Science, 1984 Dec 7, 226(4679), 1161 - 5 Characterization and molecular cloning of a human parvovirus genome; Cotmore SF et al.; The genome of the small human virus serologically associated with erythrocyte aplasia and erythema infectiosum (fifth disease) is shown to be a linear, nonpermuted, single-stranded DNA molecule with self-priming hairpin termini, properties which are characteristic of the genomes of the family Parvoviridae . This human parvovirus chromosome was molecularly cloned into bacterial plasmid vectors and the cloned DNA was used to explore its relatedness to other mammalian parvovirus serotypes by DNA:DNA hybridization . It is not related to the human adeno-associated viruses but does show a distant evolutionary relationship to genomes of the helper-independent parvoviruses of rodents . This strongly suggests that it is an autonomous parvovirus, and as such is the first example of a member of this group of common animal pathogens to cause disease in man. Biochim Biophys Acta, 1984 Dec 6, 796(3), 373 - 83 Escherichia coli membrane vesicles with elevated phosphatidic acid levels . A detergent-free system for in vitro phospholipid synthesis; Sparrow CP et al.; A new method for studying phospholipid biosynthesis in Escherichia coli is described . The method makes use of the previously reported observation that E . coli cytidine auxotrophs accumulate phosphatidic acid when starved of cytidine (Ganong, B . and Raetz, C.R.H . (1982) J . Biol . Chem . 257, 389-394) . We now show that phosphatidic acid that accumulates in these cells is competent for further biosynthetic use in vivo, if cytidine is re-supplied to the cells . Furthermore, phosphatidic acid-rich membranes prepared from such cells can be used for in situ assays of the later steps of phospholipid biosynthesis . Since this system does not require detergent, our in situ assays more accurately reflect the conditions of an intact membrane . We have used this system to probe the regulation of the branch-point of the biosynthetic pathway for phospholipid polar headgroups . Phosphatidic acid-rich membranes prepared from cells that overproduce either phosphatidylserine synthase or phosphatidylglycerolphosphate synthase do not have increased rates of lipid synthesis in our in situ assays . This correlates with synthetic rates measured in vivo and, thus, our in situ assays accurately reflect conditions in a growing cell's membrane. J Mol Biol, 1984 Dec 5, 180(2), 371 - 7 Recombinational hotspot activity of Chi-like sequences; Cheng KC et al.; Chi sites, consisting of the nucleotide octamer 5' G-C-T-G-G-T-G-G 3', stimulate coliphage lambda recombination mediated by the Escherichia coli RecBC recombination pathway . In a sensitive genetic assay using phage lambda crosses, three of four Chi-like sequences tested, namely 5' A-C-T-G-G-T-G-G 3', 5' G-T-T-G-G-T-G-G 3' and 5' G-C-T-A-G-T-G-G 3', had about 6%, 11% and 38% of full Chi activity, respectively . We conclude that certain Chi-like sequences manifest a spectrum of recombinational hotspot activities and may account for RecBC-mediated generalized recombination of lambda lacking Chi sites. J Mol Biol, 1984 Dec 5, 180(2), 267 - 82 The mini-F primary origin . Sequence analysis and multiple activities; Lane D et al.; The sequence of an 897 base-pair fragment (42.1 to 43.0 kilobase co-ordinates on the F genetic map) containing the primary origin (ori-1) of mini-F replication has been determined . It contains one significant open reading frame, which probably codes for part of the C protein thought to be necessary for ori-1 replication activity . Tests of the ability of the sequenced ori-1 region to direct replication of DNA polymerase I-dependent replicons revealed that ori-1 replication requires adjacent mini-F sequences, 43.0 to 43.9 kilobase co-ordinates on the F genetic map in cis as well as a trans-acting gene product, probably the E protein, from the essential replication region of mini-F . In addition, a sequence required for control of pif gene expression has been mapped to a 160 base-pair region immediately upstream from the C (pifC) gene, and the crossover site of a specific recA-independent recombination mechanism has been mapped to a 220 base-pair region on the side of the pif control sequence distal to the C gene. J Mol Biol, 1984 Dec 5, 180(2), 239 - 50 Yeast regulatory gene PPR1 . I . Nucleotide sequence, restriction map and codon usage; Kammerer B et al.; The PPR1 gene of Saccharomyces cerevisiae controls the transcription of two unlinked structural genes URA1 and URA3 . The primary structure of this eukaryotic regulatory gene and its flanking regions has been established by the dideoxynucleotide chain termination method . Our data show an open reading frame of 2712 nucleotides, corresponding to 904 amino acid residues . The 3' untranslated messenger RNA region presents consensus yeast termination and polyadenylation sequences . The pattern of codon usage in the gene is clearly random . This result is discussed in relation to protein abundance and is compared with the codon usage in 20 yeast structural and regulatory genes and with that found for Escherichia coli genes. Biochemistry, 1984 Dec 4, 23(25), 6046 - 52 Trinitrobenzenesulfonate modification of the lysine residues in lactose repressor protein; Whitson PA et al.; Modification of the lysine residues in the lactose repressor protein has been carried out with trinitrobenzenesulfonate . Reaction of lysine residues at positions 33, 37, 108, 290, and 327 was observed . Inducer binding was increased by modification with this reagent, while both nonspecific DNA binding and operator DNA binding were diminished, although to differing degrees . The loss in operator DNA binding capacity was complete with modification of approximately 2 equiv of lysine per monomer . The extent of reaction was affected by the presence of both sugar and DNA ligands; binding activities of the modified protein and reaction pattern of the lysines were perturbed by these ligands . The presence of operator or nonspecific DNA during the reaction protected against specific and nonspecific DNA binding activity loss . This protection presumably occurs by steric restriction of reagent access to lysine residues which are essential for both nonspecific and operator binding interactions . Lysines-33 and -108 were protected from modification in the presence of DNA . These experiments suggest that the charge on the lysine residues is important for protein interaction with DNA and that steric constraints for operator DNA interaction with the protein are more restrictive than for nonspecific DNA binding . In contrast, inducer (isopropyl beta-D-thiogalactoside) presence partially protected lysine-290 from modification while significantly enhancing reaction at lysine-327 . Conformational alterations consequent to inducer binding are apparently reflected in these altered lysine reactivities. Biochemistry, 1984 Dec 4, 23(25), 6263 - 75 Mechanisms of enzymatic and acid-catalyzed decarboxylations of prephenate; Hermes JD et al.; The prephenate dehydrogenase activity of the bifunctional enzyme chorismate mutase-prephenate dehydrogenase from Escherichia coli catalyzes the oxidative decarboxylation of both prephenate and deoxoprephenate, which lacks the keto group in the side chain (V 78% and V/K 18% those of prephenate) . Hydride transfer is to the B side of NAD, and the acetylpyridine and pyridinecarboxaldehyde analogues of NAD have V/K values 40 and 9% and V values 107 and 13% those of NAD . Since the 13C isotope effect on the decarboxylation is 1.0103 with deuterated and 1.0033 with unlabeled deoxoprephenate (the deuterium isotope effect on V/K is 2.34), the mechanism is concerted, and if CO2 has no reverse commitment, the intrinsic 13C and deuterium isotope effects are 1.0155 (corresponding to a very early transition state for C-C bond cleavage) and 7.3, and the forward commitment is 3.7 . With deoxodihydroprephenate (lacking one double bond in the ring), oxidation occurs without decarboxylation, and one enantiomer has a V/K value 23-fold higher than the other (deuterium isotope effects are 3.6 and 4.1 for fast and slow isomers; V for the fast isomer is 5% and V/K 0.7% those of prephenate) . The fully saturated analogue of deoxoprephenate is a very slow substrate (V 0.07% and V/K approximately 10(-5%) those of prephenate) . pH profiles show a group with pK = 8.3 that must be protonated for substrate binding and a catalytic group with pK = 6.5 that is a cationic acid (likely histidine) . This group facilitates hydride transfer by beginning to accept the proton from the 4-hydroxyl group of prephenate prior to the beginning of C-C cleavage (or fully accepting it in the oxidation of the analogues with only one double bond or none in the ring) . In contrast with the enzymatic reaction, the acid-catalyzed decarboxylation of prephenate and deoxoprephenate (t1/2 of 3.7 min at low pH) is a stepwise reaction with a carbonium ion intermediate, since 18O is incorporated into substrate and its epi isomer during reaction in H218O . pH profiles show that the hydroxyl group must be protonated and the carboxyl (pK approximately 4.2) ionized for carbonium ion formation . The carbonium ion formed from prephenate decarboxylates 1.75 times faster than it reacts with water (giving 1.8 times as much prephenate as epi isomer) . The observed 13C isotope effect of 1.0082 thus corresponds to an intrinsic isotope effect of 1.023, indicating an early transition state for the decarboxylation step . epi-Prephenate is at least 20 times more stable to acid than prephenate because it exists largely as an internal hemiketal.(ABSTRACT TRUNCATED AT 400 WORDS) Biochemistry, 1984 Dec 4, 23(25), 6240 - 9 Chorismate mutase/prephenate dehydrogenase from Escherichia coli K12: purification, characterization, and identification of a reactive cysteine; Hudson GS et al.; The bifunctional enzyme involved in tyrosine biosynthesis, chorismate mutase/prephenate dehydrogenase, has been isolated from extracts of a regulatory mutant of Escherichia coli K12 . The pure enzyme is a homodimer of total molecular weight 78 000 and displays Michaelis-Menten kinetics for both activities . Fingerprinting and amino acid sequencing of tryptic and thermolytic peptides of the S-{14C}carboxymethylated enzyme allowed the identification of three unique cysteine-containing sequences per subunit . Chemical modification of the native enzyme with 5,5'-dithiobis(2-nitrobenzoate) or iodoacetamide showed that one sulfhydryl group per subunit was particularly reactive, and the integrity of this group was essential for both enzymic activities . This work supports previous proposals for a close spatial relationship between the active sites. Biochemistry, 1984 Dec 4, 23(25), 6228 - 34 Interactions of tryptophan synthase, tryptophanase, and pyridoxal phosphate with oxindolyl-L-alanine and 2,3-dihydro-L-tryptophan: support for an indolenine intermediate in tryptophan metabolism; Phillips RS et al.; We have examined the interaction of tryptophan synthase and tryptophanase with the tryptophan analogues oxindolyl-L-alanine and 2,3-dihydro-L-tryptophan . Since these analogues have tetrahedral geometry at carbon 3 of the heterocyclic ring, they are structurally similar to the indolenine tautomer of L-tryptophan, a proposed intermediate in reactions of L-tryptophan . Oxindolyl-L-alanine and 2,3-dihydro-L-tryptophan are potent competitive inhibitors of both tryptophan synthase and tryptophanase, with KI values (3-17 microM) 10-100-fold lower than the corresponding Km or KI values for L-tryptophan . Addition of oxindolyl-L-alanine or 2,3-dihydro-L-tryptophan to solutions of the alpha 2 beta 2 complex of tryptophan synthase results in new absorption bands at 480 or 494 nm, respectively, which are ascribed to a quinonoid or alpha-carbanion intermediate . Spectrophotometric titration data give half-saturation values of 5 and 25 microM, which are comparable to the KI values obtained in kinetic experiments . Our finding that both enzymes catalyze incorporation of tritium from 3H2O into oxindolyl-L-alanine is evidence that both enzymes form alpha-carbanion intermediates with oxindolyl-L-alanine . These results support the proposal that the indolenine tautomer of L-tryptophan is an intermediate in reactions catalyzed by both tryptophanase and tryptophan synthase . In addition, we have found that oxindolyl-L-alanine reacts irreversibly with free pyridoxal phosphate to form a covalent adduct. Biochemistry, 1984 Dec 4, 23(25), 6210 - 6 Construction of DNA sequences complementary to rat alpha 1 and alpha 2 collagen mRNA and their use in studying the regulation of type I collagen synthesis by 1,25-dihydroxyvitamin D; Genovese C et al.; Type I collagen mRNA from fetal rat calvaria was used as a template for the synthesis of a cDNA that was subsequently inserted in the PstI site of the plasmic vector pBR322 and cloned . Three recombinant plasmids containing type I collagen specific sequences were characterized: p alpha 1R1 is 1600 bp and spans approximately 500 amino acid residues within the triple helical region of alpha 1(I) and p alpha 1R2 is 900 bp in size and covers the entire 3' noncoding and about half of the C-terminal propeptide region of alpha 1(I) collagen mRNA . The third recombinant p alpha 2R2 is 1500 bp and contains alpha 2(I) sequences specific for the entire 3' noncoding and C-terminal propeptide region . Partial nucleic acid sequence data revealed that the decreasing order of amino acid and nucleotide homology to similar regions of the rat cDNA was mouse greater than human greater than chick . Northern hybridization of mRNA after electrophoresis in 0.8% agarose revealed two distinctly different molecular weight patterns characteristic of alpha 1(I) (4.7 and 5.7 kb) and alpha 2(I) (4.2 and 4.5 kb) collagen mRNA when hybridized with the corresponding cDNA probe . Despite the high degree of sequence homology, DNA probes from rat or human produced a significantly reduced hybridization signal when used as an interspecies hybridization probe than to its corresponding mRNA . The rat cDNA probes were used in a dot hybridization assay to measure the type I collagen mRNA content in the fetal rat calvaria.