH121 (the 0.45 Kb KpnI-fragment deleted), pRH122 (the 0.95 Kb SalGI-fragment deleted) and pRH123 (the 1.35 Kb XbaI-fragment deleted) . In all these cases different levels of expression were observed (but only in the presence of IPTG) . Site KpnI in the 4.7 Kb fragment was found to be localized in the -57 b . p . region in relation to the PT promoter, i . e . to lie, seemingly, in the promoter zone; for this reason, the expression of the PT genes in plasmid pRH121 proved the existence of a transcription terminator between the lac promoter and the PT operon in plasmid pRH119.(ABSTRACT TRUNCATED AT 250 WORDS) Vaccine, 1988 Apr, 6(2), 197 - 9 Progress towards a vaccine against enterotoxigenic Escherichia coli; Kaper JB et al.; A variety of approaches are being investigated in the development of a vaccine against enterotoxigenic Escherichia coli (ETEC) . These approaches include purified fimbriae vaccines, toxoid vaccines, live attenuated E . coli vaccine strains and ETEC antigens expressed in carrier organisms . Studies of the pathogenesis and immune response to ETEC indicate that development of a vaccine against human ETEC is a realistic goal but considerable work remains before this goal is realized. Vaccine, 1988 Apr, 6(2), 110 - 2 Tip proteins of pili associated with pyelonephritis: new candidates for vaccine development; Lund B et al.; Escherichia coli strains associated with extra-intestinal infections frequently express carbohydrate binding adhesins which are present as minor components of pili . The adhesin protein, PapG, and at least two other minor pilus subunits, PapE and PapF, are associated with the tips of Gal alpha (1-4)Gal-binding Pap pili or P fimbriae of uropathogenic E . coli . The structural and antigenic variation of these tip-associated proteins is discussed and evidence is presented showing that serologically identical pili may contain antigenically distinct adhesins each capable of binding to a specific receptor . One approach to the purification of these tip-associated proteins is presented and involves complex formation with the periplasmic transport protein PapD. APMIS, 1988 Apr, 96(4), 337 - 41 In vitro cytotoxic effect of alpha-hemolytic Escherichia coli on human blood granulocytes . Correlation with size of alpha-hemolysin production; Gadeberg OV et al.; The correlation of the in vitro cytotoxic effect of 107 alpha-hemolytic strains of Escherichia coli with various other bacterial characteristics was investigated . Damage to human blood granulocytes in the presence of fresh or heated autologous plasma was quantified by measuring the release of chromium-51 from labelled cells . 95 strains had a cytotoxic effect which was equal in the presence of fresh or heated plasma, whereas 12 strains showed an effect which was reduced in fresh compared with heated plasma . The cytotoxic effect increased as the number of bacteria per granulocyte was increased . The average size of the alpha-hemolysin production of the strains was 185 HU50/ml ranging from 3-2519HU50/ml . The cytotoxic effect of the strains was directly correlated with the size of the alpha-hemolysin production . The cytotoxic effect was not correlated with the O-antigen serotype or the type of infection from which the strains were derived . These results indicate that the ability to produce alpha-hemolysin is the bacterial characteristic which is of decisive importance for the cytotoxicity of alpha-hemolytic E . coli towards human blood granulocytes. Proc Natl Acad Sci U S A, 1988 Apr, 85(8), 2464 - 8 On the active site thiol of gamma-glutamylcysteine synthetase: relationships to catalysis, inhibition, and regulation; Huang CS et al.; gamma-Glutamylcysteine synthetase (glutamate-cysteine ligase; EC 6.3.2.2) was isolated from an Escherichia coli strain enriched in the gene for this enzyme by recombinant DNA techniques . The purified enzyme has a specific activity of 1860 units/mg and a molecular weight of 56,000 . Comparison of the E . coli enzyme with the well-characterized rat kidney enzyme showed that these enzymes have similar catalytic properties (apparent Km values, substrate specificities, turnover numbers) . Both enzymes are feedback-inhibited by glutathione but not by gamma-glutamyl-alpha-aminobutyrylglycine; the data indicate that glutathione binds not only at the glutamate binding site but also at a second site on the enzyme that interacts with the thiol moiety of glutathione but not with a methyl group . Both enzymes are inactivated by buthionine sulfoximine in the presence of ATP, suggesting a common gamma-glutamyl phosphate intermediate . However, unlike the rat kidney enzyme that has an active center thiol, the bacterial enzyme is insensitive to cystamine, gamma-methylene glutamate, and S-sulfo amino acids, indicating that it does not have an active site thiol . Thus, the rat kidney and E . coli enzymes share several catalytic features but differ in active site structure . If the active site thiol of the rat kidney enzyme is involved in catalysis, which seems likely, there would appear to be differences in the mechanisms of action of the two gamma-glutamylcysteine synthetases. Proc Natl Acad Sci U S A, 1988 Apr, 85(7), 2046 - 50 Identification of the second chromophore of Escherichia coli and yeast DNA photolyases as 5,10-methenyltetrahydrofolate; Johnson JL et al.; Denaturation of DNA photolyase (deoxyribodipyrimidine photolyase, EC 4.1.99.3) from Escherichia coli with guanidine hydrochloride or acidification to pH 2 released, in addition to FAD, a chromophore with the spectral and chromatographic properties of a reduced pterin . Treatment of the enzyme with iodine prior to acidification converted the chromophore to a stable, oxidized derivative, which was resolved by HPLC into four species with identical spectral properties . The same species, in the same distribution, were obtained from the yeast enzyme . The material isolated from the iodine-oxidized enzyme was shown to be a pterin by conversion to pterin-6-carboxylic acid with alkaline permanganate and was found to release glutamate upon acid hydrolysis . The presence of 10-formylfolate in the isolated, oxidized chromophore was demonstrated by absorption and fluorescence spectroscopy and by deformylation and conversion to folic acid . Analysis of the distribution of polyglutamates revealed that the four species identified by HPLC corresponded to the tri-, tetra-, penta-, and hexaglutamate derivatives of 10-formylfolate . The results were consistent with gamma linkages in the triglutamate derivative with additional glutamates linked via the alpha-carboxyl group of the preceding residue . Treatment with rat plasma hydrolase produced the monoglutamate derivative of 10-formylfolate . The native, enzyme-bound form of the folate cofactor was identified as 5,10-methenyltetrahydrofolylpolyglutamate by effecting release and isolation at low pH to protect the 5,10-methenyl bridge and preserve the reduced pyrazine ring structure. J Bacteriol, 1988 Apr, 170(4), 1887 - 94 Structure and antigenic properties of the tip-located P pilus proteins of uropathogenic Escherichia coli; Lund B et al.; Pyelonephritogenic Escherichia coli frequently expresses pili which bind to Gal alpha (1-4)Gal receptors present on the uroepithelium . Binding of these pili is mediated by a pilus-associated adhesin, PapG, and not by the major subunit which constitutes the bulk of the pilus structure . The adhesin and two pilinlike proteins, PapE and PapF, are present in only a few copies each at the pilus tip . Surface exposure of both PapF and PapG is required to achieve receptor-specific binding . The nucleotide sequences for the genes encoding the tip-associated proteins PapE, PapF, and PapG were determined for two E . coli clones expressing P pili of serotypes F11 and F7(2) and compared with the corresponding sequences established for proteins of F13 pili . Specific antisera were used to study the cross-reactivity between the F13 tip proteins and the equivalent proteins in F11 and F7(2) pili . We present data showing that, like the major pilus subunit, PapE varies its structure and antigenic properties among pili of different serotypes . In contrast, the PapF protein was highly conserved, and PapF-specific antisera raised against serotype F13 cross-reacted with the PapF proteins of both F11 and F7(2) serotypes . The PapG adhesin protein from F11 and F7(2) pili differed by