(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1984 Dec 4, 23(25), 6178 - 84 Processing of preproteins by liposomes bearing leader peptidase; Ohno-Iwashita Y et al.; Procoat, the precursor form of M13 coat protein, assembles into sealed liposomes bearing only internally oriented leader peptidase and is processed to yield transmembrane coat protein {Ohno-Iwashita, Y., & Wickner, W . (1983) J . Biol . Chem . 258, 1895-1900} . The precursors of maltose-binding protein and of outer membrane protein A (OmpA) are also processed by these liposomes, showing that these preproteins can at least partially insert across a lipid bilayer . The ability to insert into a bilayer may be a general property of preproteins . The cleavage products, mature OmpA and maltose-binding protein, are not sequestered within the liposomes, suggesting that an additional factor(s) is (are) required for complete translocation . Liposomes were also prepared with leader peptidase in a more physiological, membrane-spanning orientation . These liposomes were also active in the cleavage of externally added procoat, pro-OmpA, and pre maltose-binding protein, though the mature OmpA and maltose-binding protein were still not sequestered within the liposomes . Pretreatment of these liposomes with trypsin cleaved near the amino terminus of the leader peptidase, inactivating the enzyme . The function of this amino-terminal domain, on the opposite side of the membrane from the catalytic domain, is unknown. Biochemistry, 1984 Dec 4, 23(25), 6171 - 8 Elongation factor Tu ternary complex binds to small ribosomal subunits in a functionally active state; Langer JA et al.; A complex between elongation factor Tu (EF-Tu), GTP, phenylalanyl-tRNA (Phe-tRNA), oligo(uridylic acid) {oligo(U)}, and the 30S ribosomal subunit of Escherichia coli has been formed and isolated . Binding of the EF-Tu complex appears to be at the functionally active 30S site, by all biochemical criteria that were examined . The complex can be isolated with 0.25-0.5 copy of EF-Tu bound per ribosome . The binding is dependent upon the presence of both the aminoacyl-tRNA and the cognate messenger RNA . Addition of 50S subunits to the preformed 30S-EF-Tu-GTP-Phe-tRNA-oligo(U) complex ("30S-EF-Tu complex") causes a rapid hydrolysis of GTP . This hydrolysis is coordinated with the formation of 70S ribosomes and the release of EF-Tu . Both the release of EF-Tu and the hydrolysis of GTP are stoichiometric with the amount of added 50S subunits . 70S ribosomes, in contrast to 50S subunits, neither release EF-Tu nor rapidly hydrolyze GTP when added to the 30S-EF-Tu complexes . The inability of 70S ribosomes to react with the 30S-EF-Tu complex argues that the 30S-EF-Tu complex does not dissociate prior to reaction with the 50S subunit . The requirements of the 30S reaction for Phe-tRNA and oligo(U) and the consequences of the addition of 50S subunits resemble the reaction of EF-Tu with 70S ribosomes, although EF-Tu binding to isolated 30S subunits does not occur during the elongation microcycle . This suggests that the EF-Tu ternary complex binds to isolated 30S subunits at the same 30S site that is occupied during ternary complex interaction with the 70S ribosome.(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1984 Dec 4, 23(25), 6132 - 7 Escherichia coli single-stranded DNA binding protein is mobile on DNA: 1H NMR study of its interaction with oligo- and polynucleotides; Romer R et al.; The interaction of the Escherichia coli single-stranded DNA binding protein (SSB) with oligo- and poly-nucleotides has been studied by 270-MHz 1H NMR spectroscopy and fast kinetic techniques . d(pT)8 and poly(dT) were used to study noncooperative and cooperative binding, respectively . The H6, H1', and CH3 resonances of d(pT)8 are high-field shifted by less than 0.05 ppm, and H8 and H2 of poly(dA) are low-field shifted upon complexation . The protein resonances remain virtually unshifted . The small shifts upon complexation provide no evidence for extensive stacking interactions between the nucleotide bases and aromatic amino acid side chains of SSB . The d(pT)8 and poly(dA) signals are broadened to about 30 Hz whereas the resonances of poly(dT) are broadened beyond detection upon stoichiometric complexation . Continuous broadening of all poly(dT) signals even at a 10-fold excess of poly(dT) indicates fast exchange of SSB between different binding sites . Dissociation and reassociation rates determined from stopped-flow experiments are too slow by at least 2 orders of magnitude to account for the experimental line widths . Therefore, we conclude that SSB translocates without dissociation from the DNA template . A model for the translocation is outlined . It is based on partial dissociation of octamer sections of poly(dT) from the complex with a rate constant as previously published for the dissociation of d(pT)8 from SSB. Eur J Biochem, 1984 Dec 3, 145(2), 403 - 11 Phosphorylation in vivo and in vitro of the arginine-ornithine periplasmic transport protein of Escherichia coli; Celis RT; The arginine-ornithine periplasmic binding protein, an essential component of the arginine-ornithine transport system of Escherichia coli, was isolated in a phosphorylated form and in a non-phosphorylated form from the periplasmic fluid, after incubation of intact cells with (32P)orthophosphate under conditions similar to those used for arginine transport studies . The binding protein could also be labeled with 32Pi by incubation in vitro of the periplasmic fluid with {gamma-32P}ATP, or by incubation in vitro of the purified binding protein with radioactive ATP, Mg2+ and a phosphokinase enzyme released by osmotic-shock treatment . The two forms of the protein were separated by DEAE-Sephacel chromatography . By several different criteria, which included binding studies, analyses of the amino acid composition of the two forms of the protein, analysis by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and testing for other components of the periplasmic space with affinities for inorganic phosphate, it was concluded that the 32P-labeled protein corresponds to a phosphorylated form of the arginine-ornithine-binding protein . The phosphorylation reaction required Mg2+ and a phosphokinase from the periplasmic fluid . The dissociation constant of the phosphorylated protein for arginine was 5.0 microM (dissociation constant of the unmodified protein equals 0.1 microM), suggesting that the chemically modified protein is the active form of the molecule which releases the ligand for its translocation through the cytoplasmic membrane . The pH-stability profile of the phosphoprotein has a 'U'-shape characteristic of acyl phosphates . Reaction of the phosphorylated binding protein with hydroxylamine at pH 5.4, also released Pi from the phosphoprotein . These properties suggest that the phosphoryl group of the phosphoprotein is linked covalently to a carboxyl function of the protein . This information indicates that ATP is a direct energy donor for the active transport of arginine and ornithine in E . coli, and a step of phosphorylation of the arginine-ornithine-binding protein appears to be involved in the utilization of the phosphate bond energy by the arginine-ornithine transport system. Eur J Biochem, 1984 Dec 3, 145(2), 397 - 402 Beta-D-galactoside transport in Escherichia coli . Solubilization in organic solvent and reconstitution of binding; Konig B et al.; The beta-D-galactoside transport protein (y-gene product) of Escherichia coli, strain T 206, was solubilized in 85-95% yield using the organic solvents hexamethylphosphoric triamide at pH 7.5 or butan-1-ol at pH 4.2 . The transport protein obtained with the former solvent could be incorporated into a defined lipid/protein aggregate of density 1.12 g/ml, but no beta-D-galactoside binding was restored . Diacylglycerol kinase regained activity in the same lipid/protein aggregates . In control experiments, liposomes formed from hexamethylphosphoric triamide were found to be active in the valinomycin-mediated uptake of Rb+ ions . beta-D-Galactoside binding (3.6-5.7 nmol/mg protein) as well as diacylglycerol kinase activity {7 nmol min-1 (mg protein)-1} was reconstituted into proteoliposomes from butan-1-ol solution by adaptation of a published procedure {Wright, J . K . et al . (1982) Eur . J . Biochem . 124, 545-552} . A microparticulate nature of the butan-1-ol-solubilized transport protein could be excluded by gel permeation chromatography on a newly synthesized matrix, hydroxypropyl-Sephacryl S-300. Proc Natl Acad Sci U S A, 1984 Dec, 81(23), 7651 - 5 Molecular cloning and sequence determination of rat preproenkephalin cDNA: sensitive probe for studying transcriptional changes in rat tissues; Howells RD et al.; A cDNA probe was prepared to investigate the regulation of proenkephalin biosynthesis in the rat . This was necessary because human and bovine proenkephalin cDNA were not sensitive enough for the accurate detection of preproenkephalin mRNA in tissues that contain low copy numbers of this message, such as the adrenal gland . The rat probe was prepared in the following manner . Preproenkephalin mRNA was enriched by sucrose gradient centrifugation of poly(A)-containing mRNA from rat brain and was used as a template for double-stranded cDNA synthesis . The resulting cDNA was inserted into the plasmid pBR322, and recombinant plasmids were used to transform Escherichia coli RR1 cells . A synthetic oligodeoxyribonucleotide (30 bases long) with a sequence that had previously been shown to be identical in bovine and human preproenkephalin cDNA was prepared to screen the clone bank . The plasmid with the longest cDNA insert (about 1200 bases) from the positive clones was isolated, and the sequence of the entire protein coding region was determined . Like the bovine and human gene products, rat preproenkephalin contains four {Met}enkephalin sequences and one copy each of {Leu}enkephalin, {Met}enkephalin-Arg6-Gly7-Leu8, and {Met}enkephalin-Arg6-Phe7 . Rat preproenkephalin is 80% and 83% homologous to the bovine and human forms, respectively, at the nucleotide level and is 82% homologous to both species at the amino acid level . Rat preproenkephalin contains 269 amino acid residues, making it larger than the human (267 residues) and bovine (263 residues) precursors . The sensitivity for detection of rat preproenkephalin mRNA with the rat cDNA was several times greater than with the corresponding cDNAs from bovine and human sources. Mol Cell Biol, 1984 Dec, 4(12), 2876 - 82 cDNA cloning and in vitro transcription of the complete brome mosaic virus genome; Ahlquist P et al.; Complete cDNA copies of each of the brome mosaic virus genomic RNAs (3.2, 2.8, and 2.1 kilobases in length) were cloned in a novel transcription vector, pPM1, designed to provide exact control of the transcription initiation site . After cleavage at a unique EcoRI site immediately downstream of the inserted cDNA, these clones can be transcribed in vitro by Escherichia coli RNA polymerase to yield complete copies of the brome mosaic virus RNAs . Dideoxy sequencing of 5' transcript cDNA runoff products and direct sequencing of 32P-3'-end-labeled transcripts show that such transcripts initiate at the same 5' position as natural viral RNA and terminate within the EcoRI runoff site after copying the entire viral RNA sequence . When synthesized in the presence of m7GpppG, the transcripts bear the natural capped 5' terminus of brome mosaic virus RNAs . Such transcripts direct the in vitro translation of proteins which coelectrophorese with the translation products of natural brome mosaic virus RNAs . pPM1 should facilitate in vitro production of other viral and nonviral RNAs. J Appl Bacteriol, 1984 Dec, 57(3), 531 - 2 A note on xenogeneic transfusion of macrophages in experimental septicaemia; Fakhri O et al.; Human peritoneal macrophages (M phi) were given intravenously to normal rabbits and rabbits with Escherichia coli septicaemia . No harmful effects were observed during a period of three months following macrophage transfusions and the septicaemia was eradicated in four of five animals . Thus xenogeneic M phi transfusion is feasible and can combat serious bacterial infections. Gene, 1984 Dec, 32(3), 487 - 92 Cloning gene ura5 for the orotidylic acid pyrophosphorylase of the filamentous fungus Podospora anserina: transformation of protoplasts; Begueret J et al.; From a genomic library of the filamentous fungus Podospora anserina, we have cloned a 4.9-kb fragment which complements an Escherichia coli mutant strain deficient for orotidylic acid pyrophosphorylase (pyrE gene) . The recombinant plasmid pPAura5 also transforms to prototrophy a mutant strain of P . anserina carrying a mutation in the ura5 gene and lacking OMPppase activity. Mol Biol Rep, 1984 Dec, 10(2), 99 - 104 Double-stranded RNA specific nuclease from germinating embryos of Pennisetum typhoides; Maran A et al.; A double-stranded RNA specific nuclease (ds RNase) has been purified from the pearl millet Pennisetum typhoides . The purification involved S-30 preparation from the germinating embryos, DEAE-cellulose and DNA-cellulose chromatography . The partially pure enzyme preferentially solubilized the synthetic double-stranded polynucleotide {3H}poly(rA) . poly(rU); the degradation of {3H}poly(rC) was fourteen fold lower under the same assay conditions . Furthermore, the ds RNase activity was inhibited to an extent of 58% by ethidium bromide, which is known to intercalate with double-stranded RNAs . Active sulfhydryl groups were found to be necessary for the ds RNase activity since the enzyme action was inhibited by N-ethylmaleimide . Ethidium bromide and N-ethyl-maleimide did not significantly inhibit the ss RNase activity . In contrast, diethyl pyrocarbonate inhibited ss RNase activity completely and ds RNase by 58% . Heating the enzyme for 20 min at 50 degrees C resulted