only five amino acids out of 316 residues . However, the F13 adhesin showed only 45% amino acid homology with the other two variants. J Bacteriol, 1988 Apr, 170(4), 1666 - 71 Regulation of fatty acid degradation in Escherichia coli: fadR superrepressor mutants are unable to utilize fatty acids as the sole carbon source; Hughes KT et al.; Localized mutagenesis of the fadR region of the Escherichia coli chromosome resulted in the isolation of two classes of fadR regulatory mutants . The first class was constitutive for the fatty acid degradative enzymes and presumably defective for fadR function . The second class was rarer and resulted in the inability to utilize fatty acids as a sole carbon source (Fad-) . These fadR superrepressor mutants {fadR(S)} had greatly reduced levels of the beta-oxidative enzymes required for growth on fatty acids . The fadR(S) mutants reverted to Fad+ at a high frequency (10(-5}, and the resulting Fad+ revertants were constitutive for expression of the fad enzymes (fadR) . Merodiploid analysis showed the fadR(S) allele to be dominant to both fadR+ and fadR alleles. J Bacteriol, 1988 Apr, 170(4), 1610 - 6 Polymorphisms in the umuDC region of Escherichia species; Sedgwick SG et al.; The umuDC operon of Escherichia coli encodes mutagenic DNA repair . The umuDC regions of multiple isolates of E . coli, E . alkalescens, and E . dispar and a single stock of E . aurescens were mapped by nucleotide hybridization . umuDC is located at one end of a conserved tract of restriction endonuclease sites either 12.5 or 14 kilobase pairs long . Rearrangements, including possible deletions, were seen in the polymorphic DNA flanking the conserved tract . Restriction site polymorphisms were not found around the DNA repair gene recA or polA . The junctions of the conserved region contain direct repeats of nucleotide sequences resembling the termini of the Tn3 group of transposons . Possible mechanisms for the generation of these variants are discussed. Infect Immun, 1988 Apr, 56(4), 815 - 22 Type 1 fimbriate Escherichia coli stimulates a unique pattern of degranulation by human polymorphonuclear leukocytes; Steadman R et al.; Uropathogenic strains of Escherichia coli bearing mannose-sensitive (type 1) fimbriae promote a unique pattern of degranulation from human polymorphonuclear leukocytes (PMN) . Significant quantities of the primary (1 degree) and tertiary (3 degree) granule markers, neutral protease-myeloperoxidase and N-acetyl-beta-D-glucosaminidase, respectively, were released by PMN in a dose- and time-dependent manner when stimulated by these defined bacterial strains . Organisms bearing mannose-resistant (P) fimbriae promoted release of only the secondary (2 degree) granule marker, vitamin B12-binding protein . When this pattern of degranulation was compared to that produced by PMN in response to a variety of soluble and particulate stimuli, only the calcium ionophore A23187 similarly triggered 1 degree and 3 degree granule marker release . All the other stimuli tested--zymosan, serum-treated and unopsonized; n-formylmethionyl-leucyl-phenylalanine; and phorbol myristate acetate--promoted release of only the 2 degree granule marker . These results demonstrate selectivity of PMN degranulation in response to a number of transmembrane signals . In addition, the capacity of E . coli to promote PMN degranulation is dependent on its phenotypic fimbrial expression, a surface characteristic which correlates significantly with its relative surface hydrophobicity as measured by binding to octyl Sepharose . Those bacteria demonstrating the greatest hydrophobicity were capable of triggering discharge of all three granule marker proteins . Thus, the mannose-sensitive fimbriae of uropathogenic E . coli may contribute significantly to their potential pathophysiologic role in renal scarring. EMBO J, 1988 Apr, 7(4), 1211 - 8 Cellular factors required for multiple stages of SV40 DNA replication in vitro; Fairman MP et al.; Plasmids containing the SV40 origin replicate in the presence of SV40 T antigen and a cell free extract derived from human 293 cells . Upon fractionation of this extract, two essential replication factors have been identified . One