in drastic loss of both enzyme activities . The ds RNase showed maximum activity in the pH range of 6.5 to 7.5 . The enzyme acts in vitro on E . coli 30S precursor ribosomal RNA and the cleavage products migrated in the region of mature 23S and 16S rRNAs. Nippon Geka Gakkai Zasshi, 1984 Dec, 85(12), 1558 - 69 {Replacement of portal vein with E-PTFE vascular graft; experimental study on process of the endothelialization}; Ohkuma T; Expanded polytetrafluoroethylene (E-PTFE) vascular graft was been exposed to intestinal content and evaluated the process of endothelialization and as to whether or not the material was feasible for its use under condition similar to clinical pancreatic surgery . In group A, E-PTFE tubular graft was inserted into the portal vein defect . In group B, choledochojejunostomy was also performed combined with the E-PTFE graft interposition . In other groups, bile (group C) or suspension of Escherichia Coli (group D) was put on graft implanted in the portal vein . The grafts were removed from the dogs for histological and scanning electronmicroscopic evaluation at intervals varying from 12 hours to 4 years and 8 months after surgery . Thirty one of 38 grafts were patent (patency rate; 82%) . Complete formation of the inner capsule of the graft was essential to endothelialization . Direct extension of the endothelium was seen from the anastomotic site . Sporadic endothelialization was observed, however, communications among islet formed endothelialized portions . It were remarkable and endothelium derived from the host vein . These results demonstrated the feasibleness of clinical application of the E-PTFE graft to portal vein reconstruction with combination of the digestive surgery. Arch Biochem Biophys, 1984 Dec, 235(2), 562 - 70 Thylakoid membranes: the translational site of chloroplast DNA-regulated thylakoid polypeptides; Minami E et al.; Stromal ribosomes and those bound to thylakoid membranes were prepared from intact spinach chloroplasts which were purified on Percoll gradients . The products of read-out translation of these ribosomes supplemented with an Escherichia coli extract were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis . Striking similarity was found between the polypeptides labeled in the read-out translation of the chloroplastic ribosomes and those synthesized in isolated chloroplasts . Among the polypeptides translated on thylakoid-bound ribosomes, apoprotein of chlorophyll-protein complex I, alpha and beta subunits of coupling factor 1, and 32,000-Da membrane polypeptide were identified from their mobility on the polyacrylamide gel . The large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase and other several stromal proteins were translated exclusively from stromal ribosomes . However, when the translation was programmed in cell-free systems from either E . coli, wheat germ, or rabbit reticulocytes by RNAs isolated separately from stroma and thylakoids, no qualitative difference was found between the products from those RNAs . These results suggest that thylakoid-bound ribosomes are the main sites of synthesis of thylakoid proteins and stromal-free ribosomes are that of stromal proteins, and that thylakoids and stroma contain mRNAs for the stromal and the thylakoid proteins, respectively, in a form not functioning in the chloroplasts. Clin Exp Immunol, 1984 Dec, 58(3), 737 - 44 A functional comparison of IIIindium-labelled elicited peripheral blood neutrophils and peritoneal neutrophils in the rat; Savige JA et al.; A functional comparison between elicited peripheral blood neutrophils has been made in vivo and in vitro . Preliminary experiments showed that separation of peripheral blood cells on a metrizamide gradient yielded too few neutrophils for efficient radiolabelling with indium (In): hence a mixed cell preparation comprising 80% neutrophils was elicited in the peripheral blood of adult male rats by the administration of endotoxin (0.25 mg i.a.) and cobra venom factor (200 microliter i.p.) 20 h before . Peritoneal neutrophils were collected 4 h after the i.p . injection of 6 ml thioglycollate . Both populations differed markedly from normal peripheral neutrophils on the in vitro testing of random locomotion, chemotaxis and phagocytosis of Candida . After labelling with IIIIn-tropolonate, a greater proportion (mean = 8%) of peripheral blood cells localized to an E . coli/Freund's complete adjuvant-induced abscess compared with peritoneal neutrophils (mean = 3%) . The abscess could be visualized externally by scanning with both cell preparations, but the distribution of activity differed markedly . The greater hepatic sequestration of peritoneal neutrophils suggested cell damage or activation . To overcome the difficulty of harvesting normal peripheral blood neutrophils in the rat, either of these populations can be used to follow the kinetics of inflammation . However, elicited peripheral blood cells yield a higher proportion of responding cells. J Cell Biol, 1984 Dec, 99(6), 1936 - 43 Legionella pneumophila inhibits acidification of its phagosome in human monocytes; Horwitz MA et al.; We used quantitative fluorescence microscopy to measure the pH of phagosomes in human monocytes that contain virulent Legionella pneumophila, a bacterial pathogen that multiplies intracellularly in these phagocytes . The mean pH of phagosomes that contain live L . pneumophila was 6.1 in 14 experiments . In the same experiments, the mean pH of phagosomes containing dead L . pneumophila averaged 0.8 pH units lower than the mean pH of phagosomes containing live L . pneumophila, a difference that was highly significant (P less than 0.01 in all 14 experiments) . In contrast, the mean pH of phagosomes initially containing live E . coli, which were then killed by monocytes, was the same as for phagosomes initially containing dead E . coli . The mean pH of L . pneumophila phagosomes in activated monocytes, which inhibit L . pneumophila intracellular multiplication, was the same as in nonactivated monocytes . To simultaneously measure the pH of different phagosomes within the same monocyte, we digitized and analyzed fluorescence images of monocytes that contained both live L . pneumophila and sheep erythrocytes . Within the same monocyte, live L . pneumophila phagosomes had a pH of approximately 6.1 and sheep erythrocyte phagosomes had a pH of approximately 5.0 or below . This study demonstrates that L . pneumophila is capable of modifying the pH of its phagocytic vacuole . This capability may be critical to the intracellular survival and multiplication of this and other intracellular pathogens. J Biochem (Tokyo), 1984 Dec, 96(6), 1727 - 35 Preparation of a monomeric 2,4-dinitrophenyl Fab'-beta-D-galactosidase conjugate for immunoenzymometric assay; Imagawa M et al.; A method is described for the preparation of a monomeric Fab'-beta-D-galactosidase conjugate, which is required for the development of a sensitive immunoenzymometric assay . Anti-human IgG F(ab')2 was labeled with 2,4-dinitrophenyl groups, split into Fab' by reduction and reacted with excess maleimide groups which had been introduced into beta-D-galactosidase through thiol groups using N,N'-o-phenylenedimaleimide . The monomeric 2,4-dinitrophenyl Fab'-beta-D-galactosidase conjugate was subsequently separated from unconjugated beta-D-galactosidase by affinity chromatography on a column of (anti-2,4-dinitrophenyl) IgG-Sepharose 4B . In the monomeric conjugate preparation, 98% of beta-D-galactosidase activity was associated with Fab' and 90% was associated with specific (anti-human IgG) Fab' . This conjugate allowed the measurement of 0.1 fmol of human IgG by an immunoenzymometric assay technique. Proc Natl Acad Sci U S A, 1984 Dec, 81(24), 7688 - 92 Isolation of the gene encoding the human T-lymphocyte differentiation antigen Leu-2 (T8) by gene transfer and cDNA subtraction; Kavathas P et al.; We report the isolation of genomic and cDNA clones encoding the human T-lymphocyte cell-surface differentiation antigen, Leu-2 (T8), by use of a combination of transfection, fluorescence-activated cell-sorting, and subtractive cDNA hybridization . We constructed a cDNA library with mRNA from a mouse L-cell transfectant in which the human Leu-2 gene is expressed and amplified . We identified Leu-2 cDNA clones by screening with a selected cDNA probe from a second amplified Leu-2 transfectant . This probe contained cDNA species not removed by hybridization with L-cell mRNA . A Leu-2 cDNA clone was used to isolate a genomic clone . Transfection with DNA from this clone resulted in a high number of Leu-2 transfectants . This approach can be used to clone genes coding for other cell-surface molecules. Proc Natl Acad Sci U S A, 1984 Dec, 81(24), 7669 - 73 Essential site for growth rate-dependent regulation within the Escherichia coli gnd structural gene; Baker HV 2nd et al.; Expression of gnd of Escherichia coli, which encodes the hexose monophosphate shunt enzyme, 6-phosphogluconate dehydrogenase (6PGD; EC 1.1.1.44), is subject to growth rate-dependent regulation: the level of the enzyme is directly proportional to growth rate under a variety of growth conditions . Previous results obtained with strains carrying transcriptional fusions of gnd to the structural genes of the lactose operon suggested that the growth rate-dependent regulation of gnd expression is at the post-transcriptional level . To characterize the regulation further, we prepared with phage MudII a set of eight independent gnd-lac gene (protein) fusions . We showed through genetic analysis and DNA sequencing that each fusion joint was located within the 6PGD-coding sequence between the first and second base pair of a codon, the reading frame required for production of a hybrid 6PGD-beta-galactosidase . Strains harboring the gnd-lac fusion plasmids produced proteins whose mobility in a NaDodSO4/polyacrylamide gel agreed with the molecular weights predicted from the DNA sequence for the respective hybrid proteins . The level of beta-galactosidase was high and relatively growth rate-independent in the fusion whose fusion joint was at codon 48 . The level of beta-galactosidase in the other seven fusion strains whose fusion joints were located further downstream in the 6PGD-coding sequence showed the same dependence on growth rate as 6PGD in a normal strain . beta-Galactosidase levels were not affected by the presence of a gnd+ gene in trans to any of the fusions . The results suggest that all sites necessary for growth rate-dependent regulation of 6PGD level lie in gnd upstream from codon 118 and that an essential site of negative control lies within the coding sequence, between codons 48 and 118. Proc Natl Acad Sci U S A, 1984 Dec, 81(23), 7525 - 8 Anti-mRNA: specific inhibition of translation of single mRNA molecules; Pestka S et al.; A plasmid was constructed to generate RNA complementary to the beta-galactosidase mRNA under control of the phage lambda PL promoter . When this anti-mRNA was produced, synthesis of beta-galactosidase was dramatically inhibited (98%) . Syntheses of galactoside permease and transacetylase, whose coding sequences are downstream of the beta-galactosidase coding region, are inhibited to a lesser degree, 80% and 55%, respectively . The generation of anti-mRNA that can be targeted to inhibit a single species of mRNA molecule within cells provides a potent mechanism by which specific transcripts can be translationally inactivated . This can be used to determine the function of proteins as well as to select cloned genes in a single rapid and convenient step. Horm Metab Res, 1984 Dec, 16 Suppl 1, 92 - 6 L-Asparaginase diabetes mellitus in rabbits: differing effects of two different schedules of L-asparaginase administration; Lavine RL et al.; The diabetogenic effect of daily injections of 1000 i.u./kg body wt . E coli L-asparaginase was studied in male New Zealand white rabbits and compared with the diabetogenic effect of a single bolus of 10,000 I.U . E coli L-asparaginase/kg body wt . to determine whether the schedule of administration of the drug altered the diabetic syndrome produced . A daily injection of 1000 i.u . L-asparaginase/kg . body wt . was continued for 30 days . During this time glucose levels in rabbits allowed free access to food rose steadily, reaching levels of 717 +/- 63 mg/dl the day after the last injection . Levels of immunoreactive insulin fell, reaching their nadir, 53 +/- 4 pg/ml (approximately 50% of baseline) at 25 days . Glucose levels declined when therapy was discontinued, but remained significantly above control levels 46 days after insulin injections were stopped . (Glucose levels in L-asparaginase-treated groups vs . those in controls on day 46 after discontinuation: 116 +/- 3 vs . 104 +/- 1 mg/dl; P less than 0.0025.) Levels of immunoreactive insulin rose when therapy ended, reaching control levels 17 days after discontinuation . In contrast, a single bolus injection of 10,000 I.U . L-asparaginase/kg resulted in hyperglycemia with hyperinsulinemia . These data suggest that L-asparaginase can induce either a hypoinsulinemic or a hyperinsulinemic diabetic syndrome depending on the schedule of administration of the L-asparaginase and that a mild abnormality in glucose homeostasis persists after discontinuation of L-asparaginase therapy. Anal Biochem, 1984 Dec, 143(2), 341 - 9 Primer-directed mutagenesis of linearized plasmids; Hollenberg SM et al.; Two novel mutagenesis techniques to specifically alter the sequence of plasmid DNA have been developed . In contrast to other primer-directed mutagenesis methods which require a single-stranded, closed-circular template, a linearized single strand was used as the mutagenesis template . The template is prepared by restriction enzyme digestion of covalently-closed-circular plasmid DNA . These methods are simple, require small amounts of plasmid DNA, and can result in a relatively high frequency of mutagenesis. Zentralbl Bakteriol Mikrobiol Hyg {A}, 1984 Dec, 258(2-3), 316 - 26 Stable insertion of C5b-9 complement complexes into the outer membrane of serum treated, susceptible Escherichia coli cells as a prerequisite for killing; Kroll HP et al.; Escherichia coli 17, a K12 derivative, was rapidly killed by human serum following a short lag period of 10 min . Stable binding of terminal C5b-9 complement complexes was investigated in time course experiments . Serum treated E . coli cells were lysed osmotically and the resulting outer and cytoplasmic membrane vesicles separated by sucrose gradient centrifugation . Exposure of E . coli 17 to serum rapidly reduced the degree of recoverability of cytoplasmic membrane vesicles . Electron microscopy revealed no interaction of C5b-9 complexes with CM vesicles . In contrast there was a clear time-dependent deposition of terminal complement complexes onto OM-vesicles . Very few complexes were detected during the prekilling phase of the reaction; initiation of the active killing phase was accompanied by a large increase in complement lesions . In contrast, no C5b-9 complexes could be visualised on outer or cytoplasmic membrane vesicles of a smooth, serum-resistant E . coli strain . We conclude that complement-mediated killing is a consequence of stable binding of C5b-9 complexes to the outer membrane of susceptible strains. Gene, 1984 Dec, 32(3), 349 - 56 Role of DNA regions flanking the tryptophan promoter of Escherichia coli . II . Insertion of lac operator fragments; Herrin GL Jr et al.; To examine the effect of introducing protein-binding sites around a promoter upon expression from that promoter, a series of altered tryptophan promoter plasmids have been constructed . In these derivatives of pKO-1 (a galK-expression, promoter-assay vector), the trp promoter has been introduced into the vector, such that the galK expression is dependent on the trp promoter . Unique restriction sites have been introduced adjacent to the trp promoter for the insertion of other DNA fragments . The DNA-binding site (a lac operator (lacO) fragment of 33 bp) for the lactose repressor was inserted into the trp promoter-pKO plasmids at various defined locations from -52 to +58 relative to the start site of transcription . Strains bearing tryptophan promoter-lactose operator plasmid derivatives were assayed for galactokinase activity in Escherichia coli C600 and JM103 (a lacIQ strain) to observe the effect of lac repressor binding and removal . In those plasmids where the lac repressor was downstream from the promoter, expression was diminished by the presence of the lactose repressor and galactokinase activity could be induced by addition of IPTG . An analogous lac promoter plasmid was repressed over 90%, and the plasmid containing the lacO fragment at +2 exhibited up to 80% repression; 40-50% repression was observed when the lacO was placed at positions +27 and +58 . Placement of the lacO at the upstream locations -39 and -52 produced lower repression . To examine the effects of more than one operator surrounding the promoter, plasmids were constructed that had a lacO at -39 or -52 and, in addition, had an operator downstream.+2 Gene, 1984 Dec, 32(1-2), 41 - 8 Nucleotide sequence and transcriptional start point of the phosphomannose isomerase gene (manA) of Escherichia coli; Miles JS et al.; A 1.6-kb MspI-HindIII chromosomal DNA segment, carrying the complete coding region of the 6-phosphomannose isomerase (PMI) structural gene, manA, and the 5' end of the gene encoding the major fumarase activity, fumA, of Escherichia coli K-12, has been sequenced using the chain termination method . The start points of manA and fumA transcripts were located by S1 mapping using 32P-labelled M13 ssDNA probes, and the two genes were shown to be transcribed divergently . The sequence of the 390 amino acid residues comprising the PMI monomer has been deduced, and the predicted Mr of 42 716 agrees with that for the protein of Mr 42 000 identified previously by the maxicell procedure. Gene, 1984 Dec, 32(1-2), 243 - 9 Structural and functional organization of the gpt gene region of Escherichia coli; Nuesch J et al.; The nucleotide sequence was determined for the Escherichia coli region containing the gpt gene, which encodes the enzyme xanthine-guanine phosphoribosyl transferase (XGPRT; EC 2.4.2.22) . Restriction enzyme and sequence analyses have allowed us to locate precisely the gpt gene (16.9-kDal XGPRT) with respect to other genes in this region, notably phoE . Genes gpt and phoE are pointing towards each other and are separated by about 1840 bp . Available sequence data and protein analyses {Overbeeke et al., J . Mol . Biol . 163 (1983) 513-522, and this paper} indicate the presence, between gpt and phoE, of two additional genes . These genes are oriented the same way as gpt and code for proteins of 49 and 15.7 kDal, respectively . By in vitro transcription with E . coli RNA polymerase and nuclease S1 analysis, we have identified a promoter upstream of gpt . The short intercistronic region between gpt and the 49-kDal protein gene contains a rho-independent termination signal that closely precedes and partially overlaps another promoter . It appears from these data that gpt transcription is essentially monocistronic, giving rise to RNA of approx . 555 nucleotides, whereas the 49-kDal and 15.7-kDal protein genes are transcribed from their own promoter. Biochem Soc Trans, 1984 Dec, 12(6), 993 - 6 Microcomputers to control the pH of growing micro-organisms; Bangham JA et al.; Negative feedback control can usefully be used in biochemistry for a variety of purposes . Two examples are presented which utilize microcomputers to generate the transfer function of the feedback loop . The first demonstrates the control of NADH concentration during an enzymic reaction, and the second shows that if the pH is held constant whilst growing E . coli the yield can be increased . In both these cases it is convenient, even essential, to use a computer because computers are both very stable over the long term and the feedback transfer function can be accurately and quickly specified. Mol Biol Rep, 1984 Dec, 10(2), 115 - 21 Detection of a 16S rRNA . initiator-tRNA complex by a new selective labelling method; Cunningham C et al.; Cupric-ion induced hydrolysis of {35S}Met-tRNA but not of N-formyl-Met-tRNAMetf permitted the specific terminal labelling of initiator tRNA . Initiator tRNA, labeled in this way, was suitable for sequence analysis without the need for further purification . By probing labeled initiator tRNA with specific RNases, changes in this molecule during its interaction with the 30S particle or with 16S rRNA were investigated . Initiation complexes were resistant to the action of single-strand, base-specific nucleases Bc and Phy M and, except for one base of the anticodon stem, were also resistant to digestion by the double-strand-specific V1 nuclease of Naja venom . In contrast, T1 RNase digestion of the initiator tRNA in the presence of 16S rRNA enhanced cleavage of bases in the T stem of the molecule. Int J Radiat Biol Relat Stud Phys Chem Med, 1984 Dec, 46(6), 771 - 8 Effect of hyperthermia and gamma-radiation on Escherichia coli K1060 D-lactate dehydrogenase; Tamba M et al.; The response of E . coli K1060 D-lactate dehydrogenase (D-LDH), an enzyme located in the cytoplasmic membrane, was studied following 42.5 degrees C hyperthermia and/or gamma-irradiation . The inactivation of D-LDH following the above treatment was used as a tool to probe the role of membrane proteins in the radiation and/or heat sensitivity of cells . No correlation between loss of enzyme activity and cell killing was found, suggesting that D-LDH does not play an important role in hyperthermic cell survival . The results obtained in combined hyperthermia and gamma-irradiation treatments on loss of D-LDH activity and E . coli cell killing suggest that an interaction between heat and radiation occurs at the membrane structure level . Moreover, when cells were heated at 42.5 degrees C in the presence of 10 mM procaine-HCl, both cell killing and loss of D-LDH activity were enhanced . The involvement of membrane structure in the heat sensitivity of cells is strongly indicated by the latter observations . The opposite effect was observed when procaine was present during irradiation in oxic conditions, suggesting that procaine itself can also act as a scavenger towards OH-induced radicals. EMBO J, 1984 Dec 1, 3(12), 2905 - 9 DNA sequencing with direct blotting electrophoresis; Beck S et al.; A method for transferring the DNA molecules of sequencing reaction mixtures onto an immobilizing matrix during electrophoresis has been developed . A blotting membrane moves with constant speed across the end of a very short, denaturing gel and collects the molecules according to size . A constant distance between bands for molecules differing in length by one nucleotide is obtained over a large range (approximately 600 nucleotides with a 5% gel), simplifying the determination of DNA sequences considerably . Reliable sequences of 500 nucleotides can be read and sequence features up to greater than 1000 nucleotides are revealed in a single experiment . The sequencing of a potential Z-DNA-forming fragment from Escherichia coli DNA is given as an example and possible further developments are discussed. EMBO J, 1984 Dec 1, 3(12), 2895 - 8 Escherichia coli ribosomes translate in vivo with variable rate; Pedersen S; The question of whether or not 'rare' codons are translated with the same rate as 'common' codons was investigated by measuring the translation time for two genes, lacI and bla, rich in rare codons, and comparing the results with the translation times measured on fus, tsf, tuf and rpsA which have very few rare codons . The rate of synthesis of the lac repressor was first measured with the up-promoter mutation lacIq1 present on the high copy number plasmid pBR322 . In such a strain the average translation times for lacI and bla were 50% slower than the rate calculated from the translation time for the four ribosomal proteins . In a strain having lacIq1 on an F'lac episome this difference was much smaller, thus slow translation of genes rich in rare codons is exaggerated in strains with increased drain on the rare codon tRNAs . The data do not exclude that only a subset of the rare codons is translated more slowly . Translation times were also measured in cells growing in different media, and the translation chain growth rate was found to increase by approximately 40% going from acetate medium to a fully supplemented medium. EMBO J, 1984 Dec 1, 3(12), 2873 - 8 The change of DNA structure by specific binding of the cAMP receptor protein from rotation diffusion and dichroism measurements; Porschke D et al.; The structure of complexes formed between cAMP receptor protein (CRP) and various restriction fragments from the promoter region of the lactose operon has been analysed by measurements of electrodichroism . Binding of CRP to a 62-bp fragment containing the major site leads to an increase of the rotation time constant from 0.33 to 0.43 microseconds; addition of cAMP to the complex induces a decrease to 0.25 microseconds . Similar data are obtained for a 80-bp fragment containing the operator site; however, in this case the decrease of the rotation time for the specific complex is only observed when the salt concentration is increased from 3 to 13 mM . A 203-bp fragment containing both sites showed a corresponding change after pre-incubation at 50 mM salt . The salt dependence of the rotation time for the specific complex formed with the 203-bp fragment also indicates that a compact structure is formed at 13 mM salt, whereas the structure is not as compact at 3 mM salt . A 98-bp fragment without specific CRP sites did not reveal changes corresponding to those observed for the specific fragments . The rotation time constants together with the dichroism amplitudes indicate that binding of CRP to specific sites in the presence of cAMP leads to the formation of compact structures, which are consistent with bending of DNA helices . The observed strong salt dependence of the structure is apparently due to electrostatic repulsion between adjoining helix segments. Am J Vet Res, 1984 Dec, 45(12), 2495 - 7 Influence of endotoxin-induced fever on the pharmacokinetics of gentamicin in ewes; Wilson RC et al.; The pharmacokinetics of gentamicin (3 mg/kg of body weight) were evaluated in 6 adult ewes before and after fever was induced with Escherichia coli endotoxin (1 micrograms/kg) . In the ewes with normal rectal temperature, significant (P less than 0.05) increases in rectal temperature occurred before gentamicin injection and during the first 2 hours . Other mild clinical signs of fever also were present . In the same ewes with endotoxin-induced fever, statistically (P less than 0.05) increased gentamicin concentrations occurred at 15 and 40 minutes and at 6 hours after injection of gentamicin . Changes were not observed in the apparent volume of distribution calculated by the area method, the volume of distribution at steady state, the overall biological half-life, or body clearance . Significant (P less than 0.05) reductions occurred in the zero time intercept for distribution, the distribution rate constant, the concentration in plasma at time of injection, the volume of the peripheral compartment, and the first order transfer rate constants; only the volume of the central compartment was increased . Total amounts of gentamicin were increased in the central compartment and decreased in the peripheral or tissue compartment. Biochem J, 1984 Dec 1, 224(2), 379 - 88 The inhibition of ornithine transcarbamoylase from Escherichia coli W by phaseolotoxin; Templeton MD et al.; The mechanism of inhibition of ornithine transcarbamoylase by the bacterial toxin phaseolotoxin {N-delta-(phosphosulphamyl)ornithylalanylhomoarginine} was investigated . Ornithine transcarbamoylase was purified by affinity chromatography from Escherichia coli W argR- by using N-delta-(phosphonoacetyl)ornithine as the ligand . Under steady-state conditions phaseolotoxin inhibition was reversible and exhibited mixed