of these is a multi-subunit DNA binding protein containing polypeptides of 70,000, 34,000 and 11,000 daltons which may function as a eukaryotic single strand DNA binding protein (SSB) . The other partially purified fraction is required with T antigen for the first stage of DNA replication, the formation of a pre-synthesis complex at the replication origin . These results, and others, define multiple stages of SV40 DNA replication in vitro which are analogous to multiple stages of Escherichia coli and phage lambda replication, and may reflect similar events in the replication of cellular chromosomes. EMBO J, 1988 Apr, 7(4), 1191 - 6 The expression of novel antigens from the Epstein-Barr virus large internal repeat; Walls D et al.; A large Epstein-Barr virus (EBV) B95-8 genomic bank has been prepared in an Escherichia coli expression vector and screened with a pool of sera from human infectious mononucleosis patients . Four immunopositive clones which also contained sequences from the viral large internal repeat were selected . DNA sequence analysis has located them on the repeat sequence and shown that they come from three potential open reading frames and that two of them consist of overlapping reading frames . This must imply extensive intron/exon splicing or that the repeat itself encodes several different proteins . The four expressed epitopes were shown to be present simultaneously in independent cases of infectious mononucleosis . These have not been previously described and based on the experimental design, they must reflect the situation in vivo. Mol Biol Med, 1988 Apr, 5(2), 107 - 22 Cloning and characterization of a cDNA encoding human galactose-1-phosphate uridyl transferase; Reichardt JK et al.; We report the cloning and characterization of a cDNA that encodes a functional human galactose-1-phosphate uridyl transferase (GALT) . The cDNA is 1400 bases in length and encodes a 43,000 Mr protein . The cloning strategy involved the identification of short peptide sequences conserved between the homologous enzymes from Escherichia coli and yeast, and the construction of oligonucleotide pools corresponding to the conserved patches . These patches of conserved amino acids tend to be conserved in humans as well. Biochem J, 1988 Apr 1, 251(1), 111 - 4 Use of progress curves to estimate the co-substrate-to-substrate flow ratio of a symport mechanism . Application to the isoleucine-Na+ symport of mouse ascites-tumour cells and to the lactose-proton symport; Eddy AA et al.; The model envisages two components in the process, whereby Ht equivalents of co-substrate and St equivalents of substrate accumulate in the cellular compartment in time t . The first is the flow through the symport, n equivalents of co-substrate entering or leaving with each substrate equivalent . The second is the basal flow of co-substrate outside the symport . In certain specific circumstances n can be derived by plotting Ht/t against St/t . The principal requirement is that, whereas the ratio of the component flows must change in the interval t, the magnitude of the basal flow must either be zero or constant . The procedure is applied to published observations {West & Mitchell (1973) Biochem . J . 132, 587-592} on the lactose-proton symport of Escherichia coli {n = 1.075 +/- 0.064(7)} and to new observations on the isoleucine-Na+ symport of mouse ascites-tumour cells {n = 1.136 +/- 0.120(18)}. Biotechnol Appl Biochem, 1988 Apr, 10(2), 107 - 17 Immobilized polyphosphate kinase: preparation, properties, and potential for use in adenosine 5'-triphosphate regeneration; Hoffman RC Jr et al.; Polyphosphate kinase (ATP:polyphosphate phosphotransferase; EC 2.7.4.1), partially purified from Escherichia coli, has been immobilized on glutaraldehyde-activated aminoethyl cellulose with a 10% retention of enzymatic activity . The immobilized enzyme can carry out the synthesis of ATP from ADP, using long-chain inorganic polyphosphate as a phosphoryl donor . Chromatographic analyses of the product mixture produced from ADP and {32P}polyphosphate demonstrated that 98% of the 32P was incorporated into ATP, indicating that the immobilized polyphosphate kinase is substantially free from contaminating polyphosphate phosphohydrolase (EC 3.6.1.11), adenosine triphosphatase (EC 3.6.1.4), and adenylate kinase (EC 2.7.4.3) . Immobilized polyphosphate kinase loses no activity when stored in an aqueous suspension for 2 months at 5 degrees C or for 1-2 weeks at 25 degrees C . It may be stored indefinitely as a lyophilized powder at -10 degrees C . Michaelis constants for ADP and polyphosphate were determined to be 160 and 120 microM, respectively, for the immobilized enzyme . A small-batch reactor was found to produce ATP linearly with time up to 65% conversion of polyphosphate into ATP and to attain greater than 85% conversion to ATP at equilibrium . The ease of purification and immobilization of E . coli polyphosphate kinase, its storage stability, the purity and yield of its ATP product, and the low values of the Michaelis constants for its substrates make it a highly promising enzyme for ATP regeneration. Mol Cell Biol, 1988 Apr, 8(4), 1509 - 17 Replication from a proximal simian virus 40 origin is severely inhibited by multiple reiterations of the 72-base-pair repeat enhancer sequence; Kumar R et al.; In a previous study in our laboratory, the effect of the reiteration frequency of the simian virus 40 (SV40) 72-base-pair (bp) repeat enhancer on transcription from the proximal SV40 early promoter was investigated (R . Kumar, T . A . Firak, C . T . Schroll, and K . N . Subramanian, Proc . Natl . Acad . Sci . USA 83:3199-3203, 1986) . Increasing the enhancer copy number to four increased transcription proportionately; further increments in enhancer copy number reversed this effect, resulting in a decrease in the transcriptional activation . In the present study, the effect of enhancer reiteration on the replication efficiency of plasmids containing the SV40 origin of replication was investigated in transient replication assays in vivo in COS-1 monkey kidney cells producing the SV40 large tumor antigen required for replication . A plasmid containing the SV40 core origin and three copies of the replication-activating, G+C-rich 21-bp repeat promoter element replicated efficiently . Plasmids containing multiple copies of the 72-bp repeat enhancer cloned in head-to-tail linkage adjacent to the 21-bp repeat and the core origin replicated less efficiently; the decrease in replication efficiency could be correlated with the number of copies of the 72-bp repeat; replication was severely curtailed when 10 or more copies of the 72-bp repeat were present . Replication was not significantly inhibited by an increase in the number of copies of the 21-bp repeat to 15 or by the presence of three copies of a 360-bp pBR322 sequence in the immediate vicinity . Multiple copies of the 72-bp enhancer in cis were unable to inhibit replication from a second SV40 origin of replication situated 2 kilobase pairs away from the enhancer reiteration . Replication of four different test plasmids was not inhibited in trans by cotransfection of an excess of a potential competitor plasmid containing a 24-copy reiteration of the 72-bp enhancer . These results indicate that multiple tandem reiterations of the 72-bp enhancer inhibit replication only when they are present in cis adjacent to the origin of replication . Possible explanations for this inhibitory effect, such as an unfavorable local chromatin structure induced by the multimeric enhancer region or reduced or improper communications between factors bound to the multimeric region and the adjacent replication origin, are discussed. Mol Gen Genet, 1988 Apr, 212(1), 99 - 104 AsnC, a multifunctional regulator of genes located around the replication origin of Escherichia coli, oriC; Kolling R et al.; The expression of the gidA gene which is located immediately counterclockwise of the replication origin of Escherichia coli, oriC, was found to be negatively regulated by the AsnC protein in an in vitro transcription-translation system . This effect is not due to simple repression of transcription originating at the gidA promoter, because the AsnC protein did not change the level of gidA promoter dependent transcription as analysed by promoter-galK fusions and by S1 mapping . From these data we conclude that the AsnC protein