kinetics with respect to carbamoyl phosphate . The apparent Ki and apparent K'i were 0.2 microM and 10 microM respectively . Inhibition with respect to ornithine was noncompetitive, with an apparent Ki of 0.9 microM . These data are consistent with competitive binding of phaseolotoxin to the carbamoyl phosphate-binding site of the enzyme . The toxin also appears to be able to bind to the enzyme-carbamoyl phosphate complex, although, since K'i is 50 times greater than Ki, this event is kinetically much less significant . In the presence of phaseolotoxin ornithine transcarbamoylase exhibited a transient phase of activity before a steady state . This is consistent with low rates of association and dissociation for the toxin with enzyme and the enzyme-toxin complex . Rate constants of 2.5 X 10(4)M-1 X s-1 and 5 X 10(-3)s-1 were estimated for the association and dissociation constants respectively. Arch Biochem Biophys, 1984 Dec, 235(2), 571 - 8 Comparative analysis of translation accuracy in an Escherichia coli and a mammalian cell-free system; Laughrea M et al.; The effect of environmental stress on the accuracy of protein synthesis in an Escherichia coli and a rat brain cell-free system was investigated . Poly-U was translated in a rat brain and an E . coli cell-free extract under identical ionic conditions . The fidelity of translation, both in the E . coli and the rat brain extracts, was commensurate with what is known about the accuracy of translation in vivo . The incorporation of phenylalanine (code: UUU) and leucine (code: CUU, UUG or A) was measured at various Mg2+ concentrations (3 to 22 mM), various pH's (6.6 to 8.6), various temperatures (23 to 42 degrees C), and in the presence or absence of 2.4% (v/v) ethanol . It was observed that (i) the accuracy of translation was generally higher in extracts from E . coli than from rat brain, and (ii) relative to that in E . coli, the translation fidelity in rat brain extracts was about 2 times more sensitive to ethanol, at least 5 times more sensitive to temperature, and at least 50 times more sensitive to pH . It was found that this differential sensitivity was not due to a differential behavior of the bacterial and the mammalian aminoacyl-tRNA synthetases under stress, but rather to the process of chain elongation itself . It is concluded that the accuracy of protein synthesis is more resistant to environmental stress in E . coli extracts than in extracts from at least one mammalian tissue. Appl Environ Microbiol, 1984 Dec, 48(6), 1072 - 5 Aspartase-hyperproducing mutants of Escherichia coli B; Nishimura N et al.; When a wild-type strain of Escherichia coli B was cultured on a medium containing L-aspartic acid as the sole carbon source (Asp-C medium), aspartase formation was higher than that observed in minimal medium . Addition of glucose to Asp-C medium decreased aspartase formation . When also cultured in a medium containing L-aspartic acid as the sole nitrogen source (Asp-N medium), E . coli B showed a low level of aspartase formation and an elongated doubling time . To obtain aspartase-hyperproducing strains, we enriched cells growing faster than cells of the wild-type strain in Asp-N medium by continuous cultivation of mutagenized cells . After plate selection, the doubling times of these mutants were measured . Thereafter, fast-growing mutants were tested for aspartase formation . One of these mutants, strain EAPc7, had a higher level of aspartase formation than did the wild-type strain in medium containing L-aspartic acid as the carbon source, however; addition of glucose to this medium decreased aspartase formation . The other mutant, strain EAPc244, had a higher level of aspartase activity than did the wild-type strain in both media . Therefore, aspartase formation in mutant EAPc244 was released from catabolite repression . In strain EAPc244 the other catabolite-repressible enzymes, beta-galactosidase, tryptophanase, and the three tricarboxylic acid cycle enzymes, were also released from catabolite repression . Both mutants had sevenfold the aspartase formation of the wild-type strain in a medium which contained fumaric acid as the main carbon source and which has been used for industrial production of E . coli B aspartase . However, strain EAPc244 had 2.5-fold the fumarase activity of strain EAPc7.(ABSTRACT TRUNCATED AT 250 WORDS) Proc Natl Acad Sci U S A, 1984 Dec, 81(24), 7850 - 4 A new class of Escherichia coli recBC mutants: implications for the role of RecBC enzyme in homologous recombination; Chaudhury AM et al.; Mutants of Escherichia coli sensitive to phage T4 gene 2 mutants were obtained following ethyl methanesulfonate mutagenesis . By mapping and complementation analysis, the mutations in each of the six mutants are in recB and recC . By both in vivo and in vitro analyses, the nuclease activity of RecBC enzyme is undetectable in these mutants . However, by several other criteria, such as proficiency in recombination, relative resistance to UV radiation, and viability of the cells in the culture, these mutants are almost identical to their recBC+ parent . The properties of these mutants indicate that the ATP-dependent double-stranded DNA exonuclease activity of RecBC enzyme is not required for recombination . Chi recombinational hotspots, which stimulate recombination by the RecBC pathway, have no detectable activity in the mutants . This result suggests that the nuclease activity of RecBC enzyme is required for Chi activity and is consistent with the hypothesis that Chi stimulates recombination by directing RecBC enzyme to cut DNA at or near Chi. Proc Natl Acad Sci U S A, 1984 Dec, 81(24), 7777 - 81 Identification of a higher molecular weight DNA polymerase alpha catalytic polypeptide in monkey cells by monoclonal antibody; Karawya E et al.; A monoclonal antibody against purified calf DNA polymerase alpha (deoxynucleosidetriphosphate:DNA deoxynucleotidyltransferase, EC 2.7.7.7) was used to immunoprecipitate proteins from a crude soluble extract of growing monkey BSC-1 cells . Immunoprecipitates contained familiar DNA polymerase alpha catalytic polypeptides of Mrs approximately equal to 115,000 and 70,000 and also a Mr 40,000 catalytic polypeptide; the major component in the immunoprecipitates, however, was a polypeptide of Mr approximately equal to 190,000 not previously identified as a DNA polymerase . This protein was capable of DNA polymerase activity after electroelution from NaDodSO4/polyacrylamide gels and renaturation . The highly purified enzyme so obtained was active with poly(dT).oligo(rA) as template.primer, resistant to dideoxy TTP (ddTTP), and inhibited by aphidicolin and butylphenyldeoxyguanosine 5'-triphosphate, thus identifying it as a DNA polymerase alpha . The results indicate that a polypeptide of Mr approximately equal to 190,000 is an abundant component among DNA polymerase alpha catalytic polypeptides in growing monkey cells. Mutat Res, 1984 Dec, 129(3), 319 - 25 Effect of L-ethionine on the expression of the SOS system in Escherichia coli; Barbe J et al.; The effect of L-ethionine, the ethyl analog of the essential amino acid methionine, on the SOS system of Escherichia coli was studied . This compound does not induce either inhibition of cell division nor cessation of cell respiration in a RecA+ Met+ RelA+ strain, nor in RecA+ Met- RelA+ or RecA+ Met- RelA- mutants . Nevertheless, L-ethionine blocks the expression of both cited SOS functions in a recA441 mutant when it is growing at the restrictive temperature of 42 degrees C . Furthermore, the inhibitory effect of the L-ethionine on the induction of the SOS system in this mutant is increased when the cells are preincubated for several hours in the presence of the analog, before the temperature shift . Moreover, cultures of the recA441 mutant incubated at 42 degrees C in the presence of both L-ethionine and L-methionine present the same behaviour as the cultures of this mutant growing at the same temperature but without either amino acid . On the other hand, L-ethionine does not have any effect on the expression of the two mentioned SOS functions when these are induced by UV-irradiation in a RecA+ strain even if this compound is added to the cells several hours before irradiation. J Ultrasound Med, 1984 Dec, 3(12), 533 - 7 Sonography of emphysematous pyelonephritis; Allen HA 3rd et al.; Emphysematous pyelonephritis is a rare gas-producing bacterial infection of the renal parenchyma seen primarily in patients with diabetes mellitus . Sonographic findings in four patients with this disease are described . Sonographic features consist of multiple high-amplitude echoes within the renal parenchyma, renal sinus, and/or perirenal space accompanied by acoustic shadowing . Computed tomography confirmed bilateral involvement in one case . Plain abdominal radiographs demonstrated abnormal extraluminal gas in three of four cases . The role of sonography in diagnosis is discussed. Genetics, 1984 Dec, 108(4), 765 - 72 Specific deletion occurring in the directed evolution of 6-phosphogluconate dehydrogenase in Escherichia coli; Miller RD et al.; A novel genetic change leading to increased activity of 6-phosphogluconate dehydrogenase (6PGD) in E . coli has been observed . The mutation is a deletion of approximately 0.4 kilobase pairs occurring between the structural gene of 6PGD (gnd) and one copy of an insertion element (IS5) found normally in E . coli K12 a few hundred base pairs upstream (counterclockwise) from gnd at 44 minutes on the conventional genetic map . The deletion is associated with a threefold higher activity of 6PGD and a 57% increase in the maximum growth rate when cells are grown in gluconate. Am Surg, 1984 Dec, 50(12), 653 - 5 Protective effect of previous burn on murine endotoxemia; Spillert CR et al.; Major body burns suppress the immune system, at least temporarily . However, it has not been demonstrated that following recovery from burn injury, the burned subject can become immunocompetent . This study was designed to test this hypothesis . Swiss-Webster mice (25 +/- 2 gm) anesthetized with intraperitoneal pentobarbital sodium were given full thickness burns on a depilated area of the lower back with a stainless steel tube (2 cm diameter) at 100 C for 10 seconds . Control mice were anesthetized, depilated in the same manner, but sham burned . Six weeks later, (the burn wound was completely healed, and the mice weighed 33 +/- 4 gm) each animal was given 0.3 mg of Escherichia coli endotoxin (055:B5) in saline intraperitoneally . The 24- and 72-hour survivals for the control animals were 31/45 (69%) and 13/45 (29%), respectively; while the 24- and 72-hour survivals for the post burn mice was 26/27 (96%) and 14/27 (52%), respectively (P less than 0.05) . All animals surviving 72 hours recovered . These data clearly demonstrate that the survival from endotoxemia is significantly increased in animals that have been previously burned . This observation indicates that the immune system is stimulated 6 weeks postburn. Am Rev Respir Dis, 1984 Dec, 130(6), 1140 - 6 Modification of pulmonary responses to endotoxemia in awake sheep by steroidal and nonsteroidal anti-inflammatory agents; Begley CJ et al.; The effects of intravenous infusions of Escherichia coli endotoxin on white blood cell counts, hemodynamics, gas exchange, body temperature, and lung lymph flow were studied in chronically instrumented unanesthetized sheep . Six sheep received endotoxin (0.5 micrograms/kg) in the presence and absence of methylprednisolone treatment . Six sheep received the same dose of endotoxin with and without meclofenamate and methylprednisolone infusion . Endotoxemia caused an early increase in pulmonary artery pressure from 15.5 +/- 1.3 (mean +/- SEM) to 52.7 +/- 2.1 cmH2O (p less than 0.05), an initial phase of high flow of protein-poor lung lymph, an elevation in core body temperature, severe leukopenia, and an early increase in the alveolar to arterial oxygen difference (AaPO2) from 7.4 +/- 2.5 mmHg to 35.9 +/- 2.5 mmHg (p less than 0.05) . From 2 to 5 h after endotoxin infusion, lung lymph flow averaged 4.8 times that of the baseline measurement, although lymph protein concentration relative to plasma was not different from the baseline measurement . Peripheral leukopenia and significantly increased AaPO2 (28.8 +/- 4.5 mmHg) persisted at 2 to 5 h . Methylprednisolone attenuated the early pulmonary artery hypertension (43.0 +/- 3.4 cmH2O), but did not inhibit the initial febrile response, severe leukopenia, or the early increase in AaPO2 (29.2 +/- 1.6 mmHg) in response to endotoxemia . Methylprednisolone did prevent the late phase increase in lung lymph flow, significantly attenuated the late phase leukopenia and hypoxemia (AaPO2, 13.3 +/- 5.9 mmHg), and attenuated accumulation of granulocytes in peripheral lung tissue throughout the endotoxin reaction.(ABSTRACT TRUNCATED AT 250 WORDS) Am Rev Respir Dis, 1984 Dec, 130(6), 1065 - 71 Neutrophil alveolitis following endotoxemia . Enhancement by previous exposure to hyperoxia; Rinaldo JE et al.; We injected Escherichia coli endotoxin, 2.5 mg/kg, intraperitoneally in rats, sequentially quantified alveolar inflammation during a 6-day period by several techniques, and observed the effect of previous exposure to hyperoxia on the intensity of alveolitis in this model . As noted in other models of endotoxemia, we found intravascular sequestration of leukocytes and an increase in the retention of 125I albumin in the lung 4 to 6 h after the injection of endotoxin . Bronchoalveolar lavage fluid (BALF) obtained at this time only slightly stimulated the migration of neutrophils in vitro, and the numbers and types of cells recovered by lavage were normal . Fifteen h after the injection of endotoxin, however, bronchoalveolar lavage fluid stimulated both random and directed migration of neutrophils in vitro, although recovery of neutrophils by lavage was increased only slightly . By 24 h, 125I albumin retention had returned to normal levels, but the chemotactic activity of BALF remained high, and the percentage and absolute number of neutrophils recovered by lung lavage were increased markedly . The recovery of neutrophils remained significantly elevated for 3 days but declined to control levels by 6 days, whereas the recovery of alveolar macrophages was increased at this time . Exposure to 100% O2 for 36 h prior to endotoxemia accelerated and intensified neutrophil influx into the lung and increased the stimulatory effect of BALF on neutrophil migration in vitro . We conclude that a single episode of endotoxemia in the rat causes a multi-phasic alveolar inflammatory response, and that this response is accelerated and intensified by prior, mild exposure to hyperoxia.