controls gidA gene expression at a post-transcriptional level . gidA is the third gene in the oriC region, besides asnA and asnC, whose expression is under AsnC control . However, the mechanisms involved are different: regulation of transcription in the case of asnA and asnC and post-transcriptional control of gidA . The gidA promoter was mapped by deletion analysis and by S1 mapping . We defined two regions that affect promoter activity negatively . Additional transcripts, regulated by AsnC, started more than 300 bp upstream of the gidA promoter and were found to enter the gidA region . These transcripts, originating either at the mioC and/or the ansC promoter traverse the replication origin. Mol Gen Genet, 1988 Apr, 212(1), 1 - 5 Nucleotide sequence of the cybB gene encoding cytochrome b561 in Escherichia coli K12; Nakamura H et al.; The complete nucleotide sequence of the Escherichia coli cybB gene for diheme cytochrome b561 and its flanking region was determined . The cybB gene comprises 525 nucleotides and encodes a 175 amino acid polypeptide with a molecular weight of 20,160 . From its deduced amino acid sequence, cytochrome b561 is predicted to be very hydrophobic (polarity 33.7%) and to have three membrane spanning regions . Histidines, canonical ligand residues for protohemes, are localized in these regions, and the heme pockets are thought to be in the cytoplasmic membrane . No significant homology of the primary structure of cytochrome b561 with those of other bacterial b-type cytochromes was observed. DNA, 1988 Apr, 7(3), 193 - 201 Cloning and expression in Escherichia coli of the gene for mouse tumor necrosis factor; Shirai T et al.; The mouse tumor necrosis factor (TNF) gene was isolated from a mouse genomic library . The entire sequence of the gene was determined using both an automated DNA sequencer with improved primer extension reaction conditions as well as the standard radioisotopic method . Comparison of the nucleotide sequence of the gene with that of mouse TNF cDNA showed that the mouse gene consists of four exons, like rabbit and human TNF genes . There is strong nucleotide sequence homology in the 5'-flanking region among the mouse, rabbit, and human TNF genes, suggesting that the mechanisms regulating TNF gene expression are highly conserved . Direct expression of mature mouse TNF was achieved using a plasmid constructed by site-directed mutagenesis . Purified mouse TNF produced in Escherichia coli showed cytotoxicity to mouse L cells. DNA, 1988 Apr, 7(3), 173 - 9 Effect of ribosome binding site on gene expression in Escherichia coli; Curry KA et al.; Using the expression of human renin gene in Escherichia coli as a model, we have observed that a distance of 7-10 nucleotides between the Shine-Dalgarno site to the initiation codon ATG is optimal for translation initiation . The sequence ATA as the triplet preceding the ATG gives the best expression among those tested . These observations agree with the statistical bias observed for the genes of E . coli and its phages . We have also compared gene expression with three different Shine-Dalgarno sites . Expression of a given gene can be increased by as much as 1000-fold with slight modifications in the Shine-Dalgarno sequence. Genetics, 1988 Apr, 118(4), 561 - 70 Different reading frames are responsible for IS1-dependent deletions and recombination; Braedt G; Two classes of plasmids in addition to the parent become apparent when plasmids that contain direct repeats of IS1 . One class of plasmids has deleted sequences from the end of IS1 to nonrandom sites within the plasmid . The appearance of these plasmids in the population requires intact insA and insB reading frames, but not insC . The other class of plasmids has undergone an exchange within the direct repeats of IS1 on the plasmid . Their appearance requires InsC but neither InsA nor InsB . The two reactions may represent two distinguishable steps in IS1 transposition . The InsC-catalyzed exchange is independent of RecA and resembles hologous recombination since the frequency of recombinants arising from exchanges in different regions of IS1 appears to be roughly proportional to the size of the region . InsC can