(ABSTRACT TRUNCATED AT 250 WORDS) Transplant Proc, 1984 Dec, 16(6), 1470 - 2 Ineffective cellular interaction and interleukin 2 deficiency causing T cell defects in human allogeneic marrow recipients early after grafting and in those with chronic graft-versus-host disease; Tsoi MS et al.; Impairment in T cell proliferation in response to E . coli and in CML to unrelated alloantigens was usually observed in patients early after marrow grafting and persisted in long-term patients with chronic GVHD but not in those without chronic GVHD . We analyzed various cellular functions in the pathway of T cell activation and found that in patients with immunodeficiency, (1) their M phi usually could process and present antigens to normal T cells, (2) their T cells did not proliferate even in the presence of normal antigen-pulsed M phi, (3) IL-2 production by T cells was deficient, and (4) exogenous IL-2 restored CML activity in cells of most patients early after grafting but not in cells of most patients with chronic GVHD . Therefore, failure to induce proliferation and cytotoxicity in T cells of marrow recipients is not likely due to M phi defects but because of ineffective communication among T cell subsets, probably related to impaired IL-2 production and/or unresponsiveness to IL-2. Proc Natl Acad Sci U S A, 1984 Dec, 81(23), 7539 - 43 Involvement of the activated form of RecA protein in SOS mutagenesis and stable DNA replication in Escherichia coli; Witkin EM et al.; DNA damage activates RecA protein of E . coli to a form (RecA*) that promotes proteolytic cleavage of LexA protein, the repressor of at least 17 DNA damage-inducible genes, resulting in expression of the SOS response . In addition to this known role, RecA* performs another function necessary for expression of SOS mutagenesis {Blanco, M., Herrera, G., Collado, P., Rebollo, J . & Botella, L . M . (1982) Biochimie 64, 633-636} . The additional role of RecA* could be (i) cleavage of another repressor, (ii) proteolytic processing of one or more proteins, or (iii) mechanistic interaction with DNA or with one or more other proteins . We describe experiments designed to test the first possibility . Our results suggest that neither SOS mutator activity nor ultraviolet mutagenesis requires induction by RecA* of any gene(s) outside the LexA regulon and that the additional role of RecA* is not cleavage of another repressor . We show that stable DNA replication, another DNA damage-inducible function {Kogoma, T., Torrey, T . A . & Connaughton, M . J . (1979) Mol . Gen . Genet . 176, 1-9}, shares with SOS mutagenesis the requirement for RecA* activity, even in a strain constitutively expressing all LexA-controlled genes . In this strain, conditions that activate RecA initiate expression of stable DNA replication in the presence of chloramphenicol, without an intervening period of protein synthesis . We conclude that the additional function of RecA* in stable DNA replication is not another antirepressor activity. Proc Natl Acad Sci U S A, 1984 Dec, 81(23), 7397 - 401 Escherichia coli DNA photolyase stimulates uvrABC excision nuclease in vitro; Sancar A et al.; Pyrimidine dimers are the major photoproducts produced in cellular DNA upon UV irradiation . In Escherichia coli there are dark and photorepair mechanisms that eliminate the dimers from DNA and prevent their lethal and mutagenic effects . To determine whether these repair mechanisms act cooperatively or competitively in repairing DNA, we investigated the effects upon one another of DNA photolyase, which mediates photorepair, and uvrABC excision nuclease, an enzyme complex of the uvrABC gene products, which catalyzes nucleotide excision repair . We found that photolyase stimulates the removal of pyrimidine dimers but not other DNA adducts by uvrABC excision nuclease . The two subunits of uvrABC excision nuclease, the uvrA and uvrB proteins which together bind to the dimer region of DNA, had no effect on the activity of photolyase . T4 endonuclease V, which like photolyase is specific for pyrimidine dimers, was inhibited by photolyase, suggesting that these two proteins recognize the same or similar chemical structures in UV-irradiated DNA that are different from those recognized by uvrABC excision nuclease. Proc Natl Acad Sci U S A, 1984 Dec, 81(23), 7274 - 8 Molecular basis of DNA sequence recognition by the catabolite gene activator protein: detailed inferences from three mutations that alter DNA sequence specificity; Ebright RH et al.; Previously, we reported that substitution of Glu-181 of the catabolite gene activator protein (CAP) by lysine, leucine, or valine results in a protein that has specificity for A X T base pairs at positions 7 and 16 of the DNA recognition site, rather than G X C base pairs as is the case with the wild-type CAP . In this paper, we deduce from these genetic data both (i) the specific chemical interactions by which amino acid side chains at position 181 interact with base pairs 7 and 16 and (ii) the precise alignment between the structures of the CAP and DNA in the intermolecular CAP-DNA complex . Our analysis supports the idea that the two symmetry-related F alpha-helices of the CAP dimer interact with successive major grooves of right-handed B-type DNA {Pabo, C . & Lewis, M . (1982) Nature (London) 298, 443-447; and Steitz, T., Weber, I . & Matthew, J . (1983) Cold Spring Harbor Symp . Quant . Biol . 47, 419-426}. J Infect Dis, 1984 Dec, 150(6), 925 - 34 A role for hemolysin in Escherichia coli-induced inflammation in granulocytopenic rabbits; Issekutz AC et al.; Inflammation induced in the skin of granulocytopenic rabbits by Escherichia coli was examined . Protein exudation and platelet deposition in lesions were measured with 125I-labeled albumin and 111In-labeled platelets . In granulocytopenic rabbits 10(4)-10(7) live serum-resistant E . coli induced protein exudation and platelet deposition beginning at 3 hr and then progressing over the next 24 hr to much higher levels than in normal rabbits . These responses were associated with interstitial edema and progressive venous thrombosis in the absence of leukocytes; no such reactions were observed in normal rabbits . No reactions were induced in granulocytopenic rabbits by killed E . coli . Of six E . coli strains tested, all three hemolytic strains induced lesions with four to five times more thrombosis (platelet deposition) than did nonhemolytic strains . Two hemolysin-negative mutants lost most of their thrombogenic activity . All three hemolytic strains had cell-associated hemolysin, but only one of these elaborated appreciable free, filterable hemolysin as well. J Bacteriol, 1984 Dec, 160(3), 1188 - 90 Tetracycline resistance element of pBR322 mediates potassium transport; Dosch DC et al.; The tetracycline resistance element of plasmid pBR322 partially complements the potassium transport defect of Escherichia coli K-12 mutants having markedly impaired K+ transport . The plasmid increases K+ transport . The Tn10 element does not result in increased transport, demonstrating that the effect is not general for elements that increase resistance to tetracycline. J Bacteriol, 1984 Dec, 160(3), 1151 - 7 Phase separation between nucleoid and cytoplasm in Escherichia coli as defined by immersive refractometry; Valkenburg JA et al.; The refractive indices of nucleoid and cytoplasm in Escherichia coli were derived theoretically and experimentally . For the theoretical estimates, we made use of the known macromolecular composition of E . coli B/r (G . Churchward and H . Bremer, J . Theor . Biol . 94:651-670, 1982) and of estimates of cell and nucleoid volumes . These were obtained from micrographs of living bacteria made with a confocal scanning light microscope . The theoretical values were calculated, assuming that all DNA occurred in the nucleoid and that all protein and RNA occurred in the cytoplasm . Comparison with experimental refractive index values directly obtained by immersive refractometry showed that, besides its DNA, the nucleoid must contain an additional amount of solids equivalent to 8.6% (wt/vol) protein . With the nucleoid containing 6.8% (wt/vol) DNA and 8.6% (wt/vol) protein and the cytoplasm containing 21% (wt/vol) protein and 4% (wt/vol) RNA, a mass difference is obtained, which accounts for the phase separation observed between the nucleoid and cytoplasm in living cells by phase-contrast microscopy . The decrease in the refractive index of the nucleoid relative to that of the cytoplasm observed upon, for instance, OsO4 fixation was interpreted as being indicative of the loss of protein content in the nucleoid. J Bacteriol, 1984 Dec, 160(3), 1101 - 4 Purine-mediated growth inhibition caused by a pyrE mutation in Escherichia coli K-12; Shimosaka M et al.; A purine-sensitive phenotype results from a previously described mutation in the structural gene (pyrE) for orotate phosphoribosyltransferase (OPT) in Escherichia coli K-12 . OPT from both the mutant and the wild-type was partially inhibited by adenine and adenosine, although other purine derivatives were not effective for this inhibition . The Km values of the mutant OPT were 580 and 760 microM for orotate and 5'-phosphoribosyl-1'-pyrophosphate (PRib-PP), respectively, whereas the corresponding values for the wild-type OPT were 40 and 60 microM . The intracellular level of PRib-PP was decreased to less than 15% of the normal level when purine derivatives were added to exponentially growing cultures of both the parent and mutant strains . However, this decrease of the PRib-PP level was not found in strains derived from the mutant, in which the purine-sensitive phenotype was suppressed by a secondary mutation . The purine-sensitive phenotype was caused by retardation of the pyrimidine de novo pathway, when the intracellular level of PRib-PP was diminished by exogenously supplied purine derivatives. J Bacteriol, 1984 Dec, 160(3), 1017 - 21 Relaxation of supercoiled plasmid DNA by oxidative stresses in Escherichia coli; Horiuchi H et al.; The relaxation of plasmid DNA was observed after the visible light irradiation of Escherichia coli AB1157 harboring plasmid pBR322 or some other plasmids in the presence of a photosensitizing dye, such as toluidine blue or acridine orange, and molecular oxygen . Treatment of the cells with hydroperoxides, such as tert-butyl hydroperoxide, cumene hydroperoxide, and hydrogen peroxide, also caused the plasmid DNA relaxation in vivo . Relaxation was not observed in these treatments of purified pBR322 DNA in vitro . Plasmid DNA relaxation was also detected after near-UV irradiation . Far-UV irradiation did not induce such relaxation. J Bacteriol, 1984 Dec, 160(3), 1010 - 6 Mutationally altered ribonucleotide reductase from Escherichia coli: characterization of mutations isolated on multicopy plasmids; Platz A et al.; The Escherichia coli ribonucleotide reductase genes (nrd genes) were mutagenized at random . Point mutations were introduced in vitro into a recombinant nrd plasmid . Transformants were initially screened for altered tolerance toward the drug hydroxyurea and further characterized by enzymatic and immunological methods . The screening procedure could pick out defects in either of the two subunits of ribonucleotide reductase . Cells carrying the nrd plasmid pPS2 were earlier shown to have levels of ribonucleotide reductase molecules that were 10 to 20 times higher than those in wild-type cells . We now demonstrate that the enzymatic activity in gently lysed pPS2-containing cells on cellophane disks is six times higher than in wild-type cells . Supplementation of the pPS2-containing lysates with a purified thioredoxin system results in a further 4.5-fold stimulation of the enzymatic activity, which implies a functional shortage of the electron donor system(s) for ribonucleotide reduction in pPS2-containing cells. Infect Immun, 1984 Dec, 46(3), 740 - 6 A plasmid-encoded outer membrane protein, TraT, enhances resistance of Escherichia coli to phagocytosis; Aguero ME et al.; The presence of the outer membrane protein TraT, encoded by plasmid R6-5, reduces the sensitivity of Escherichia coli cells to phagocytosis by macrophages . This effect is independent of the bacterial capsule and is more evident in the presence of adsorbed normal human serum . The property of inhibiting phagocytosis is specifically abolished by anti-TraT protein antiserum and anti-TraT immunoglobulin G but not by Fab fragments . These results indicate that the TraT protein is a passive inhibitor of phagocytosis . Inhibition of phagocytosis is produced because the TraT protein antagonizes opsonization by complement, such that C3 deposition is reduced and altered in distribution. Infect Immun, 1984 Dec, 46(3), 690 - 6 Pathogenicity of attaching effacing enteropathogenic Escherichia coli isolated from diarrheic suckling and weanling rabbits for newborn rabbits; Peeters JE et al.; The pathogenicity of six strains of Escherichia coli originating from different commercial rabbitries was tested in neonatal rabbits . Two strains isolated from healthy weaned rabbits (O7:H6 and O9:H?) did not induce any clinical sign or lesion . Two strains (O109:H2) isolated from diarrheic suckling rabbits caused yellow diarrhea 36 to 60 h after inoculation and high mortality between 60 and 72 h after infection . At 12 h after infection, light and electron microscopy showed attachment to epithelial cells and