also catalyze an exchange between direct repeats of non-IS1 DNA. Leukemia, 1988 Apr, 2(4), 211 - 5 Binding of iodinated recombinant human GM-CSF to the blast cells of acute myeloblastic leukemia; Kelleher CA et al.; Granulocyte/macrophage-colony-stimulating factor (GM-CSF) is an effective growth factor for the blasts of acute myeloblastic leukemia (AML) . Radioiodinated Chinese hamster ovary (CHO)-cell derived GM-CSF was prepared using Bolton-Hunter reagent to label free amino groups on the protein . Normal human neutrophils and the blast cells from AML patients were examined for binding . We found that there were fewer receptors of higher affinity on blast cells compared with neutrophils . After brief culture in suspension, receptor number increased and affinity decreased . Experiments provided evidence that GM-CSF from Escherichia coli had a higher affinity for neutrophils (kd = 20 pM) than the CHO-cell derived protein (kd = 500 pM-1 nM) . This difference was reflected in the increased effectiveness of the E . coli protein over the CHO protein to stimulate colony formation in both normal bone marrow cells and AML blasts. Proc Natl Acad Sci U S A, 1988 Apr, 85(8), 2563 - 7 A unique deoxyguanosine triphosphatase is responsible for the optA1 phenotype of Escherichia coli; Beauchamp BB et al.; Escherichia coli optA1, a mutant unable to support the growth of T7 phage containing mutations in gene 1.2, contains reduced amounts of dGTP . Extracts of E . coli optA1 catalyze the hydrolysis of dGTP at a rate 50-fold greater than do extracts of E . coli optA+ . The dGTPase responsible for the increased hydrolysis has been purified to apparent homogeneity . Purification of the protein is facilitated by its high affinity for single-stranded DNA . By using this purification scheme an identical dGTPase has been purified from E . coli optA+ . The purified proteins catalyze the hydrolysis of dGTP to yield deoxyguanosine and tripolyphosphate . The products of hydrolysis, chromatographic properties, denatured molecular mass of 56 kDa, N-terminal amino acid sequence, substrate specificity, and heat inactivation indicate that the proteins purified from optA1 and from optA+ cells are identical and identify the enzyme as the deoxyguanosine 5'-triphosphate triphosphohydrolase purified to homogeneity from wild-type E . coli {Seto, D., Bhatnagar, S . K . & Bessman, M . J . (1988) J . Biol . Chem . 263, 1494-1499} . OptA1 cells contain approximately equal to 50-fold more active molecules of the 56-kDa dGTPase than do E . coli optA+ cells. Virology, 1988 Apr, 163(2), 330 - 40 A poliovirus mutant defective for self-cleavage at the COOH-terminus of the 3C protease exhibits secondary processing defects; Kean KM et al.; By in vitro recombination between the wild-type full-length infectious cDNA of poliovirus and a clone generated by the construction of a cDNA bank from a chemically derived temperature-sensitive plurimutant, we obtained a mutant cDNA with a T to C change at nucleotide 5658 . This mutation replaces the isoleucine at residue 74 of the viral protease 3C by a threonine . The mutant virus recovered after transfection exhibited a small-plaque phenotype, and was deficient for viral RNA synthesis . Both these defects were more marked at 39 than at 37 degrees . The mutation was introduced into a bacterial plasmid which expresses the 3C protease along with its flanking autocatalytic cleavage sites . Analysis of the cleavage products expressed in Escherichia coli provided direct evidence that the modification impaired cleavage at the COOH-terminus of 3C . Cleavage at this same site was partially defective in mutant virus-infected HeLa cells, reducing the production of mature 3C and the viral replicase, 3D . Cleavage of P1, the precursor to the capsid polypeptides, was apparently unaffected by this defect, whereas cleavage events within the P2 region of the genome occurred inefficiently . This is indicative of differential strategies for 3C-specific cleavage events in vivo. Proc Natl Acad Sci U S A, 1988 Apr, 85(7), 2224 - 8 Mutational analysis of insertion sequence 50 (IS50) and transposon 5 (Tn5) ends; Makris JC et al.; Insertion sequence 50 (IS50) transposition utiliz