effacement of microvilli from proximal small intestine to colon . Bacteria were often present in the apical cytoplasm of epithelial cells . The two strains isolated from diarrheic weanling rabbits (O109:H2 and O15:H-) did not induce any clinical sign . Attachment to epithelial cells and effacement of microvilli was observed 48 h after inoculation in distal small intestine, cecum, and colon . These data are further evidence for the existence of two groups of attaching effacing enteropathogenic E . coli in rabbits, showing different preferences for age group and intestinal compartment. Fed Proc, 1984 Dec, 43(15), 2987 - 90 Size polymorphism and the structure of aminoacyl-tRNA synthetases; Schimmel P et al.; Although aminoacyl-tRNA synthetases catalyze the same chemical reaction, the individual enzymes have a wide range of sizes . Proteolytic digestion has yielded active catalytic fragments of two synthetases . A set of gene deletions in a large synthetase has been used successfully in the creation of a variety of enzyme fragments that have been studied individually; a fragment with about half of the total polypeptide is sufficient to aminoacylate tRNA in vivo . The results suggest that size polymorphism is caused by fusion, to a core catalytic segment, of variable amounts of additional polypeptide sequences . These sequences may serve to impart additional functions . For example, in one case, a synthetase binds to its own gene promoter and regulates transcription. Fed Proc, 1984 Dec, 43(15), 2972 - 6 Misaminoacylation by glutaminyl-tRNA synthetase: relaxed specificity in wild-type and mutant enzymes; Hoben P et al.; Escherichia coli glutaminyl-tRNA synthetase (GlnRS) (EC 6.1.1.18) is a monomeric polypeptide of 553 amino acids . Its amino acid sequence and its gene (glnS) sequence are known . A structural gene mutation, glnS7, codes for a mischarging GlnRS, which acylates some noncognate tRNA species (e.g., su+3 tRNATyr) with glutamine . The mutant enzyme was shown to catalyze in vitro the acylation of glutamine to su+3 tRNATyr, but not to wild-type tRNATyr . The mutation responsible produces an amino acid change in the amino-terminal half of the enzyme . Unexpectedly, overproduction of wild-type GlnRS also leads to in vivo mischarging of su+3 tRNATyr . In vitro and in vivo studies have not revealed evidence for an attenuation or autogenous regulation mechanism for GlnRS, but have implicated transcriptional and translational control in the expression of this enzyme. Zh Mikrobiol Epidemiol Immunobiol, 1984 Dec, (12), 62 - 5 {Action of dicaine on the efficiency of plasmid transformation and transfection in Escherichia coli K12}; Likhacheva NA et al.; The action of tetracaine hydrochloride, a local anesthetic, on the effectiveness of plasmid transformation and transfection in variants of E . coli K-12 has been studied . The concentrations of tetracaine hydrochloride used in the experiment (0.002 M) affect the level of the synthesis of most proteins in the outer membrane of these bacteria . This seems to be one of the causes of changes in the effectiveness of plasmid transformation and transfection in E . coli K-12. Can J Genet Cytol, 1984 Dec, 26(6), 706 - 9 Effect of ozone on prophage induction in different strains of Escherichia coli K-12 lysogenic for lambda; L'Herault P et al.; Ozone was tested for its effect upon induction of lambda prophage in two different strains of Escherichia coli K-12 . Based on the induction index and when compared to ultraviolet light, ozone appeared to be a weak, if any at all, inducer of the lytic cycle in E . coli . This is in agreement with other studies which have suggested that this agent is a weak inducer of the SOS functions. Genetics, 1984 Dec, 108(4), 795 - 808 Does Chi give or take? Stahl FW, Lieb M, Stahl MM. In lytic cycle crosses with Red-Gam-lambda phage, particles were examined that had undergone an Int-mediated exchange . It was assumed that this exchange dimerized the circular lambda, making it packageable . Among these Int-mediated recombinants, particles were identified that had, in addition, enjoyed a close double exchange mediated by the RecBC pathway . Such close double exchanges indicate localized negative interference and are analogous to eukaryotic conversions that have retained parental configuration of flanking markers . These events are stimulated by Chi, a recombinator specific to the RecBC pathway . When Chi is present in only one parent in the cross, the complementary double exchange recombinants are Chi stimulated to the same degree . This behavior of Chi contrasts with that of characterized eukaryotic recombinators. Cell, 1984 Dec, 39(3 Pt 2), 699 - 706 Determinants of directionality in lambda site-specific recombination; Bushman W et al.; The DNA structural features governing directionality in lambda site-specific recombination are shown to reside in regions of the phage attachment site more than 70 bp to the left and more than 40 bp to the right of the cross-over region . Disposition of these sequences on the same attachment site in integration, and on different attachment sites in excision, determines the opposite effects of Xis protein upon the two reactions (stimulation of excision and inhibition of integration) . The binding of Xis to two adjacent directly repeated sequences in the left phage arm is shown to occur in a highly cooperative manner, to alter the conformation of the DNA, and to produce a 32-fold stimulation of Int binding to an adjacent locus. J Bacteriol, 1984 Dec, 160(3), 1055 - 60 In vivo evidence for the role of the epsilon subunit as an inhibitor of the proton-translocating ATPase of Escherichia coli; Klionsky DJ et al.; The function of the epsilon subunit of the Escherichia coli proton-translocating ATPase has been examined by using a mutant defective in the uncC gene . Strains with a defective uncC gene show a reduction in both growth yield and growth rate that is more severe than for other unc mutants; this deleterious effect is shown to be a result of the ATPase activity of the F1 complex which is missing the epsilon subunit . In addition, the epsilon-deficient F1 is bound less tightly to the membrane . These data suggest that, in vivo, the epsilon subunit is capable of inhibiting the ATPase activity of F1 and also functions in the binding of F1 to F0. Proc Natl Acad Sci U S A, 1984 Dec, 81(23), 7520 - 4 Circumsporozoite gene of plasmodium cynomolgi (Gombak):cDNA cloning and expression of the repetitive circumsporozoite epitope; Enea V et al.; We report the identification, sequence, and expression in Escherichia coli of the immunodominant epitope of the circumsporozoite (CS) gene of Plasmodium cynomolgi (Gombak), a simian malaria parasite . This epitope is encoded by a DNA sequence that is tandemly repeated 10 times in the cDNA clone . Subclones that contain and express only repeats and in variable number have been constructed . We show that the binding of a specific anti-CS protein monoclonal antibody correlates positively with the number of repeats in each subclone . The CS gene of another strain of P . cynomolgi (NIH) encodes an immunodominant epitope that is immunologically distinct from that of the Gombak strain . We present evidence that these two CS genes share extensive overall homology, although the nucleotide sequences that encode the epitopes appear to be unrelated. Proc Natl Acad Sci U S A, 1984 Dec, 81(23), 7288 - 92 Human U1 RNA genes contain an unusually sensitive nuclease S1 cleavage site within the conserved 3' flanking region; Htun H et al.; We find that the cloned DNAs of human U1 small nuclear RNA genes contain two nuclease S1-sensitive sites, one about 1.8 kilobases downstream of the U1 RNA coding region and the other around 0.3 kilobase upstream . The downstream site is unusually sensitive to the nuclease, being cleaved in both linear and negatively supercoiled DNAs . The extent of cleavage at this site is enhanced at lower pH and reduced concentrations of NaCl; the effects of salt are more apparent on linear than supercoiled DNAs . The nuclease S1 sensitivity of this downstream site is dependent on the presence of the sequence (dC-dT)n X (dA-dG)n, where n = 15-25 . (One gene with n = 5 is resistant to nuclease S1 cleavage in this region.) In contrast, the nuclease S1 site upstream of the coding region is cleaved only when the DNA is supercoiled . This site also has a homopyrimidine X homopurine bias in the DNA strands, but the sequence is less regular . In the course of these studies, we detected several discrepancies between our restriction maps of some U1 RNA genes and those published by others . Our maps demonstrate that all seven cloned human U1 RNA genes are very similar in sequence for as much as 2.3 kilobases downstream of the U1 RNA coding region. Proc Natl Acad Sci U S A, 1984 Dec, 81(23), 7279 - 83 Subunit b of the membrane moiety (F0) of ATP synthase (F1F0) from Escherichia coli is indispensable for H+ translocation and binding of the water-soluble F1 moiety; Schneider E et al.; The ATP synthase complex, designated F1F0, of Escherichia coli is composed of a water-soluble portion (F1; membrane-associated ATPase, EC 3.6.1.3) with ATP-hydrolyzing activity and a membrane-integrated part (F0) with H+-translocating activity . F0 is built up from three kinds of subunits (a, b, and c) . We have isolated the F0 portion directly from membranes of an E . coli strain (KY 7485) that overproduces the enzyme several fold . Subunit b was extracted from purified F0 by two methods . One method included prolonged incubation of the F0 complex in the presence of trichloroacetate (2.5 M) and the separation of subunit b and an a-c complex by gel filtration . Alternatively, subunit b was extracted by deoxycholate and separated from the a-c complex by hydrophobic-interaction chromatography . Integrated into liposomes, the a-c complex exhibited neither H+ uptake nor binding of F1 . However, a functional F0 complex was reconstituted by adding stoichiometric amounts of subunit b to the a-c complex. J Bacteriol, 1984 Dec, 160(3), 1088 - 92 Molecular cloning of the gene (poxB) encoding the pyruvate oxidase of Escherichia coli, a lipid-activated enzyme; Grabau C et al.; The pyruvate oxidase structural gene (poxB) of Escherichia coli was cloned into derivatives of plasmid pBR322 . The gene was first cloned into a cosmid vector by selection for the tetracycline resistance determinant of a closely linked Tn10 insertion (no direct selection for the gene was available) . Subsequent subcloning resulted in localization of the gene to a 3.1-kilobase-pair DNA segment . Two of the smaller poxB plasmids were shown to cause the overproduction of oxidase activity (by six- to eightfold), and one of these plasmids was shown to encode a protein having the size and antigenic determinants of pyruvate oxidase . Introduction of poxB plasmids into strains (aceEF) lacking pyruvate dehydrogenase activity relieved the aerobic growth requirement of the strains for exogenous acetate. Infect Immun, 1984 Dec, 46(3), 759 - 64 Monoclonal antibodies with an expanded repertoire of specificities and potent neutralizing activity for Escherichia coli heat-labile enterotoxin; Belisle BW et al.; Nine selected hybridoma cell lines that produced monoclonal antibodies against the heat-labile enterotoxin encoded by a plasmid from an Escherichia coli strain of human origin (LTh) were characterized . Hybridomas that produced anti-LTh antibodies with previously unrecognized specificities or reactivities were selected for cloning . Each monoclonal antibody was tested for isotype and for binding to LTh holotoxin, the A and B subunits derived from LTh (LTh-A and LTh-B), holotoxin encoded by a plasmid from an E . coli strain of porcine origin (LTp), and cholera enterotoxin (CT) . Binding was also tested in Western blots with the following antigens: pentameric LTh-B, monomeric LTh-B, LTh-A, and the A1 and A2 fragments produced from LTh-A by treatment with trypsin . These monoclonal anti-LTh antibodies and selected anti-LTh and anti-CT monoclonal antibodies described previously were tested for neutralization of LTh, LTp, and CT . Five of the nine new monoclonal antibodies gave detectable cross-reactions with LTp and CT . Four reacted with determinants of LTh that were not present on CT; one of these four did not react with LTp and was specific for a unique epitope of LTh . Three antibodies were specific for LTh-B . All three reacted with pentameric LTh-B, but only one reacted in Western blots with monomeric LTh-B . Six antibodies were specific for LTh-A . Three reacted in Western blots with LTh-A and its A1 fragment; the other three did not react in Western blots . All nine of the new monoclonal antibodies neutralized LTh but not CT; the eight that cross-reacted with LTp in binding assays also neutralized LTp . Of four neutralizing anti-CT monoclonal antibodies that bound to LTh, none had significant neutralizing activity against LTh. Gene, 1984 Dec, 32(1-2), 83 - 90 Nucleotide sequence of the gene encoding the F72 fimbrial subunit of a uropathogenic Escherichia coli strain; van Die I et al.; The cloned DNA fragment encoding the F72 fimbrial subunit from the uropathogenic Escherichia coli strain AD110 has been identified . The nucleotide sequence of the structural gene and of 196 bp of the noncoding region preceding the gene was determined . The structural gene codes for a polypeptide of 188 amino acid residues, including a 21-residue N-terminal signal sequence . The nucleotide sequence and the deduced amino acid sequence of the F72 gene were compared with the reported sequences of the papA gene (Baga et al., 1984) . Both genes code for subunits of fimbriae that are involved in mannose-resistant hemagglutination (MRHA) of human erythrocytes . The available data show that there is absolute homology between the noncoding regions preceding both genes over 129 bp . The two proteins are homologous at the N terminus and C terminus; there is less, but significant, homology in the region between the N and C termini. J Gen Microbiol, 1984 Dec, 130 ( Pt 12), 3071 - 7 Nitrogen regulation of synthesis of the high affinity methylammonium transport system of Escherichia coli; Servin-Gonzalez L et al.; Uptake of 14CH3NH+3 (methylammonium) was measured as a probe of NH+4 transport in intact Escherichia coli cells and derivatives impaired in the Ntr regulatory system . The results suggest that expression of the high affinity 14CH3NH+3 transport system (a) requires de novo polypeptide synthesis, (b) is activated by the glnG and glnF regulatory products under nitrogen limitation, and (c) is repressed under nitrogen excess by the glnL product . Cells deficient in glutamate synthase activity by virtue of their harbouring the gltB31 mutation were unable to activate synthesis of 14CH3NH+3 transport . This could explain the inability of cells carrying gltB mutations to grow on low concentrations of NH+4. J Bioenerg Biomembr, 1984 Dec, 16(5-6), 477 - 89 Activation studies by phospholipids on the purified cytochrome c4:o oxidase of Azotobacter vinelandii; Wong TY et al.; A modified procedure is described that was used to solubilize and purify the TMPD-dependent cytochrome c4:o oxidase from Azotobacter vinelandii . Two functional components (Fractions I and V) were obtained after DEAE-cellulose chromatography . Fraction V contained both cytochrome c4 (3.6 nmol/mg protein) and cytochrome o (1.6 nmol/mg protein) . This cytochrome oxidase complex oxidized TMPD at "moderate" rates . Fraction I, a clear greenish-yellow fraction, contained primarily phosphatidylethanolamine with some phosphatidylglycerol . Fraction I itself could not oxidize TMPD, but when it was preincubated with Fraction V, a 2-4-fold stimulation in TMPD oxidase activity occurred . Other "authentic" micellar phospholipids also readily activated TMPD oxidase activity in Fraction V . The maximum activation effect obtained with Fraction I was in essence duplicated with purified phosphatidylethanolamine. Gene, 1984 Dec, 32(3), 481 - 5 The pIC plasmid and phage vectors with versatile cloning sites for recombinant selection by insertional inactivation; Marsh JL et al.; The versatility of insertional inactivation of beta-galactosidase activity for subcloning and sequencing has been enhanced by combining a chemically synthesized oligonucleotide which specifies nine 6-bp-cutter restriction sites including BglII, XhoI, NruI, ClaI, SacI and EcoRV in various configurations with existing polylinkers to create a set of highly versatile cloning sites . These improved polylinkers have been inserted into plasmids (the pICs) for routine cloning of double-stranded DNA, and into chimeric phage/plasmids (the pICEMs) for biological production of single stranded DNA . The most versatile polylinker specifies 17 restriction sites in the beta-galactosidase alpha-complementing gene fragment . One of the new polylinkers was inserted into M13 DNA to produce a vector (M13mIC7) with nine cloning sites. Gene, 1984 Dec, 32(3), 399 - 408 Genetic and physical analysis of the thioredoxin (trxA) gene of Escherichia coli K-12; Wallace BJ et al.; The trxA gene of Escherichia coli K-12 has been cloned into multicopy plasmids on DNA fragments of varying sizes . The smallest of these was a 1-kb fragment resulting from partial digestion with Sau3A (pBHK10) . The complete nucleotide sequence of the trxA gene and its promoter was determined . Comparison of the DNA sequence with the published amino acid sequence revealed the inversion of two amino acid pairs and the possibility of a leader peptide 18 amino acids in length . Three-factor P1 transductional crosses and physical mapping experiments have determined a map order of ilv-trxA-uvrD-corA-metE. Gene, 1984 Dec, 32(3), 369 - 79 New Tn10 derivatives for transposon mutagenesis and for construction of lacZ operon fusions by transposition; Way JC et al.; We describe below several new variants of the tetracycline-resistance transposon Tn10 which are more useful than the wild-type transposon for many types of genetic and physical analysis of bacteria . These derivatives have one or more of the following new properties: (i) new drug resistance markers; (ii) high transposition frequencies; (iii) removal of the transposase gene to a position outside of the transposing segment; (iv) internal deletions which eliminate the ability of Tn10 to make adjacent deletion/inversions; or (v) addition of a trp-lac operon fusion segment just inside one terminus such that insertion can automatically generate a transcriptional fusion to the interrupted operon . Phage and plasmid vehicles carrying these new elements are described. Gene, 1984 Dec, 32(3), 337 - 48 Role of DNA regions flanking the tryptophan promoter of Escherichia coli . I . Insertion of synthetic oligonucleotides; Russell DR et al.; To examine the effect of altering the nucleotide sequence near the promoter on its activity, pKO-1 vector derivatives have been constructed which allow insertion of DNA fragments at specified sites upstream or downstream from the trp promoter . Oligonucleotides that might be expected to alter the melting properties, or have a tendency to form a distinctive nonstandard structure were introduced . These oligonucleotides had the repeating dinucleotide sequences GC, AT or AG . Sequence analysis of the inserts and studies of the relative galactokinase expression from the altered plasmids indicated that changes upstream from the trp promoter at -39 or beyond had little effect on trp promoter activity, whereas changes at +2 or farther downstream produced up to two-fold increases in gene expression, as compared to the control plasmid. Gene, 1984 Dec, 32(3), 311 - 20 Physical and genetic map of the organomercury resistance (Omr) and inorganic mercury resistance (Hgr) loci of the IncM plasmid R831b; Ogawa HI et al.; Tn7 insertion mutagenesis has been used to facilitate the generation of a physical (restriction endonuclease) and genetic map of the IncM plasmid, R831b . The only selectable phenotypes carried by this 90-kb conjugative plasmid are resistances to inorganic mercury {Hg(II)} and to organomercury compounds . Mutants in the Hgr locus of R831b complemented previously described mutants in the mer operon of the IncFII plasmid R100, indicating functional homology of the locus in each of these different plasmids . However, the R831b Hgr locus is not notably similar in restriction site pattern to either the mer operon of R100 or the mercury resistance transposon, Tn501 . Although the enzymes they encode are co-ordinately regulated, the Omr locus of R831b maps approx . 13.5 kb away from the Hgr locus . Three insertions which affect neither phenotype lie between the Hgr and Omr loci; thus, the loci are separated both physically and genetically . One mutant was obtained which tentatively identifies the position of the Tra locus of R831b as adjacent to the Hgr locus. Gene, 1984 Dec, 32(1-2), 99 - 106 Cloning the cyd gene locus coding for the cytochrome d complex of Escherichia coli; Green GN et al.; Two plasmids containing the two structural genes for the inner-membrane-bound cytochrome d complex (Cyd) have been isolated from the Clarke and Carbon Escherichia coli DNA bank . A 5.4-kb DNA fragment from one plasmid was subcloned in both orientations into pBR322 . The promoter(s) and both genes must have been present within this fragment since the two orientations yielded similar levels of Cyd . Recombination and transduction studies indicated that the cyd gene locus had been isolated . These results demonstrate that cyd contains all the structural information for the complex . Overproduction of Cyd has yielded a visual screening procedure for plasmids bearing cyd that is unique to colored proteins like cytochromes . Colonies of E . coli bearing the cloned cyd gene are yellow-green . The cyd gene can, therefore, be used as a vehicle for detection of inserted DNA fragments. Gene, 1984 Dec, 32(1-2), 91 - 8 Kinetics of Tn5 transposition; Rossetti OL et al.; The kinetics of Tn5 transposition and gene expression were studied . For about 2 h after infection with lambda Tn5, Tn5 transpositions accumulate, reaching a level of about 1.5% of the infected cells . After 2 h transposition is essentially turned off . In cells carrying a resident Tn5, transposition is undetectable after infection . The synthesis of the Tn5-specific proteins p58 and p54 and the kanamycin-resistance protein were studied in pre-irradiated cells infected with lambda Tn5 . The synthesis of p58 and p54 peaked early after infection and was significantly reduced, relative to pneo, by 2 h after infection . Moreover, p54 appeared to reach a maximum later than p58 . These kinetic data put new constraints on models for the regulation of Tn5 transposition. Gene, 1984 Dec, 32(1-2), 31 - 40 Analysis of the ptsH-ptsI-crr region in Escherichia coli K-12: evidence for the existence of a single transcriptional unit; De Reuse H et al.; A cosmid containing the ptsH, ptsI and crr genes of Escherichia coli coding for three components of the phosphoenolpyruvate: sugar phosphotransferase system (PTS) has been isolated . The products of these genes were identified using the maxicell technique . The cloning of the PTS region as a whole allowed us to map and order ptsH, ptsI and crr genes in a 3.8-kb DNA fragment and determine that ptsI and crr are transcribed from a common promoter . They, therefore, constitute a single transcriptional unit which is likely to include also the ptsH gene . In addition, the crr gene may have a secondary promoter located inside ptsI. Gene, 1984 Dec, 32(1-2), 251 - 4 F plasmid pif region: Tn1725 mutagenesis and polypeptide analysis; Cram HK et al.; Analysis of Tn1725 insertions in the Pif+ plasmid pRS2496 showed the maximum limits of the F pif region to be between 43.7 and 47.15 on the 100-kb map of the F plasmid . The effect of these insertions on the expression of pif polypeptides indicated that two of the pif genes, pifA and pifC, lie within a polycistronic operon. Gene, 1984 Dec, 32(1-2), 203 - 15 Expression of Herpes simplex virus type 1 glycoprotein C antigens in Escherichia coli; Amann E et al.; DNA fragments encoding structural information of the Herpes simplex virus type 1 (HSV-1) glycoprotein C (gC) gene were cloned into pUC plasmids {Vieira and Messing, Gene 19 (1982) 259-268} . None of the hybrid plasmids were able to direct the synthesis of significant amounts of gC related peptides . Several of the plasmid-bearing strains, however, exhibited inhibition characteristics which can be correlated with the presence on the plasmid of specific gC gene sequences . After insertion of gC DNA fragments into expression vector pMF2 between phage lambda repressor gene cI and lacZ, significant amounts of cI::gC::beta-galactosidase fusion proteins are synthesized . These tripartite fusion proteins are immunologically reactive with anti-HSV-1 antisera . The expression system based on pMF2 can be generally used to identify and express foreign antigens in Escherichia coli. Can J Biochem Cell Biol, 1984 Dec, 62(12), 1275 - 82 Rapid sensitive fluorescence assays for DNA endonucleases and DNA glycosylases; Evans DH et al.; Using ethidium fluorescence as a probe and the unique topological properties of covalently closed circular (CCC) DNA's picogram quantities of pancreatic DNase I can be detected . By chemically modifying CCC DNA's without the introduction of nicks, various DNA repair enzymes can be detected even in crude cell extracts . This has previously been done for AP endonucleases from yeast . In this paper we describe the formation of ultraviolet (UV) and bisulfite modified CCC DNA's for the detection of T4 UV endonuclease (a DNA glycosylase + AP endonuclease) and uracil-DNA glycosylases, respectively . These assays should be useful in the isolation of DNA repair mutants. Mol Cell Biol, 1984 Dec, 4(12), 2573 - 9 Stimuli that induce a yeast heat shock gene fused to beta-galactosidase; Brazzell C et al.; Saccharomyces cerevisiae contain a multigene family related to the Drosophila heat shock gene hsp70 . Two members of this family, YG100 and YG101, have been previously characterized (Ingolia et al., Mol . Cell . Biol . 2:1388-1398, 1982), and only YG100 was found to have elevated levels of transcription after heat shock . The yeast hsp70 genes contained on YG100 and YG101 were truncated and fused to the Escherichia coli lacZ gene contained on pMC1587 (Casadaban et al., Methods Enzymol . 100:283-308, 1983) . The resulting plasmids directed synthesis of the beta-galactosidase gene as measured by in vitro enzyme assays and by colorimetric assays on plates . The expression level from the YG101 gene was constant under all the conditions tested, whereas expression driven by the YG100 gene could be induced over 50-fold . Other stimuli besides heat, including recovery from anoxia and high cell density, were found to strongly induce YG100 gene expression . Most physical and chemical stimuli tested, including UV irradiation, zymolyase treatment, and ethanol, did not stimulate expression of this heat shock gene. EMBO J, 1984 Dec 1, 3(12), 2917 - 22 Covalently closed circles of human adenovirus DNA are infectious; Graham FL; Replication of the linear adenovirus DNA molecule is thought to result from semiconservative synthesis off linear templates, starting from origins at either end of the genome . Recently, however, it has been shown that in cells infected with adenovirus type 5 (Ad5) a significant fraction of the ends of viral DNA molecules become joined head-to-tail due at least in part to the formation of covalently closed circles . Circular DNA is not present in virions but joining of the ends of viral DNA is detectable shortly after infection, well before the onset of viral DNA replication . To learn more about the structure and possible function of these circular forms of viral DNA, I have cloned Ad5 circles as plasmids replicating in Escherichia coli . Two plasmids have been analyzed in detail and shown to generate infectious virus with an efficiency comparable with that of virion DNA following transfection into human cells . These results suggest that circles are